register interest

Professor Colin R Goding

Research Area: Cell and Molecular Biology
Technology Exchange: Immunohistochemistry, In situ hybridisation and Transcript profiling
Scientific Themes: Cancer Biology and Physiology, Cellular & Molecular Biology
Keywords: melanocytes, melanoma, stem cells, transcription, Signalling and cancer
Web Links:
Heterogeneity of melanoma cells within a tumour stained with anti-Brn2 (red) and anti-Mitf (green)

Heterogeneity of melanoma cells within a tumour stained with anti-Brn2 (red) and anti-Mitf (green)

Tbx2 associated with small subnuclear heterochromatin foci stained with anti-Tbx2 (green) and tri-methyl K9 histone H3 (red)

Tbx2 associated with small subnuclear heterochromatin foci stained with anti-Tbx2 (green) and ...

Left: melanoma cell; right: invasive melanoma stem cell-like cell stained for Diaph1 (green) and actin red

Left: melanoma cell; right: invasive melanoma stem cell-like cell stained for Diaph1 (green) and ...

The interface between signal transduction pathways and transcription regulation acts to coordinate the programme of gene expression underlying the genesis of specific cell lineages and cellular proliferation and differentiation during development. Deregulation of transcription is a key factor in cancer. Our laboratory is interested in determining how a precise programme of transcription regulation is achieved, particularly in the transition between normal and cancer stem cells and their proliferating progeny, and the parallels with normal stem cell populations.

Over recent years we have made use of melanocytes and melanoma as a model system.

Melanocytes offer an interesting developmental system as mutations affecting the lineage result in an obvious coat colour phenotype, yet do not affect the viability of the animal. Moreover, melanoma, originating in the skin, offers a unique opportunity to examine a cancer at all stages of its development from benign naevi/moles to highly aggressive metastatic tumours.

We have recently proposed a new model for melanoma metastasis that reflects a dynamic and reversible epigenetic mechanism rather than irreversible genetic lesions, and have identified the transcription factor Mitf as a master regulator of melanoma proliferation, differentiation and invasiveness. We are currently analyzing how Mitf integrates a wide range of signals to give a variable biological output and how this relates to melanoma stem cells that are proposed to provide a therapeutically resistant sub-population of cancer cells.

Our most recent work has revealed how cancer progression, including invasion, drug- and immunotherapy-resistance and senescence bypass  can be explained by cells trying to adapt to changes in nutrient supply and demand. In this model, low supply of of nutrients leads to global suppression of translation to reduce demand, including suppression of MITF expression. At the same time cells increase translation of factors required to fix any supply demand imbalance. Invasion in response to starvation makes sense, since many organisms from bacteria, yeast and humans migrate or invade in response to starvation.

Our current work is focussed on understanding how changes in nutrient supply-demand affect melanoma progression, and how other non-nutritional signals such as inflammation hijack this response to nutritional limitation to impose a pseudo-starvation state.

To address these questions we a wide range of cutting edge techniques including real-time imaging, CRISPR-mediated gene editing, genome wide ChIP and RNA-seq, Mass Spec and metabolomics.

Name Department Institution Country
Lionel Larue Institute Curie France
Eirkur Steingrimsson University of Iceland Iceland
Luisa Lanfrancone Euorpean Instute of Oncology, Milan Italy
Dr Irwin Davidson IGBMC France
Rutao Cui Boston University United States
Sharon Prince University of Cape Town South Africa
Marie-Dominique Galibert University of Rennes France
Veronique Delmas Institut Curie France
Professor Panagis Filippakopoulos Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Professor Sarah De Val Oxford Ludwig Institute Oxford University, Old Road Campus Research Building United Kingdom
Professor Xin Lu Oxford Ludwig Institute Oxford University, Old Road Campus Research Building United Kingdom
Jane Mellor University of Oxford United Kingdom
Anne Willis MRC Toxicology Unit United Kingdom
JP Martinez-Barbera Institute of Child Health United Kingdom
Liz Patton MRC Institute of Genetics and Molecular Medicine United Kingdom
Mark Middleton Department of Oncology, University of Oxford United Kingdom
Vincenzo D'Angiolella Oncology University of Oxford United Kingdom
Herbert K, Binet R, Lambert J-P, Louphrasitthiphol P, Kalkavan H, Sesma-Sanz L, Robles-Espinoza CD, Sarkar S, Suer E, Andrews S et al. 2019. BRN2 suppresses apoptosis, reprograms DNA damage repair, and is associated with a high somatic mutation burden in melanoma. Genes Dev, 33 (5-6), pp. 310-332. | Show Abstract | Read more

Whether cell types exposed to a high level of environmental insults possess cell type-specific prosurvival mechanisms or enhanced DNA damage repair capacity is not well understood. BRN2 is a tissue-restricted POU domain transcription factor implicated in neural development and several cancers. In melanoma, BRN2 plays a key role in promoting invasion and regulating proliferation. Here we found, surprisingly, that rather than interacting with transcription cofactors, BRN2 is instead associated with DNA damage response proteins and directly binds PARP1 and Ku70/Ku80. Rapid PARP1-dependent BRN2 association with sites of DNA damage facilitates recruitment of Ku80 and reprograms DNA damage repair by promoting Ku-dependent nonhomologous end-joining (NHEJ) at the expense of homologous recombination. BRN2 also suppresses an apoptosis-associated gene expression program to protect against UVB-, chemotherapy- and vemurafenib-induced apoptosis. Remarkably, BRN2 expression also correlates with a high single-nucleotide variation prevalence in human melanomas. By promoting error-prone DNA damage repair via NHEJ and suppressing apoptosis of damaged cells, our results suggest that BRN2 contributes to the generation of melanomas with a high mutation burden. Our findings highlight a novel role for a key transcription factor in reprogramming DNA damage repair and suggest that BRN2 may impact the response to DNA-damaging agents in BRN2-expressing cancers.

Chen S, Han C, Miao X, Li X, Yin C, Zou J, Liu M, Li S, Stawski L, Zhu B et al. 2019. Targeting MC1R depalmitoylation to prevent melanomagenesis in redheads. Nat Commun, 10 (1), pp. 877. | Show Abstract | Read more

Some genetic melanocortin-1 receptor (MC1R) variants responsible for human red hair color (RHC-variants) are consequently associated with increased melanoma risk. Although MC1R signaling is critically dependent on its palmitoylation primarily mediated by the ZDHHC13 protein-acyl transferase, whether increasing MC1R palmitoylation represents a viable therapeutic target to limit melanomagenesis in redheads is unknown. Here we identify a specific and efficient in vivo strategy to induce MC1R palmitoylation for therapeutic benefit. We validate the importance of ZDHHC13 to MC1R signaling in vivo by targeted expression of ZDHHC13 in C57BL/6J-MC1RRHC mice and subsequently inhibit melanomagenesis. By identifying APT2 as the MC1R depalmitoylation enzyme, we are able to demonstrate that administration of the selective APT2 inhibitor ML349 treatment efficiently increases MC1R signaling and represses UVB-induced melanomagenesis in vitro and in vivo. Targeting APT2, therefore, represents a preventive/therapeutic strategy to reduce melanoma risk, especially in individuals with red hair.

Yin C, Zhu B, Zhang T, Liu T, Chen S, Liu Y, Li X, Miao X, Li S, Mi X et al. 2019. Pharmacological Targeting of STK19 Inhibits Oncogenic NRAS-Driven Melanomagenesis. Cell, 176 (5), pp. 1113-1127.e16. | Show Abstract | Read more

Activating mutations in NRAS account for 20%-30% of melanoma, but despite decades of research and in contrast to BRAF, no effective anti-NRAS therapies have been forthcoming. Here, we identify a previously uncharacterized serine/threonine kinase STK19 as a novel NRAS activator. STK19 phosphorylates NRAS to enhance its binding to its downstream effectors and promotes oncogenic NRAS-mediated melanocyte malignant transformation. A recurrent D89N substitution in STK19 whose alterations were identified in 25% of human melanomas represents a gain-of-function mutation that interacts better with NRAS to enhance melanocyte transformation. STK19D89N knockin leads to skin hyperpigmentation and promotes NRASQ61R-driven melanomagenesis in vivo. Finally, we developed ZT-12-037-01 (1a) as a specific STK19-targeted inhibitor and showed that it effectively blocks oncogenic NRAS-driven melanocyte malignant transformation and melanoma growth in vitro and in vivo. Together, our findings provide a new and viable therapeutic strategy for melanomas harboring NRAS mutations.

García-Jiménez C, Goding CR. 2019. Starvation and Pseudo-Starvation as Drivers of Cancer Metastasis through Translation Reprogramming. Cell Metab, 29 (2), pp. 254-267. | Show Abstract | Read more

Considerable progress has been made in identifying microenvironmental signals that effect the reversible phenotypic transitions underpinning the early steps in the metastatic cascade. However, although the general principles underlying metastatic dissemination have been broadly outlined, a common theme that unifies many of the triggers of invasive behavior in tumors has yet to emerge. Here we discuss how many diverse signals that induce invasion converge on the reprogramming of protein translation via phosphorylation of eIF2α, a hallmark of the starvation response. These include starvation as a consequence of nutrient or oxygen limitation, or pseudo-starvation imposed by cell-extrinsic microenvironmental signals or by cell-intrinsic events, including oncogene activation. Since in response to resource limitation single-cell organisms undergo phenotypic transitions remarkably similar to those observed within tumors, we propose that a starvation/pseudo-starvation model to explain cancer progression provides an integrated and evolutionarily conserved conceptual framework to understand the progression of this complex disease.

Ngeow KC, Friedrichsen HJ, Li L, Zeng Z, Andrews S, Volpon L, Brunsdon H, Berridge G, Picaud S, Fischer R et al. 2018. BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export. Proc Natl Acad Sci U S A, 115 (37), pp. E8668-E8677. | Show Abstract | Read more

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Li L, Friedrichsen HJ, Andrews S, Picaud S, Volpon L, Ngeow K, Berridge G, Fischer R, Borden KLB, Filippakopoulos P, Goding CR. 2018. A TFEB nuclear export signal integrates amino acid supply and glucose availability. Nat Commun, 9 (1), pp. 2685. | Show Abstract | Read more

How cells coordinate the response to fluctuating carbon and nitrogen availability required to maintain effective homeostasis is a key issue. Amino acid limitation that inactivates mTORC1 promotes de-phosphorylation and nuclear translocation of Transcription Factor EB (TFEB), a key transcriptional regulator of lysosome biogenesis and autophagy that is deregulated in cancer and neurodegeneration. Beyond its cytoplasmic sequestration, how TFEB phosphorylation regulates its nuclear-cytoplasmic shuttling, and whether TFEB can coordinate amino acid supply with glucose availability is poorly understood. Here we show that TFEB phosphorylation on S142 primes for GSK3β phosphorylation on S138, and that phosphorylation of both sites but not either alone activates a previously unrecognized nuclear export signal (NES). Importantly, GSK3β is inactivated by AKT in response to mTORC2 signaling triggered by glucose limitation. Remarkably therefore, the TFEB NES integrates carbon (glucose) and nitrogen (amino acid) availability by controlling TFEB flux through a nuclear import-export cycle.

Zhu B, Tang L, Chen S, Yin C, Peng S, Li X, Liu T, Liu W, Han C, Stawski L et al. 2018. Targeting the upstream transcriptional activator of PD-L1 as an alternative strategy in melanoma therapy. Oncogene, 37 (36), pp. 4941-4954. | Show Abstract | Read more

Programmed cell death ligand 1 (PD-L1) interacts with programmed cell death protein-1 (PD-1) as an immune checkpoint. Reactivating the immune response by inhibiting PD-L1 using therapeutic antibodies provides substantial clinical benefits in many, though not all, melanoma patients. However, transcriptional suppression of PD-L1 expression as an alternative therapeutic anti-melanoma strategy has not been exploited. Here we provide biochemical evidence demonstrating that ultraviolet radiation (UVR) induction of PD-L1 in skin is directly controlled by nuclear factor E2-related transcription factor 2 (NRF2). Depletion of NRF2 significantly induces tumor infiltration by both CD8+ and CD4+ T cells to suppress melanoma progression, and combining NRF2 inhibition with anti-PD-1 treatment enhanced its anti-tumor function. Our studies identify a critical and targetable PD-L1 upstream regulator and provide an alternative strategy to inhibit the PD-1/PD-L1 signaling in melanoma treatment.

Apps JR, Carreno G, Gonzalez-Meljem JM, Haston S, Guiho R, Cooper JE, Manshaei S, Jani N, Hölsken A, Pettorini B et al. 2018. Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target. Acta Neuropathol, 135 (5), pp. 757-777. | Show Abstract | Read more

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

Zhu B, Chen S, Wang H, Yin C, Han C, Peng C, Liu Z, Wan L, Zhang X, Zhang J et al. 2018. The protective role of DOT1L in UV-induced melanomagenesis. Nat Commun, 9 (1), pp. 259. | Show Abstract | Read more

The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.

Goding CR. 2018. Cell and molecular biology: a new section joins the fight against cancer. Br J Cancer, 118 (1), pp. 1-2. | Read more

Louphrasitthiphol P, Goding CR. 2017. Macrophage Cytoplasmic Transfer in Melanoma Invasion. Dev Cell, 43 (5), pp. 543-544. | Show Abstract | Read more

Within tumors, macrophage infiltration can promote cancer cell invasiveness and, consequently, metastatic dissemination. In this issue of Developmental Cell, Roh-Johnson et al. (2017) reveal that cytoplasmic transfer from macrophages to melanoma cells correlates with melanoma invasion and arises as a result of intimate cell-cell contact.

Gonzalez-Meljem JM, Haston S, Carreno G, Apps JR, Pozzi S, Stache C, Kaushal G, Virasami A, Panousopoulos L, Mousavy-Gharavy SN et al. 2017. Stem cell senescence drives age-attenuated induction of pituitary tumours in mouse models of paediatric craniopharyngioma. Nat Commun, 8 (1), pp. 1819. | Show Abstract | Read more

Senescent cells may promote tumour progression through the activation of a senescence-associated secretory phenotype (SASP), whether these cells are capable of initiating tumourigenesis in vivo is not known. Expression of oncogenic β-catenin in Sox2+ young adult pituitary stem cells leads to formation of clusters of stem cells and induction of tumours resembling human adamantinomatous craniopharyngioma (ACP), derived from Sox2- cells in a paracrine manner. Here, we uncover the mechanisms underlying this paracrine tumourigenesis. We show that expression of oncogenic β-catenin in Hesx1+ embryonic precursors also results in stem cell clusters and paracrine tumours. We reveal that human and mouse clusters are analogous and share a common signature of senescence and SASP. Finally, we show that mice with reduced senescence and SASP responses exhibit decreased tumour-inducing potential. Together, we provide evidence that senescence and a stem cell-associated SASP drive cell transformation and tumour initiation in vivo in an age-dependent fashion.

Chen S, Zhu B, Yin C, Liu W, Han C, Chen B, Liu T, Li X, Chen X, Li C et al. 2017. Palmitoylation-dependent activation of MC1R prevents melanomagenesis. Nature, 549 (7672), pp. 399-403. | Show Abstract | Read more

The melanocortin-1 receptor (MC1R), a G-protein-coupled receptor, has a crucial role in human and mouse pigmentation. Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH) stimulates cAMP signalling and melanin production and enhances DNA repair after ultraviolet irradiation. Individuals carrying MC1R variants, especially those associated with red hair colour, fair skin and poor tanning ability (denoted as RHC variants), are associated with higher risk of melanoma. However, how MC1R activity is modulated by ultraviolet irradiation, why individuals with red hair are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit are unknown. Here we demonstrate a potential MC1R-targeted intervention strategy in mice to rescue loss-of-function MC1R in MC1R RHC variants for therapeutic benefit by activating MC1R protein palmitoylation. MC1R palmitoylation, primarily mediated by the protein-acyl transferase ZDHHC13, is essential for activating MC1R signalling, which triggers increased pigmentation, ultraviolet-B-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo. Using C57BL/6J-Mc1re/eJ mice, in which endogenous MC1R is prematurely terminated, expressing Mc1r RHC variants, we show that pharmacological activation of palmitoylation rescues the defects of Mc1r RHC variants and prevents melanomagenesis. The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.

Phung B, Kazi JU, Lundby A, Bergsteinsdottir K, Sun J, Goding CR, Jönsson G, Olsen JV, Steingrímsson E, Rönnstrand L. 2017. KITD816V Induces SRC-Mediated Tyrosine Phosphorylation of MITF and Altered Transcription Program in Melanoma. Mol Cancer Res, 15 (9), pp. 1265-1274. | Show Abstract | Read more

The oncogenic D816V mutation of the KIT receptor is well characterized in systemic mastocytosis and acute myeloid leukemia. Although KITD816V has been found in melanoma, its function and involvement in this malignancy is not understood. Here we show that KITD816V induces tyrosine phosphorylation of MITF through a triple protein complex formation between KIT, MITF, and SRC family kinases. In turn, phosphorylated MITF activates target genes that are involved in melanoma proliferation, cell-cycle progression, suppression of senescence, survival, and invasion. By blocking the triple protein complex formation, thus preventing MITF phosphorylation, the cells became hypersensitive to SRC inhibitors. We have therefore delineated a mechanism behind the oncogenic effects of KITD816V in melanoma and provided a rationale for the heightened SRC inhibitor sensitivity in KITD816V transformed cells.Implications: This study demonstrates that an oncogenic tyrosine kinase mutant, KITD816V, can alter the transcriptional program of the transcription factor MITF in melanoma Mol Cancer Res; 15(9); 1265-74. ©2017 AACR.

Falletta P, Sanchez-Del-Campo L, Chauhan J, Effern M, Kenyon A, Kershaw CJ, Siddaway R, Lisle R, Freter R, Daniels MJ et al. 2017. Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma. Genes Dev, 31 (1), pp. 18-33. | Show Abstract | Read more

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

Fane ME, Chhabra Y, Hollingsworth DEJ, Simmons JL, Spoerri L, Oh TG, Chauhan J, Chin T, Harris L, Harvey TJ et al. 2017. NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF. EBioMedicine, 16 pp. 63-75. | Show Abstract | Read more

While invasion and metastasis of tumour cells are the principle factor responsible for cancer related deaths, the mechanisms governing the process remain poorly defined. Moreover, phenotypic divergence of sub-populations of tumour cells is known to underpin alternative behaviors linked to tumour progression such as proliferation, survival and invasion. In the context of melanoma, heterogeneity between two transcription factors, BRN2 and MITF, has been associated with phenotypic switching between predominantly invasive and proliferative behaviors respectively. Epigenetic changes, in response to external cues, have been proposed to underpin this process, however the mechanism by which the phenotypic switch occurs is unclear. Here we report the identification of the NFIB transcription factor as a novel downstream effector of BRN2 function in melanoma cells linked to the migratory and invasive characteristics of these cells. Furthermore, the function of NFIB appears to drive an invasive phenotype through an epigenetic mechanism achieved via the upregulation of the polycomb group protein EZH2. A notable target of NFIB mediated up-regulation of EZH2 is decreased MITF expression, which further promotes a less proliferative, more invasive phenotype. Together our data reveal that NFIB has the ability to promote dynamic changes in the chromatin state of melanoma cells to facilitate migration, invasion and metastasis.

Savitsky P, Krojer T, Fujisawa T, Lambert J-P, Picaud S, Wang C-Y, Shanle EK, Krajewski K, Friedrichsen H, Kanapin A et al. 2016. Multivalent Histone and DNA Engagement by a PHD/BRD/PWWP Triple Reader Cassette Recruits ZMYND8 to K14ac-Rich Chromatin. Cell Rep, 17 (10), pp. 2724-2737. | Show Abstract | Read more

Elucidation of interactions involving DNA and histone post-translational-modifications (PTMs) is essential for providing insights into complex biological functions. Reader assemblies connected by flexible linkages facilitate avidity and increase affinity; however, little is known about the contribution to the recognition process of multiple PTMs because of rigidity in the absence of conformational flexibility. Here, we resolve the crystal structure of the triple reader module (PHD-BRD-PWWP) of ZMYND8, which forms a stable unit capable of simultaneously recognizing multiple histone PTMs while presenting a charged platform for association with DNA. Single domain disruptions destroy the functional network of interactions initiated by ZMYND8, impairing recruitment to sites of DNA damage. Our data establish a proof of principle that rigidity can be compensated by concomitant DNA and histone PTM interactions, maintaining multivalent engagement of transient chromatin states. Thus, our findings demonstrate an important role for rigid multivalent reader modules in nucleosome binding and chromatin function.

Pérez-Guijarro E, Karras P, Cifdaloz M, Martínez-Herranz R, Cañón E, Graña O, Horcajada-Reales C, Alonso-Curbelo D, Calvo TG, Gómez-López G et al. 2016. Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers. Nat Commun, 7 (1), pp. 13418. | Show Abstract | Read more

Nuclear 3'-end-polyadenylation is essential for the transport, stability and translation of virtually all eukaryotic mRNAs. Poly(A) tail extension can also occur in the cytoplasm, but the transcripts involved are incompletely understood, particularly in cancer. Here we identify a lineage-specific requirement of the cytoplasmic polyadenylation binding protein 4 (CPEB4) in malignant melanoma. CPEB4 is upregulated early in melanoma progression, as defined by computational and histological analyses. Melanoma cells are distinct from other tumour cell types in their dependency on CPEB4, not only to prevent mitotic aberrations, but to progress through G1/S cell cycle checkpoints. RNA immunoprecipitation, sequencing of bound transcripts and poly(A) length tests link the melanoma-specific functions of CPEB4 to signalling hubs specifically enriched in this disease. Essential in these CPEB4-controlled networks are the melanoma drivers MITF and RAB7A, a feature validated in clinical biopsies. These results provide new mechanistic links between cytoplasmic polyadenylation and lineage specification in melanoma.

Lu M, Breyssens H, Salter V, Zhong S, Hu Y, Baer C, Ratnayaka I, Sullivan A, Brown NR, Endicott J et al. 2016. Restoring p53 Function in Human Melanoma Cells by Inhibiting MDM2 and Cyclin B1/CDK1-Phosphorylated Nuclear iASPP. Cancer Cell, 30 (5), pp. 822-823. | Show Abstract | Read more

© 2016 Elsevier Inc. (Cancer Cell 23, 618–633; May 13, 2013) In the original Figure 4G, two of the upper panels (His-iASPP(625-828)-FITC; the two rightmost panels) were inadvertently duplicated in the lower set of panels (His-ASPP2(905-1128)-FITC; third and fourth from left). This was a mistake made by the authors during the assembly of the figure. This error does not affect any of the findings reported in the paper. The corrected Figure 4 is presented below. The authors apologize for any confusion that this error may have caused. [figure presented]

Faião-Flores F, Alves-Fernandes DK, Pennacchi PC, Sandri S, Vicente ALSA, Scapulatempo-Neto C, Vazquez VL, Reis RM, Chauhan J, Goding CR et al. 2017. Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells. Oncogene, 36 (13), pp. 1849-1861. | Show Abstract | Read more

BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.

Goding CR. 2016. Targeting the lncRNA SAMMSON Reveals Metabolic Vulnerability in Melanoma. Cancer Cell, 29 (5), pp. 619-621. | Show Abstract | Read more

In a recent study, Leucci et al. report a role for the long non-coding RNA SAMMSON in driving mitochondrial function in melanoma. Targeting SAMMSON, the gene of which is frequently co-amplified with MITF, highlights a new cell-type-specific therapeutic vulnerability in melanoma irrespective of BRAF, NRAS, or p53 status.

Stracquadanio G, Wang X, Wallace MD, Grawenda AM, Zhang P, Hewitt J, Zeron-Medina J, Castro-Giner F, Tomlinson IP, Goding CR et al. 2016. The importance of p53 pathway genetics in inherited and somatic cancer genomes. Nat Rev Cancer, 16 (4), pp. 251-265. | Show Abstract | Read more

Decades of research have shown that mutations in the p53 stress response pathway affect the incidence of diverse cancers more than mutations in other pathways. However, most evidence is limited to somatic mutations and rare inherited mutations. Using newly abundant genomic data, we demonstrate that commonly inherited genetic variants in the p53 pathway also affect the incidence of a broad range of cancers more than variants in other pathways. The cancer-associated single nucleotide polymorphisms (SNPs) of the p53 pathway have strikingly similar genetic characteristics to well-studied p53 pathway cancer-causing somatic mutations. Our results enable insights into p53-mediated tumour suppression in humans and into p53 pathway-based cancer surveillance and treatment strategies.

Vlahov N, Scrace S, Soto MS, Grawenda AM, Bradley L, Pankova D, Papaspyropoulos A, Yee KS, Buffa F, Goding CR et al. 2015. Alternate RASSF1 Transcripts Control SRC Activity, E-Cadherin Contacts, and YAP-Mediated Invasion. Curr Biol, 25 (23), pp. 3019-3034. | Show Abstract | Read more

Tumor progression to invasive carcinoma is associated with activation of SRC family kinase (SRC, YES, FYN) activity and loss of cellular cohesion. The hippo pathway-regulated cofactor YAP1 supports the tumorigenicity of RAS mutations but requires both inactivation of hippo signaling and YES-mediated phosphorylation of YAP1 for oncogenic activity. Exactly how SRC kinases are activated and hippo signaling is lost in sporadic human malignancies remains unknown. Here, we provide evidence that hippo-mediated inhibition of YAP1 is lost upon promoter methylation of the RAS effector and hippo kinase scaffold RASSF1A. We find that RASSF1A promoter methylation reduces YAP phospho-S127, which derepresses YAP1, and actively supports YAP1 activation by switching RASSF1 transcription to the independently transcribed RASSF1C isoform that promotes Tyr kinase activity. Using affinity proteomics, proximity ligation, and real-time molecular visualization, we find that RASSF1C targets SRC/YES to epithelial cell-cell junctions and promotes tyrosine phosphorylation of E-cadherin, β-catenin, and YAP1. RASSF1A restricts SRC activity, preventing motility, invasion, and tumorigenesis in vitro and in vivo, with epigenetic inactivation correlating with increased inhibitory pY527-SRC in breast tumors. These data imply that distinct RASSF1 isoforms have opposing functions, which provide a biomarker for YAP1 activation and explain correlations of RASSF1 methylation with advanced invasive disease in humans. The ablation of epithelial integrity together with subsequent YAP1 nuclear localization allows transcriptional activation of β-catenin/TBX-YAP/TEAD target genes, including Myc, and an invasive phenotype. These findings define gene transcript switching as a tumor suppressor mechanism under epigenetic control.

Riesenberg S, Groetchen A, Siddaway R, Bald T, Reinhardt J, Smorra D, Kohlmeyer J, Renn M, Phung B, Aymans P et al. 2015. MITF and c-Jun antagonism interconnects melanoma dedifferentiation with pro-inflammatory cytokine responsiveness and myeloid cell recruitment. Nat Commun, 6 (1), pp. 8755. | Show Abstract | Read more

Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood. Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment. We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun. MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression. This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction. Consistently, inflammatory MITF(low)/c-Jun(high) syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts. Our study suggests myeloid cell-directed therapies may be useful for MITF(low)/c-Jun(high) melanomas to counteract their growth-promoting and immunosuppressive functions.

Ullrich N, Löffek S, Horn S, Ennen M, Sánchez-Del-Campo L, Zhao F, Breitenbuecher F, Davidson I, Singer BB, Schadendorf D et al. 2015. MITF is a critical regulator of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in malignant melanoma. Pigment Cell Melanoma Res, 28 (6), pp. 736-740. | Show Abstract | Read more

The multifunctional Ig-like carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is neo-expressed in the majority of malignant melanoma lesions. CEACAM1 acts as a driver of tumor cell invasion, and its expression correlates with poor patient prognosis. Despite its importance in melanoma progression, how CEACAM1 expression is regulated is largely unknown. Here, we show that CEACAM1 expression in melanoma cell lines and melanoma tissue strongly correlates with that of the microphthalmia-associated transcription factor (MITF), a key regulator of melanoma proliferation and invasiveness. MITF is revealed as a direct and positive regulator for CEACAM1 expression via binding to an M-box motif located in the CEACAM1 promoter. Taken together, our study provides novel insights into the regulation of CEACAM1 expression and suggests an MITF-CEACAM1 axis as a potential determinant of melanoma progression.

Cao J, Dai X, Wan L, Wang H, Zhang J, Goff PS, Sviderskaya EV, Xuan Z, Xu Z, Xu X et al. 2015. The E3 ligase APC/C(Cdh1) promotes ubiquitylation-mediated proteolysis of PAX3 to suppress melanocyte proliferation and melanoma growth. Sci Signal, 8 (392), pp. ra87. | Show Abstract | Read more

The anaphase-promoting complex or cyclosome with the subunit Cdh1 (APC/C(Cdh1)) is an E3 ubiquitin ligase involved in the control of the cell cycle. Here, we identified sporadic mutations occurring in the genes encoding APC components, including Cdh1, in human melanoma samples and found that loss of APC/C(Cdh1) may promote melanoma development and progression, but not by affecting cell cycle regulatory targets of APC/C. Most of the mutations we found in CDH1 were those associated with ultraviolet light (UV)-induced melanomagenesis. Compared with normal human skin tissue and human or mouse melanocytes, the abundance of Cdh1 was decreased and that of the transcription factor PAX3 was increased in human melanoma tissue and human or mouse melanoma cell lines, respectively; Cdh1 abundance was further decreased with advanced stages of human melanoma. PAX3 was a substrate of APC/C(Cdh1) in melanocytes, and APC/C(Cdh1)-mediated ubiquitylation marked PAX3 for proteolytic degradation in a manner dependent on the D-box motif in PAX3. Either mutating the D-box in PAX3 or knocking down Cdh1 prevented the ubiquitylation and degradation of PAX3 and increased proliferation and melanin production in melanocytes. Knocking down Cdh1 in melanoma cells in culture or before implantation in mice promoted doxorubicin resistance, whereas reexpressing wild-type Cdh1, but not E3 ligase-deficient Cdh1 or a mutant that could not interact with PAX3, restored doxorubicin sensitivity in melanoma cells both in culture and in xenografts. Thus, our findings suggest a tumor suppressor role for APC/C(Cdh1) in melanocytes and that targeting PAX3 may be a strategy for treating melanoma.

Zhang T, Zhou Q, Ogmundsdottir MH, Möller K, Siddaway R, Larue L, Hsing M, Kong SW, Goding CR, Palsson A et al. 2015. Mitf is a master regulator of the v-ATPase, forming a control module for cellular homeostasis with v-ATPase and TORC1. J Cell Sci, 128 (15), pp. 2938-2950. | Show Abstract | Read more

The v-ATPase is a fundamental eukaryotic enzyme that is central to cellular homeostasis. Although its impact on key metabolic regulators such as TORC1 is well documented, our knowledge of mechanisms that regulate v-ATPase activity is limited. Here, we report that the Drosophila transcription factor Mitf is a master regulator of this holoenzyme. Mitf directly controls transcription of all 15 v-ATPase components through M-box cis-sites and this coordinated regulation affects holoenzyme activity in vivo. In addition, through the v-ATPase, Mitf promotes the activity of TORC1, which in turn negatively regulates Mitf. We provide evidence that Mitf, v-ATPase and TORC1 form a negative regulatory loop that maintains each of these important metabolic regulators in relative balance. Interestingly, direct regulation of v-ATPase genes by human MITF also occurs in cells of the melanocytic lineage, showing mechanistic conservation in the regulation of the v-ATPase by MITF family proteins in fly and mammals. Collectively, this evidence points to an ancient module comprising Mitf, v-ATPase and TORC1 that serves as a dynamic modulator of metabolism for cellular homeostasis.

Langendonk JG, Balwani M, Anderson KE, Bonkovsky HL, Anstey AV, Bissell DM, Bloomer J, Edwards C, Neumann NJ, Parker C et al. 2015. Afamelanotide for Erythropoietic Protoporphyria. N Engl J Med, 373 (1), pp. 48-59. | Show Abstract | Read more

BACKGROUND: Erythropoietic protoporphyria is a severe photodermatosis that is associated with acute phototoxicity. Patients with this condition have excruciating pain and a markedly reduced quality of life. We evaluated the safety and efficacy of an α-melanocyte-stimulating hormone analogue, afamelanotide, to decrease pain and improve quality of life. METHODS: We conducted two multicenter, randomized, double-blind, placebo-controlled trials of subcutaneous implants containing 16 mg of afamelanotide. Patients in the European Union (74 patients) and the United States (94 patients) were randomly assigned, in a 1:1 ratio, to receive a subcutaneous implant containing either afamelanotide or placebo every 60 days (a total of five implants in the European Union study and three in the U.S study). The type and duration of sun exposure, number and severity of phototoxic reactions, and adverse events were recorded over the respective 180-day and 270-day study periods. Quality of life was assessed with the use of validated questionnaires. A subgroup of U.S. patients underwent photoprovocation testing. The primary efficacy end point was the number of hours of direct exposure to sunlight without pain. RESULTS: In the U.S. study, the duration of pain-free time after 6 months was longer in the afamelanotide group (median, 69.4 hours, vs. 40.8 hours in the placebo group; P=0.04). In the European Union study, the duration of pain-free time after 9 months was also longer in the afamelanotide group than in the placebo group (median, 6.0 hours vs. 0.8 hours; P=0.005), and the number of phototoxic reactions was lower in the the afamelanotide group (77 vs. 146, P=0.04). In both trials, quality of life improved with afamelanotide therapy. Adverse events were mostly mild; serious adverse events were not thought to be related to the study drug. CONCLUSIONS: Afamelanotide had an acceptable side-effect and adverse-event profile and was associated with an increased duration of sun exposure without pain and improved quality of life in patients with erythropoietic protoporphyria. (Funded by Clinuvel Pharmaceuticals and others; ClinicalTrials.gov numbers, NCT01605136 and NCT00979745.).

Grawenda AM, Møller EK, Lam S, Repapi E, Teunisse AFAS, Alnæs GIG, Børresen-Dale A-L, Kristensen VN, Goding CR, Jochemsen AG et al. 2015. Interaction between p53 mutation and a somatic HDMX biomarker better defines metastatic potential in breast cancer. Cancer Res, 75 (4), pp. 698-708. | Show Abstract | Read more

TP53 gene mutation is associated with poor prognosis in breast cancer, but additional biomarkers that can further refine the impact of the p53 pathway are needed to achieve clinical utility. In this study, we evaluated a role for the HDMX-S/FL ratio as one such biomarker, based on its association with other suppressor mutations that confer worse prognosis in sarcomas, another type of cancer that is surveilled by p53. We found that HDMX-S/FL ratio interacted with p53 mutational status to significantly improve prognostic capability in patients with breast cancer. This biomarker pair offered prognostic utility that was comparable with a microarray-based prognostic assay. Unexpectedly, the utility tracked independently of DNA-damaging treatments and instead with different tumor metastasis potential. Finally, we obtained evidence that this biomarker pair might identify patients who could benefit from anti-HDM2 strategies to impede metastatic progression. Taken together, our work offers a p53 pathway marker, which both refines our understanding of the impact of p53 activity on prognosis and harbors potential utility as a clinical tool.

Müller J, Krijgsman O, Tsoi J, Robert L, Hugo W, Song C, Kong X, Possik PA, Cornelissen-Steijger PDM, Geukes Foppen MH et al. 2014. Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma. Nat Commun, 5 (1), pp. 5712. | Show Abstract | Read more

Increased expression of the Microphthalmia-associated transcription factor (MITF) contributes to melanoma progression and resistance to BRAF pathway inhibition. Here we show that the lack of MITF is associated with more severe resistance to a range of inhibitors, while its presence is required for robust drug responses. Both in primary and acquired resistance, MITF levels inversely correlate with the expression of several activated receptor tyrosine kinases, most frequently AXL. The MITF-low/AXL-high/drug-resistance phenotype is common among mutant BRAF and NRAS melanoma cell lines. The dichotomous behaviour of MITF in drug response is corroborated in vemurafenib-resistant biopsies, including MITF-high and -low clones in a relapsed patient. Furthermore, drug cocktails containing AXL inhibitor enhance melanoma cell elimination by BRAF or ERK inhibition. Our results demonstrate that a low MITF/AXL ratio predicts early resistance to multiple targeted drugs, and warrant clinical validation of AXL inhibitors to combat resistance of BRAF and NRAS mutant MITF-low melanomas.

Wansleben S, Peres J, Hare S, Goding CR, Prince S. 2014. T-box transcription factors in cancer biology. Biochim Biophys Acta, 1846 (2), pp. 380-391. | Show Abstract | Read more

The evolutionarily conserved T-box family of transcription factors have critical and well-established roles in embryonic development. More recently, T-box factors have also gained increasing prominence in the field of cancer biology where a wide range of cancers exhibit deregulated expression of T-box factors that possess tumour suppressor and/or tumour promoter functions. Of these the best characterised is TBX2, whose expression is upregulated in cancers including breast, pancreatic, ovarian, liver, endometrial adenocarcinoma, glioblastomas, gastric, uterine cervical and melanoma. Understanding the role and regulation of TBX2, as well as other T-box factors, in contributing directly to tumour progression, and especially in suppression of senescence and control of invasiveness suggests that targeting TBX2 expression or function alone or in combination with currently available chemotherapeutic agents may represent a therapeutic strategy for cancer.

Li J, Ballim D, Rodriguez M, Cui R, Goding CR, Teng H, Prince S. 2014. The anti-proliferative function of the TGF-β1 signaling pathway involves the repression of the oncogenic TBX2 by its homologue TBX3. J Biol Chem, 289 (51), pp. 35633-35643. | Show Abstract | Read more

A growing body of work has shown that the highly homologous T-box transcription factors TBX2 and TBX3 play critical but distinct roles in embryonic development and cancer progression. For example, TBX2 and TBX3 are up-regulated in several cancers and recent evidence suggests that whereas TBX2 functions as a pro-proliferative factor, TBX3 inhibits cell proliferation but promotes cancer cell migration and invasion. While the molecular mechanisms regulating these functions of TBX2 and TBX3 are poorly understood we recently reported that the TGF-β1 signaling pathway up-regulates TBX3 expression to mediate, in part, its well described anti-proliferative and pro-migratory roles. The TBX3 targets responsible for these functions were however not identified. Here we reveal for the first time that the TGF-β1 signaling pathway represses TBX2 transcriptionally and we provide a detailed mechanism to show that this is mediated by TBX3. Furthermore, we implicate the down-regulation of TBX2 in the anti-proliferative function of the TGF-β1-TBX3 axis. These findings have important implications for our understanding of the regulation of TBX2 and TBX3 and shed light on the mechanisms involved in the anti-proliferative and pro-migratory roles of TGF-β1.

Kalkavan H, Goding CR. 2015. Sensitivity to anti-BRAF therapy: lost in translation. Pigment Cell Melanoma Res, 28 (1), pp. 4-5. | Read more

Bastonini E, Jeznach M, Field M, Juszczyk K, Corfield E, Dezfouli M, Ahmat N, Smith A, Womersley H, Jordan P et al. 2014. Chromatin barcodes as biomarkers for melanoma. Pigment Cell Melanoma Res, 27 (5), pp. 788-800. | Show Abstract | Read more

The major barrier to effective cancer therapy is the presence of genetic and phenotypic heterogeneity within cancer cell populations that provides a reservoir of therapeutically resistant cells. As the degree of heterogeneity present within tumours will be proportional to tumour burden, the development of rapid, robust, accurate and sensitive biomarkers for cancer progression that could detect clinically occult disease before substantial heterogeneity develops would provide a major therapeutic benefit. Here, we explore the application of chromatin conformation capture technology to generate a diagnostic epigenetic barcode for melanoma. The results indicate that binary states from chromatin conformations at 15 loci within five genes can be used to provide rapid, non-invasive multivariate test for the presence of melanoma using as little as 200 μl of patient blood.

Goding CR, Pei D, Lu X. 2014. Cancer: pathological nuclear reprogramming? Nat Rev Cancer, 14 (8), pp. 568-573. | Show Abstract | Read more

The ability of stem cells to self-renew and generate different lineages during development and organogenesis is a fundamental, tightly controlled, and generally unidirectional process, whereas the 'immortality' of cancer cells could be regarded as pathological self-renewal. The molecular mechanisms that underpin the generation of induced pluripotent stem cells are remarkably similar to those that are deregulated in cancer - so much so that aberrant reprogramming is tumorigenic. The similarities also suggest that mutations in genes implicated in DNA methylation dynamics might represent a hallmark of cancers with a stem cell origin, and they highlight an alternative view of cancer that may be of clinical benefit.

Jayachandran A, Anaka M, Prithviraj P, Hudson C, McKeown SJ, Lo P-H, Vella LJ, Goding CR, Cebon J, Behren A. 2014. Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma. Oncotarget, 5 (14), pp. 5782-5797. | Show Abstract | Read more

Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance.

Falletta P, Bagnato P, Bono M, Monticone M, Schiaffino MV, Bennett DC, Goding CR, Tacchetti C, Valetti C. 2014. Melanosome-autonomous regulation of size and number: the OA1 receptor sustains PMEL expression. Pigment Cell Melanoma Res, 27 (4), pp. 565-579. | Show Abstract | Read more

Little is known as to how cells ensure that organelle size and number are coordinated to correctly couple organelle biogenesis to the demands of proliferation or differentiation. OA1 is a melanosome-associated G-protein-coupled receptor involved in melanosome biogenesis during melanocyte differentiation. Cells lacking OA1 contain fewer, but larger, mature melanosomes. Here, we show that OA1 loss of function reduces both the basal expression and the α-melanocyte-stimulating hormone/cAMP-dependent induction of the microphthalmia-associated transcription factor (MITF), the master regulator of melanocyte differentiation. In turn, this leads to a significant reduction in expression of PMEL, a major melanosomal structural protein, but does not affect tyrosinase and melanin levels. In line with its pivotal role in sensing melanosome maturation, OA1 expression rescues melanosome biogenesis, activates MITF expression and thereby coordinates melanosome size and number, providing a quality control mechanism for the organelle in which resides. Thus, resident sensor receptors can activate a transcriptional cascade to specifically promote organelle biogenesis.

Goding CR. 2014. Fishful thinking: the rise and fall of MITF in melanoma. Pigment cell & melanoma research, 27 (1), pp. 7-8. | Read more

Goding CR. 2013. Three BRNs are better than two. Pigment cell & melanoma research, 26 (6), pp. 789-790.

Goding CR. 2014. Fishful thinking: the rise and fall of MITF in melanoma. Pigment Cell Melanoma Res, 27 (1), pp. 7-8. | Read more

Cao J, Wan L, Hacker E, Dai X, Lenna S, Jimenez-Cervantes C, Wang Y, Leslie NR, Xu GX, Widlund HR et al. 2013. MC1R is a potent regulator of PTEN after UV exposure in melanocytes. Mol Cell, 51 (4), pp. 409-422. | Show Abstract | Read more

The individuals carrying melanocortin-1 receptor (MC1R) variants, especially those associated with red hair color, fair skin, and poor tanning ability (RHC trait), are more prone to melanoma; however, the underlying mechanism is poorly defined. Here, we report that UVB exposure triggers phosphatase and tensin homolog (PTEN) interaction with wild-type (WT), but not RHC-associated MC1R variants, which protects PTEN from WWP2-mediated degradation, leading to AKT inactivation. Strikingly, the biological consequences of the failure of MC1R variants to suppress PI3K/AKT signaling are highly context dependent. In primary melanocytes, hyperactivation of PI3K/AKT signaling leads to premature senescence; in the presence of BRAF(V600E), MC1R deficiency-induced elevated PI3K/AKT signaling drives oncogenic transformation. These studies establish the MC1R-PTEN axis as a central regulator for melanocytes' response to UVB exposure and reveal the molecular basis underlying the association between MC1R variants and melanomagenesis.

Goding CR. 2013. Three BRNs are better than two. Pigment Cell Melanoma Res, 26 (6), pp. 789-790. | Read more

Sacilotto N, Monteiro R, Fritzsche M, Becker PW, Sanchez-Del-Campo L, Liu K, Pinheiro P, Ratnayaka I, Davies B, Goding CR et al. 2013. Analysis of Dll4 regulation reveals a combinatorial role for Sox and Notch in arterial development. Proc Natl Acad Sci U S A, 110 (29), pp. 11893-11898. | Show Abstract | Read more

The mechanisms by which arterial fate is established and maintained are not clearly understood. Although a number of signaling pathways and transcriptional regulators have been implicated in arterio-venous differentiation, none are essential for arterial formation, and the manner in which widely expressed factors may achieve arterial-specific gene regulation is unclear. Using both mouse and zebrafish models, we demonstrate here that arterial specification is regulated combinatorially by Notch signaling and SoxF transcription factors, via direct transcriptional gene activation. Through the identification and characterization of two arterial endothelial cell-specific gene enhancers for the Notch ligand Delta-like ligand 4 (Dll4), we show that arterial Dll4 expression requires the direct binding of both the RBPJ/Notch intracellular domain and SOXF transcription factors. Specific combinatorial, but not individual, loss of SOXF and RBPJ DNA binding ablates all Dll4 enhancer-transgene expression despite the presence of multiple functional ETS binding sites, as does knockdown of sox7;sox18 in combination with loss of Notch signaling. Furthermore, triple knockdown of sox7, sox18 and rbpj also results in ablation of endogenous dll4 expression. Fascinatingly, this combinatorial ablation leads to a loss of arterial markers and the absence of a detectable dorsal aorta, demonstrating the essential roles of SoxF and Notch, together, in the acquisition of arterial identity.

Sáez-Ayala M, Montenegro MF, Sánchez-Del-Campo L, Fernández-Pérez MP, Chazarra S, Freter R, Middleton M, Piñero-Madrona A, Cabezas-Herrera J, Goding CR, Rodríguez-López JN. 2013. Directed phenotype switching as an effective antimelanoma strategy. Cancer Cell, 24 (1), pp. 105-119. | Show Abstract | Read more

Therapeutic resistance in melanoma and other cancers arises via irreversible genetic, and dynamic phenotypic, heterogeneity. Here, we use directed phenotype switching in melanoma to sensitize melanoma cells to lineage-specific therapy. We show that methotrexate (MTX) induces microphthalmia-associated transcription factor (MITF) expression to inhibit invasiveness and promote differentiation-associated expression of the melanocyte-specific Tyrosinase gene. Consequently, MTX sensitizes melanomas to a tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), that inhibits the essential enzyme DHFR with high affinity. The combination of MTX and TMECG leads to depletion of thymidine pools, double-strand DNA breaks, and highly efficient E2F1-mediated apoptosis in culture and in vivo. Importantly, this drug combination delivers an effective and tissue-restricted antimelanoma therapy in vitro and in vivo irrespective of BRAF, MEK, or p53 status.

Lu M, Breyssens H, Salter V, Zhong S, Hu Y, Baer C, Ratnayaka I, Sullivan A, Brown NR, Endicott J et al. 2013. Restoring p53 function in human melanoma cells by inhibiting MDM2 and cyclin B1/CDK1-phosphorylated nuclear iASPP. Cancer Cell, 23 (5), pp. 618-633. | Show Abstract | Read more

Nearly 90% of human melanomas contain inactivated wild-type p53, the underlying mechanisms for which are not fully understood. Here, we identify that cyclin B1/CDK1-phosphorylates iASPP, which leads to the inhibition of iASPP dimerization, promotion of iASPP monomer nuclear entry, and exposure of its p53 binding sites, leading to increased p53 inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most wild-type p53-expressing melanoma cell lines coexpress high levels of phosphorylated nuclear iASPP, MDM2, and cyclin B1. Inhibition of MDM2 and iASPP phosphorylation with small molecules induced p53-dependent apoptosis and growth suppression. Concurrent p53 reactivation and BRAFV600E inhibition achieved additive suppression in vivo, presenting an alternative for melanoma therapy.

Cited:

31

Scopus

Boyd SC, Mijatov B, Pupo GM, Tran SL, Gowrishankar K, Shaw HM, Goding CR, Scolyer RA, Mann GJ, Kefford RF et al. 2013. Oncogenic B-RAF V600E signaling induces the T-Box3 transcriptional repressor to repress E-cadherin and enhance melanoma cell invasion Journal of Investigative Dermatology, 133 (5), pp. 1269-1277. | Show Abstract | Read more

Approximately 50% of melanomas require oncogenic B-RAF V600E signaling for proliferation, survival, and metastasis, and the use of highly selective B-RAF inhibitors has yielded remarkable, although short-term, clinical responses. Reactivation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may therefore provide new strategies for managing melanoma. In this report, we applied whole-genome expression analyses to reveal that oncogenic B-RAF V600E regulates genes associated with epithelial-mesenchymal transition in normal cutaneous human melanocytes. Most prominent was the B-RAF-mediated transcriptional repression of E-cadherin, a keratinocyte-melanoma adhesion molecule whose loss is intimately associated with melanoma invasion and metastasis. Here we identify a link between oncogenic B-RAF, the transcriptional repressor Tbx3, and E-cadherin. We show that B-RAF V600E induces the expression of Tbx3, which potently represses E-cadherin expression in melanocytes and melanoma cells. Tbx3 expression is normally restricted to developmental embryonic tissues and promoting cell motility, but it is also aberrantly increased in various cancers and has been linked to tumor cell invasion and metastasis. We propose that this B-RAF/Tbx3/E-cadherin pathway has a critical role in promoting the metastasis of B-RAF-mutant melanomas. © 2013 The Society for Investigative Dermatology.

Goding CR. 2013. The path of least resistance: enhancing the effectiveness of BRAF inhibitors. Pigment Cell Melanoma Res, 26 (3), pp. 296-297. | Read more

Liu F, Cao J, Lv J, Dong L, Pier E, Xu GX, Wang RA, Xu Z, Goding C, Cui R. 2013. TBX2 expression is regulated by PAX3 in the melanocyte lineage Pigment Cell and Melanoma Research, 26 (1), pp. 67-77. | Show Abstract | Read more

The paired box homeotic gene 3 (PAX3) is a crucial regulator for the maintenance of melanocytic progenitor cells and has a poorly defined role in melanoma. To understand how PAX3 affects melanocyte and melanoma proliferation, we identified potential PAX3 downstream targets through gene expression profiling. Here, we identify T-box 2 (TBX2), a key developmental regulator of cell identity and an antisenescence factor in melanoma, as a directly regulated PAX3 target. We also found that TBX2 is involved in the survival of melanoma cells and is overexpressed in some melanoma specimens. The identification of TBX2 as a target for PAX3 provides a key insight into how PAX3 may contribute to melanoma evolution and may provide opportunities for prosenescence therapeutic intervention aimed at disrupting the ability of PAX3 to regulate TBX2. © 2012 John Wiley & Sons A/S.

Goding CR. 2013. Three BRNs are better than two. Pigment cell & melanoma research, 26 (6), pp. 789-790. | Read more

Boyd SC, Mijatov B, Pupo GM, Tran SL, Gowrishankar K, Shaw HM, Goding CR, Scolyer RA, Mann GJ, Kefford RF et al. 2013. Oncogenic B-RAF(V600E) signaling induces the T-Box3 transcriptional repressor to repress E-cadherin and enhance melanoma cell invasion. J Invest Dermatol, 133 (5), pp. 1269-1277. | Show Abstract | Read more

Approximately 50% of melanomas require oncogenic B-RAF(V600E) signaling for proliferation, survival, and metastasis, and the use of highly selective B-RAF inhibitors has yielded remarkable, although short-term, clinical responses. Reactivation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may therefore provide new strategies for managing melanoma. In this report, we applied whole-genome expression analyses to reveal that oncogenic B-RAF(V600E) regulates genes associated with epithelial-mesenchymal transition in normal cutaneous human melanocytes. Most prominent was the B-RAF-mediated transcriptional repression of E-cadherin, a keratinocyte-melanoma adhesion molecule whose loss is intimately associated with melanoma invasion and metastasis. Here we identify a link between oncogenic B-RAF, the transcriptional repressor Tbx3, and E-cadherin. We show that B-RAF(V600E) induces the expression of Tbx3, which potently represses E-cadherin expression in melanocytes and melanoma cells. Tbx3 expression is normally restricted to developmental embryonic tissues and promoting cell motility, but it is also aberrantly increased in various cancers and has been linked to tumor cell invasion and metastasis. We propose that this B-RAF/Tbx3/E-cadherin pathway has a critical role in promoting the metastasis of B-RAF-mutant melanomas.

Cited:

21

European Pubmed Central

Bonvin E, Falletta P, Shaw H, Delmas V, Goding CR. 2012. A phosphatidylinositol 3-kinase-Pax3 axis regulates Brn-2 expression in melanoma. Mol Cell Biol, 32 (22), pp. 4674-4683. | Show Abstract | Read more

Deregulation of transcription arising from mutations in key signaling pathways is a hallmark of cancer. In melanoma, the most aggressive and lethal form of skin cancer, the Brn-2 transcription factor (POU3F2) regulates proliferation and invasiveness and lies downstream from mitogen-activated protein kinase (MAPK) and Wnt/β-catenin, two melanoma-associated signaling pathways. In vivo Brn-2 represses expression of the microphthalmia-associated transcription factor, MITF, to drive cells to a more stem cell-like and invasive phenotype. Given the key role of Brn-2 in regulating melanoma biology, understanding the signaling pathways that drive Brn-2 expression is an important issue. Here, we show that inhibition of phosphatidylinositol 3-kinase (PI3K) signaling reduces invasiveness of melanoma cells in culture and strongly inhibits Brn-2 expression. Pax3, a transcription factor regulating melanocyte lineage-specific genes, directly binds and regulates the Brn-2 promoter, and Pax3 expression is also decreased upon PI3K inhibition. Collectively, our results highlight a crucial role for PI3K in regulating Brn-2 and Pax3 expression, reveal a mechanism by which PI3K can regulate invasiveness, and imply that PI3K signaling is a key determinant of melanoma subpopulation diversity. Together with our previous work, the results presented here now place Brn-2 downstream of three melanoma-associated signaling pathways.

Liu F, Cao J, Lv J, Dong L, Pier E, Xu GX, Wang R-A, Xu Z, Goding C, Cui R. 2013. TBX2 expression is regulated by PAX3 in the melanocyte lineage. Pigment Cell Melanoma Res, 26 (1), pp. 67-77. | Show Abstract | Read more

The paired box homeotic gene 3 (PAX3) is a crucial regulator for the maintenance of melanocytic progenitor cells and has a poorly defined role in melanoma. To understand how PAX3 affects melanocyte and melanoma proliferation, we identified potential PAX3 downstream targets through gene expression profiling. Here, we identify T-box 2 (TBX2), a key developmental regulator of cell identity and an antisenescence factor in melanoma, as a directly regulated PAX3 target. We also found that TBX2 is involved in the survival of melanoma cells and is overexpressed in some melanoma specimens. The identification of TBX2 as a target for PAX3 provides a key insight into how PAX3 may contribute to melanoma evolution and may provide opportunities for prosenescence therapeutic intervention aimed at disrupting the ability of PAX3 to regulate TBX2.

Berlin I, Denat L, Steunou A-L, Puig I, Champeval D, Colombo S, Roberts K, Bonvin E, Bourgeois Y, Davidson I et al. 2012. Phosphorylation of BRN2 modulates its interaction with the Pax3 promoter to control melanocyte migration and proliferation. Mol Cell Biol, 32 (7), pp. 1237-1247. | Show Abstract | Read more

MITF-M and PAX3 are proteins central to the establishment and transformation of the melanocyte lineage. They control various cellular mechanisms, including migration and proliferation. BRN2 is a POU domain transcription factor expressed in melanoma cell lines and is involved in proliferation and invasion, at least in part by regulating the expression of MITF-M and PAX3. The T361 and S362 residues of BRN2, both in the POU domain, are conserved throughout the POU protein family and are targets for phosphorylation, but their roles in vivo remain unknown. To examine the role of this phosphorylation, we generated mutant BRN2 in which these two residues were replaced with alanines (BRN2TS→BRN2AA). When expressed in melanocytes in vitro or in the melanocyte lineage in transgenic mice, BRN2TS induced proliferation and repressed migration, whereas BRN2AA repressed both proliferation and migration. BRN2TS and BRN2AA bound and repressed the MITF-M promoter, whereas PAX3 transcription was induced by BRN2TS but repressed by BRN2AA. Expression of the BRN2AA transgene in a Mitf heterozygous background and in a Pax3 mutant background enhanced the coat color phenotype. Our findings show that melanocyte migration and proliferation are controlled both through the regulation of PAX3 by nonphosphorylated BRN2 and through the regulation of MITF-M by the overall BRN2 level.

Luciani F, Champeval D, Herbette A, Denat L, Aylaj B, Martinozzi S, Ballotti R, Kemler R, Goding CR, De Vuyst F et al. 2011. Biological and mathematical modeling of melanocyte development. Development, 138 (18), pp. 3943-3954. | Show Abstract | Read more

We aim to evaluate environmental and genetic effects on the expansion/proliferation of committed single cells during embryonic development, using melanoblasts as a paradigm to model this phenomenon. Melanoblasts are a specific type of cell that display extensive cellular proliferation during development. However, the events controlling melanoblast expansion are still poorly understood due to insufficient knowledge concerning their number and distribution in the various skin compartments. We show that melanoblast expansion is tightly controlled both spatially and temporally, with little variation between embryos. We established a mathematical model reflecting the main cellular mechanisms involved in melanoblast expansion, including proliferation and migration from the dermis to epidermis. In association with biological information, the model allows the calculation of doubling times for melanoblasts, revealing that dermal and epidermal melanoblasts have short but different doubling times. Moreover, the number of trunk founder melanoblasts at E8.5 was estimated to be 16, a population impossible to count by classical biological approaches. We also assessed the importance of the genetic background by studying gain- and loss-of-function β-catenin mutants in the melanocyte lineage. We found that any alteration of β-catenin activity, whether positive or negative, reduced both dermal and epidermal melanoblast proliferation. Finally, we determined that the pool of dermal melanoblasts remains constant in wild-type and mutant embryos during development, implying that specific control mechanisms associated with cell division ensure half of the cells at each cell division to migrate from the dermis to the epidermis. Modeling melanoblast expansion revealed novel links between cell division, cell localization within the embryo and appropriate feedback control through β-catenin.

Goding CR. 2011. SL4/PASPCR Aaron Lerner Lecture Signalling and transcription in melanoma stem-like cells PIGMENT CELL & MELANOMA RESEARCH, 24 (4), pp. 761-761.

Luciani F, Champeval D, Herbette A, Denat L, Aylaj B, Martinozzi S, Ballotti R, Kemler R, Goding CR, De Vuyst F et al. 2011. Understanding melanocyte development: biological analysis associated with mathematical modeling PIGMENT CELL & MELANOMA RESEARCH, 24 (4), pp. 805-805.

Berlin I, Denat L, Steunou A-L, Puig I, Champeval D, Colombo S, Roberts K, Bonvin E, Bourgeois Y, Delmas V et al. 2011. BRN2 phosphorylation regulates melanoblast migration and proliferation through PAX3 and MITF-M PIGMENT CELL & MELANOMA RESEARCH, 24 (4), pp. 819-819.

Goding C. 2011. Many are called, few are chosen. Pigment Cell Melanoma Res, 24 (3), pp. 395. | Read more

Goding CR. 2011. Commentary. A picture of Mitf in melanoma immortality. Oncogene, 30 (20), pp. 2304-2306. | Show Abstract | Read more

The Mitf gene has a key role in melanocytes and melanoma by regulating cell cycle progression, survival and differentiation. Two papers in this issue of Oncogene (Cheli et al., 2011; Strub et al., 2011) reveal that low-Mitf cells can initiate tumors with high efficiency, and that Mitf blocks senescence by regulating genes implicated in S-phase progression and mitosis.

Dalziel M, Kolesnichenko M, das Neves RP, Iborra F, Goding C, Furger A. 2011. Alpha-MSH regulates intergenic splicing of MC1R and TUBB3 in human melanocytes. Nucleic Acids Res, 39 (6), pp. 2378-2392. | Show Abstract | Read more

Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived from a restricted number of genes. However, the possibility that functional diversity may also be augmented by splicing between adjacent genes has been largely neglected. Here, we show that the human melanocortin 1 receptor (MC1R) gene, a critical component of the facultative skin pigmentation system, has a highly complex and inefficient poly(A) site which is instrumental in allowing intergenic splicing between this locus and its immediate downstream neighbour tubulin-β-III (TUBB3). These transcripts, which produce two distinct protein isoforms localizing to the plasma membrane and the endoplasmic reticulum, seem to be restricted to humans as no detectable chimeric mRNA could be found in MC1R expressing mouse melanocytes. Significantly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key molecules associated with ultraviolet radiation dermal insult and subsequent skin tanning, result in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. We propose that these chimeric proteins serve to equip melanocytes with novel cellular phenotypes required as part of the pigmentation response.

Mowla S, Pinnock R, Leaner VD, Goding CR, Prince S. 2011. PMA-induced up-regulation of TBX3 is mediated by AP-1 and contributes to breast cancer cell migration. Biochem J, 433 (1), pp. 145-153. | Show Abstract | Read more

The T-box transcription factor TBX3 provides an important link between embryonic development and cancer. TBX3 mediates limb, mammary gland and heart development and, in humans, mutations resulting in haplo-insufficiency of TBX3 lead to ulnar-mammary syndrome. Importantly, the de-regulation of TBX3 gene expression has been linked to several cancers, where it acts to suppress senescence and promotes proliferation and tumour invasion. Despite the negative impact of de-regulated TBX3 expression as seen by developmental defects and cancer, surprisingly little is known about the regulation of the TBX3 gene. In the present paper, we show that the phorbol ester PMA increases TBX3 protein and mRNA levels in a protein kinase C-dependent manner via the AP-1 (activator protein 1) transcription factors c-Jun and JunB. Furthermore, these AP-1 factors are shown to mediate the activation of the TBX3 gene by binding a non-consensus PMA-response element in the TBX3 promoter in vitro and in vivo. We also demonstrate that TBX3 contributes to the PMA-induced migration previously observed for the MCF-7 breast epithelium cancer cell line. Our present results reveal a previously unidentified pathway that up-regulates TBX3 expression and provides additional evidence that increased levels of TBX3 contribute to metastasis.

Vance KW, Shaw HM, Rodriguez M, Ott S, Goding CR. 2010. The retinoblastoma protein modulates Tbx2 functional specificity. Mol Biol Cell, 21 (15), pp. 2770-2779. | Show Abstract | Read more

Tbx2 is a member of a large family of transcription factors defined by homology to the T-box DNA-binding domain. Tbx2 plays a key role in embryonic development, and in cancer through its capacity to suppress senescence and promote invasiveness. Despite its importance, little is known of how Tbx2 is regulated or how it achieves target gene specificity. Here we show that Tbx2 specifically associates with active hypophosphorylated retinoblastoma protein (Rb1), a known regulator of many transcription factors involved in cell cycle progression and cellular differentiation, but not with the Rb1-related proteins p107 or p130. The interaction with Rb1 maps to a domain immediately carboxy-terminal to the T-box and enhances Tbx2 DNA binding and transcriptional repression. Microarray analysis of melanoma cells expressing inducible dominant-negative Tbx2, comprising the T-box and either an intact or mutated Rb1 interaction domain, shows that Tbx2 regulates the expression of many genes involved in cell cycle control and that a mutation which disrupts the Rb1-Tbx2 interaction also affects Tbx2 target gene selectivity. Taken together, the data show that Rb1 is an important determinant of Tbx2 functional specificity.

Primot A, Mogha A, Corre S, Roberts K, Debbache J, Adamski H, Dreno B, Khammari A, Lesimple T, Mereau A et al. 2010. ERK-regulated differential expression of the Mitf 6a/b splicing isoforms in melanoma. Pigment Cell Melanoma Res, 23 (1), pp. 93-102. | Show Abstract | Read more

The master regulator of the melanocyte lineage Mitf is intimately involved in development as well as melanoma, controlling cell survival, differentiation, proliferation and metastasis/migration. Consistent with its central role, Mitf expression and Mitf post-translational modifications are tightly regulated. An additional potential level of regulation is afforded by differential splicing of Mitf exon-6 leading to the generation of two isoforms that differ by the presence of six amino-acids in the Mitf (+) isoform and which have differential effects on cell cycle progression. However, whether the ratio of the two isoforms is regulated and whether their expression correlates with melanoma progression is not known. Here, we show that the differential expression of the Mitf 6a/b isoforms is dependent on the MAPKinase signalling, being linked to the activation of MEK1-ERK2, but not to N-RAS/B-RAF mutation status. In addition, quantification of Mitf 6a/b splicing forms in 86 melanoma samples revealed substantially increased levels of the Mitf (-) form in a subset of metastatic melanomas. The results suggest that differential expression of the Mitf 6a/b isoforms may represent an additional mechanism for regulating Mitf function and melanoma biology.

Grazini U, Zanardi F, Citterio E, Casola S, Goding CR, McBlane F. 2010. The RING domain of RAG1 ubiquitylates histone H3: a novel activity in chromatin-mediated regulation of V(D)J joining. Mol Cell, 37 (2), pp. 282-293. | Show Abstract | Read more

The RAG1 and RAG2 proteins are the only lymphoid-specific factors required to perform the first step of V(D)J recombination, DNA cleavage. While the catalytic domain of RAG1, the core region, has been well characterized, the role of the noncore region in modulating chromosomal V(D)J recombination efficiency remains ill defined. Recent studies have highlighted the role of chromatin structure in regulation of V(D)J recombination. Here we show that RAG1 itself, through a RING domain within its N-terminal noncore region, preferentially interacts directly with and promotes monoubiquitylation of histone H3. Mutations affecting the RAG1 RING domain reduce histone H3 monoubiquitylation activity, decrease V(D)J recombination activity in vivo, reduce formation of both signal-joint and coding-joint products on episomal substrates, and decrease efficiency of V(D)J recombination at the endogenous IgH locus in lymphoid cells. The results reveal that RAG1-mediated histone monoubiquitylation activity plays a role in regulating the joining phase of chromosomal V(D)J recombination.

Hoek KS, Goding CR. 2010. Cancer stem cells versus phenotype-switching in melanoma. Pigment Cell Melanoma Res, 23 (6), pp. 746-759. | Show Abstract | Read more

Tumours comprise multiple phenotypically distinct subpopulations of cells, some of which are proposed to possess stem cell-like properties, being able to self-renew, seed and maintain tumours, and provide a reservoir of therapeutically resistant cells. Here, we use melanoma as a model to explore the validity of the cancer stem cell hypothesis in the light of accumulating evidence that melanoma progression may instead be driven by phenotype-switching triggered by genetic lesions that impose an increased sensitivity to changes in the tumour microenvironment. Although at any given moment cells within a tumour may exhibit differentiated, proliferative or invasive phenotypes, an ability to switch phenotypes implies that most cells will have the potential to adopt a stem cell-like identity. Insights into the molecular events underpinning phenotype-switching in melanoma highlight the close relationship between signalling pathways that generate, maintain and activate melanocyte stem cells as well as the inverse correlation between proliferation and invasive potentials. An understanding of phenotype-switching in melanoma, and in particular the signalling events that regulate the expression of the microphthalmia-associated transcription factor Mitf, points to new therapeutic opportunities aimed at eradicating therapeutically resistant stem cell-like melanoma cells.

Goding CR. 2010. Dicing with death: Mitf regulates DICER. Pigment Cell Melanoma Res, 23 (4), pp. 483-484. | Read more

Pinner S, Jordan P, Sharrock K, Bazley L, Collinson L, Marais R, Bonvin E, Goding C, Sahai E. 2009. Intravital imaging reveals transient changes in pigment production and Brn2 expression during metastatic melanoma dissemination. Cancer Res, 69 (20), pp. 7969-7977. | Show Abstract | Read more

How melanoma acquire a metastatic phenotype is a key issue. One possible mechanism is that metastasis is driven by microenvironment-induced switching between noninvasive and invasive states. However, whether switching is a reversible or hierarchical process is not known and is difficult to assess by comparison of primary and metastatic tumors. We address this issue in a model of melanoma metastasis using a novel intravital imaging method for melanosomes combined with a reporter construct in which the Brn-2 promoter drives green fluorescent protein (GFP) expression. A subpopulation of cells containing little or no pigment and high levels of Brn2::GFP expression are motile in the primary tumor and enter the vasculature. Significantly, the less differentiated state of motile and intravasated cells is not maintained at secondary sites, implying switching between states as melanoma cells metastasize. We show that melanoma cells can switch in both directions between high- and low-pigment states. However, switching from Brn2::GFP high to low was greatly favored over the reverse direction. Microarray analysis of high- and low-pigment populations revealed that transforming growth factor (TGF)beta2 was up-regulated in the poorly pigmented cells. Furthermore, TGFbeta signaling induced hypopigmentation and increased cell motility. Thus, a subset of less differentiated cells exits the primary tumor but subsequently give rise to metastases that include a range of more differentiated and pigment-producing cells. These data show reversible phenotype switching during melanoma metastasis.

Corre S, Primot A, Baron Y, Le Seyec J, Goding C, Galibert M-D. 2009. Target gene specificity of USF-1 is directed via p38-mediated phosphorylation-dependent acetylation. J Biol Chem, 284 (28), pp. 18851-18862. | Show Abstract | Read more

How transcription factors interpret the output from signal transduction pathways to drive distinct programs of gene expression is a key issue that underpins development and disease. The ubiquitously expressed basic-helix-loop-helix leucine zipper upstream stimulating factor-1 binds E-box regulatory elements (CANNTG) to regulate a wide number of gene networks. In particular, USF-1 is a key component of the tanning process. Following UV irradiation, USF-1 is phosphorylated by the p38 stress-activated kinase on threonine 153 and directly up-regulates expression of the POMC, MC1R, TYR, TYRP-1 and DCT genes. However, how phosphorylation on Thr-153 might affect the activity of USF-1 is unclear. Here we show that, in response to DNA damage, oxidative stress and cellular infection USF-1 is acetylated in a phospho-Thr-153-dependent fashion. Phospho-acetylated USF-1 is nuclear and interacts with DNA but displays altered gene regulatory properties. Phospho-acetylated USF-1 is thus proposed to be associated with loss of transcriptional activation properties toward several target genes implicated in pigmentation process and cell cycle regulation. The identification of this critical stress-dependent USF-1 modification gives new insights into understanding USF-1 gene expression modulation associated with cancer development.

Goding C. 2009. So long, and thanks for all the... PIGMENT CELL & MELANOMA RESEARCH, 22 (6), pp. 699-700. | Read more

Goding C. 2009. The eyes have it. Pigment Cell Melanoma Res, 22 (5), pp. 517. | Read more

Goding C. 2009. Upwards and onwards PIGMENT CELL & MELANOMA RESEARCH, 22 (4), pp. 375-375. | Read more

Goding C. 2009. The new black. Pigment Cell Melanoma Res, 22 (3), pp. 243. | Read more

Goding C. 2009. From mechanistic insights to therapeutic applications. Pigment Cell Melanoma Res, 22 (2), pp. 145. | Read more

Goding C. 2009. Dendritic cells and melanoma therapy. Pigment Cell Melanoma Res, 22 (1), pp. 1. | Read more

Goding C. 2009. Browsing issues Pigment Cell & Melanoma Research, 22 (1), pp. 1-1. | Read more

Goodall J, Carreira S, Denat L, Kobi D, Davidson I, Nuciforo P, Sturm RA, Larue L, Goding CR. 2008. Brn-2 represses microphthalmia-associated transcription factor expression and marks a distinct subpopulation of microphthalmia-associated transcription factor-negative melanoma cells. Cancer Res, 68 (19), pp. 7788-7794. | Show Abstract | Read more

The origin of tumor heterogeneity is poorly understood, yet it represents a major barrier to effective therapy. In melanoma and in melanocyte development, the microphthalmia-associated transcription factor (Mitf) controls survival, differentiation, proliferation, and migration/metastasis. The Brn-2 (N-Oct-3, POU3F2) transcription factor also regulates melanoma proliferation and is up-regulated by BRAF and beta-catenin, two key melanoma-associated signaling molecules. Here, we show that Brn-2 also regulates invasiveness and directly represses Mitf expression. Remarkably, in melanoma biopsies, Mitf and Brn-2 each mark a distinct subpopulation of melanoma cells, providing a striking illustration of melanoma tumor heterogeneity with implications for melanoma therapy.

Rodriguez M, Aladowicz E, Lanfrancone L, Goding CR. 2008. Tbx3 represses E-cadherin expression and enhances melanoma invasiveness. Cancer Res, 68 (19), pp. 7872-7881. | Show Abstract | Read more

The T-box transcription factors Tbx2 and Tbx3 are overexpressed in many cancers and in melanoma promote proliferation by actively suppressing senescence. Whether they also contribute to tumor progression via other mechanisms is not known. Here, we identify a novel role for these factors, providing evidence that Tbx3, and potentially Tbx2, directly repress the expression of E-cadherin, a keratinocyte-melanoma adhesion molecule whose loss is required for the acquisition of an invasive phenotype. Overexpression of Tbx2 and Tbx3 in melanoma cells down-regulates endogenous E-cadherin expression, whereas depletion of Tbx3, but not Tbx2, increases E-cadherin mRNA and protein levels and decreases melanoma invasiveness in vitro. Consistent with these observations, in melanoma tissue, Tbx3 and E-cadherin expression are inversely correlated. Depletion of Tbx3 also leads to substantial up-regulation of Tbx2. The results suggest that Tbx2 and Tbx3 may play a dual role during the radial to vertical growth phase transition by both inhibiting senescence via repression of p21(CIP1) expression, and enhancing melanoma invasiveness by decreasing E-cadherin levels.

Goding CR. 2008. T-box factors in melanocytes and melanoma PIGMENT CELL & MELANOMA RESEARCH, 21 (2), pp. 248-249.

Goding CR. 2008. Transcription regulation in melanocytes and melanoma PIGMENT CELL & MELANOMA RESEARCH, 21 (2), pp. 281-281.

Goding C. 2008. Genesis and NEMESIS. Pigment Cell Melanoma Res, 21 (6), pp. 591. | Read more

Goding C. 2008. Tanning, p53 and PCMR. Pigment Cell Melanoma Res, 21 (5), pp. 499-500. | Read more

Goding C. 2008. The impact of PCMR. Pigment Cell Melanoma Res, 21 (4), pp. 409. | Read more

Pontecorvi M, Goding CR, Richardson WD, Kessaris N. 2008. Expression of Tbx2 and Tbx3 in the developing hypothalamic-pituitary axis. Gene Expr Patterns, 8 (6), pp. 411-417. | Show Abstract | Read more

TBX2 and TBX3 are transcription factors that belong to the T-box family, members of which play important roles during mammalian embryogenesis. Mutations in T-box genes have been linked to several human genetic disorders and increasing evidence suggests that Tbx2 and Tbx3 may play a key role in cancer. The primary functions of Tbx2 and Tbx3 remain poorly defined, mainly because of their widespread expression in several tissues and their multiple potential roles in morphogenesis, organogenesis and cell-fate commitment. Here, we describe in detail the expression of Tbx2 and Tbx3 in the developing hypothalamic-pituitary axis. Localized transcripts can be detected during the early stages of pituitary commitment. Expression of Tbx2 is restricted to the infundibular region of the ventral diencephalon (VD) at all ages examined, whereas Tbx3 can be detected in both the VD and Rathke's pouch, the precursor of the anterior pituitary. Outside the developing hypophyseal organ novel sites of Tbx3 and Tbx2 expression include migrating branchiomotor (BM) and visceromotor (VM) neurons in the hindbrain, neuroepithelial cells of the developing tongue (Tbx3) as well as the developing blood vessel network (Tbx2).

Hoogaars WMH, Barnett P, Rodriguez M, Clout DE, Moorman AFM, Goding CR, Christoffels VM. 2008. TBX3 and its splice variant TBX3 + exon 2a are functionally similar. Pigment Cell Melanoma Res, 21 (3), pp. 379-387. | Show Abstract | Read more

Tbx3, a member of the conserved family of T-box developmental transcription factors, is a transcriptional repressor required during cardiogenesis for the formation and specification of the sinoatrial node, the pacemaker of the heart. Both the TBX3 and the highly related TBX2 genes are also associated with several cancers, most likely as a consequence of their powerful anti-senescence properties mediated via suppression p14(Arf) and p21(CIP) expression. In melanoma, the TBX2 gene is frequently amplified and inhibition of Tbx2 function leads to senescence and up-regulation of p21(CIP), a Tbx2 target gene. Tbx3 + 2a is a splice variant containing an extra 20 amino acids encoded by exon 2a inserted into the highly conserved T-box DNA-binding domain. We find here that Tbx3 + 2a is evolutionary conserved and that similar insertions are largely absent from the T-box domains of other T-box factors. Tbx3 + 2a has been reported to lack DNA-binding ability and act as a functional antagonist of Tbx3. By contrast, we now demonstrate that both Tbx3 and Tbx3 + 2a bind the consensus T-element, the p21(CIP1) promoter, and the Nppa cardiac target gene. Both isoforms also function as repressors of p21(CIP1) and Nppa promoter activity and interact with homeobox factor Nkx2-5. When ectopically expressed in the embryonic heart of mice, Tbx3 and Tbx3 + 2a both suppressed chamber formation and repressed expression of cardiac chamber markers Nppa and Cx40. The results suggest that in the assays used, Tbx3 and Tbx3 + 2a are functionally equivalent and that like Tbx2, Tbx3 may also function as an anti-senescence factor in melanoma.

Goding C. 2008. Environmental issues PIGMENT CELL & MELANOMA RESEARCH, 21 (3), pp. 337-337. | Read more

Goding C. 2008. E pluribus unum PIGMENT CELL & MELANOMA RESEARCH, 21 (2), pp. 109-109. | Read more

Davis E, Teng H, Bilican B, Parker MI, Liu B, Carriera S, Goding CR, Prince S. 2008. Ectopic Tbx2 expression results in polyploidy and cisplatin resistance. Oncogene, 27 (7), pp. 976-984. | Show Abstract | Read more

T-box factors play critical roles in embryonic development and have been implicated in cell cycle regulation and cancer. For example, Tbx2 can suppress senescence through a mechanism involving the repression of the cyclin-dependent kinase inhibitors, p19(ARF) and p21(WAF1/CIP1/SDII), and the Tbx2 gene is deregulated in melanoma, breast and pancreatic cancers. In this study, several transformed human lung fibroblast cell lines were shown to downregulate Tbx2. To further investigate the role of Tbx2 in oncogenesis we therefore stably reexpressed Tbx2 in one such cell line. Compared to their parental cells, the resulting Tbx2-expressing cells are larger, with binucleate and lobular nuclei containing double the number of chromosomes. Moreover, these cells had an increase in frequency of several features of genomic instability such as chromosome missegregation, chromosomal rearrangements and polyploidy. While grossly abnormal, these cells still divide and give rise to cells that are resistant to the chemotherapeutic drug cisplatin. Furthermore, this is shown to be neither species nor cell type dependent, as ectopically expressing Tbx2 in a murine melanoma cell line also induce mitotic defects and polyploidy. These results have important implications for our understanding of the role of Tbx2 in tumorigenesis because polyploidy frequently precedes aneuploidy, which is associated with high malignancy and poor prognosis.

Goding C. 2008. Its all in the name. Pigment Cell Melanoma Res, 21 (1), pp. 1-2. | Read more

Abrahams A, Mowla S, Parker MI, Goding CR, Prince S. 2008. UV-mediated regulation of the anti-senescence factor Tbx2. J Biol Chem, 283 (4), pp. 2223-2230. | Show Abstract | Read more

Several lines of evidence have implicated members of the developmentally important T-box gene family in cell cycle regulation and in cancer. Importantly, the highly related T-box factors Tbx2 and Tbx3 can suppress senescence through repressing the cyclin-dependent kinase inhibitors p19(ARF) and p21(WAF1/CIP1/SDII). Furthermore, Tbx2 is up-regulated in several cancers, including melanomas where it was shown to function as an anti-senescence factor, suggesting that this may be one of the mechanisms by which T-box proteins contribute to the oncogenic process. However, very little is known about whether Tbx2 is regulated by p21-mediated stress-induced senescence signaling pathways. In this study, using the MCF-7 breast cancer cell line known to overexpress Tbx2, we show that in response to stress induced by ultraviolet irradiation the Tbx2 protein is specifically phosphorylated by the p38 mitogen-activated protein kinase. Using site-directed mutagenesis and in vitro kinase assays, we have identified serine residues 336, 623, and 675 in the Tbx2 protein as the p38 target sites and show that these sites are phosphorylated in vivo. Importantly, we show by Western blotting, immunofluorescence, and reporter assays that this phosphorylation leads to increased Tbx2 protein levels, predominant nuclear localization of the protein, and an increase in the ability of Tbx2 to repress the p21(WAF1/CIP1/SDII) promoter. These results show for the first time that the ability of Tbx2 to repress the p21 gene is enhanced in response to a stress-induced senescence pathway, which leads to a better understanding of the regulation of the anti-senescence function of Tbx2.

Goding C. 2007. Melanoma, making a difference. Pigment Cell Res, 20 (6), pp. 457. | Read more

Delmas V, Beermann F, Martinozzi S, Carreira S, Ackermann J, Kumasaka M, Denat L, Goodall J, Luciani F, Viros A et al. 2007. Beta-catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development. Genes Dev, 21 (22), pp. 2923-2935. | Show Abstract | Read more

Tumor progression is a multistep process in which proproliferation mutations must be accompanied by suppression of senescence. In melanoma, proproliferative signals are provided by activating mutations in NRAS and BRAF, whereas senescence is bypassed by inactivation of the p16(Ink4a) gene. Melanomas also frequently exhibit constitutive activation of the Wnt/beta-catenin pathway that is presumed to induce proliferation, as it does in carcinomas. We show here that, contrary to expectations, stabilized beta-catenin reduces the number of melanoblasts in vivo and immortalizes primary skin melanocytes by silencing the p16(Ink4a) promoter. Significantly, in a novel mouse model for melanoma, stabilized beta-catenin bypasses the requirement for p16(Ink4a) mutations and, together with an activated N-Ras oncogene, leads to melanoma with high penetrance and short latency. The results reveal that synergy between the Wnt and mitogen-activated protein (MAP) kinase pathways may represent an important mechanism underpinning the genesis of melanoma, a highly aggressive and increasingly common disease.

Goding CR. 2007. Melanoma senescence: HDAC1 in focus. Pigment Cell Res, 20 (5), pp. 336-338. | Read more

Goding C. 2007. Let's stick together PIGMENT CELL RESEARCH, 20 (5), pp. 333-333. | Read more

Demay F, Bilican B, Rodriguez M, Carreira S, Pontecorvi M, Ling Y, Goding CR. 2007. T-box factors: targeting to chromatin and interaction with the histone H3 N-terminal tail. Pigment Cell Res, 20 (4), pp. 279-287. | Show Abstract | Read more

T-box transcription factors play a crucial role in development where they are implicated in patterning and cell fate decisions. Tbx2 and Tbx3 have also been implicated in several cancers including melanoma, and can act as antisenescence factors through their ability to repress p19(ARF) and p21(CIP1) expression. Although several target genes for T-box factors have been identified, it is unknown whether this family of proteins can bind chromatin, a property that would facilitate the epigenetic reprogramming that occurs in both development and cancer progression. Here, we show that Tbx2 has the potential to recognize mitotic chromatin in a DNA-dependent fashion, can interact specifically with the histone H3 N-terminal tail, a property shared with Tbx4, Tbx5 and Tbx6, and can also recognize nucleosomal DNA, with binding to nucleosomes being antagonized by the presence of the histone tails. Strikingly, in vivo Tbx2 co-localization with pericentric heterochromatin appears to be regulated and ectopic expression of Tbx2 leads to severe mitotic defects. Taken together our results suggest that Tbx2, and most likely other members of the T-box family, are able to target chromatin and may indicate a role for the T-box factors in epigenetic reprogramming events.

Goding C. 2007. Finding a niche PIGMENT CELL RESEARCH, 20 (4), pp. 257-257. | Read more

Goding C. 2007. Back to black: Pigment cell & melanoma research PIGMENT CELL RESEARCH, 20 (3), pp. 147-147. | Read more

Goding C. 2007. Melanoma: markers and epigenetics PIGMENT CELL RESEARCH, 20 (2), pp. 79-79. | Read more

Goding C. 2007. Editorial: A colourful first 20 years PIGMENT CELL RESEARCH, 20 (1), pp. 1-1. | Read more

Goding CR. 2007. Melanocytes: the new Black. Int J Biochem Cell Biol, 39 (2), pp. 275-279. | Show Abstract | Read more

Melanocytes, pigment-producing cells residing primarily in the hair follicle, epidermis and eye, are responsible for skin hair and eye pigmentation. Pigmentation is achieved by the highly regulated manufacture of the pigment melanin in specialised organelles, melanosomes that are transported along dendritic processes before being transferred to growing hair, or keratinocytes where melanin protects from UV-induced DNA damage. Because loss of melanocytes gives a clear pigmentation phenotype yet is non-lethal, over 130 genes implicated in the development or function of this cell type have been identified to date, and in humans the loss of melanocytes or their ability to produce pigment, or transport or transfer melanosomes is associated with several diseases such as vitiligo, albinism and Hermansky-Pudlak syndrome. Importantly, the effective combination of genetics, cell and molecular biology possible with this cell type is attracting an increasing number of researchers focussed on understanding how cells coordinate survival, proliferation, differentiation and stem cell maintenance.

Goding C. 2006. Untitled PIGMENT CELL RESEARCH, 19 (2), pp. 103-103. | Read more

Carreira S, Goodall J, Denat L, Rodriguez M, Nuciforo P, Hoek KS, Testori A, Larue L, Goding CR. 2006. Mitf regulation of Dia1 controls melanoma proliferation and invasiveness. Genes Dev, 20 (24), pp. 3426-3439. | Show Abstract | Read more

It is widely held that cells with metastatic properties such as invasiveness and expression of matrix metalloproteinases arise through the stepwise accumulation of genetic lesions arising from genetic instability and "clonal evolution." By contrast, we show here that in melanomas invasiveness can be regulated epigenetically by the microphthalmia-associated transcription factor, Mitf, via regulation of the DIAPH1 gene encoding the diaphanous-related formin Dia1 that promotes actin polymerization and coordinates the actin cytoskeleton and microtubule networks at the cell periphery. Low Mitf levels lead to down-regulation of Dia1, reorganization of the actin cytoskeleton, and increased ROCK-dependent invasiveness, whereas increased Mitf expression leads to decreased invasiveness. Significantly the regulation of Dia1 by Mitf also controls p27(Kip1)-degradation such that reduced Mitf levels lead to a p27(Kip1)-dependent G1 arrest. Thus Mitf, via regulation of Dia1, can both inhibit invasiveness and promote proliferation. The results imply variations in the repertoire of environmental cues that determine Mitf activity will dictate the differentiation, proliferative, and invasive/migratory potential of melanoma cells through a dynamic epigenetic mechanism.

Goding C. 2006. Editorial: beyond the pale. Pigment Cell Res, 19 (6), pp. 549. | Read more

Schepsky A, Bruser K, Gunnarsson GJ, Goodall J, Hallsson JH, Goding CR, Steingrimsson E, Hecht A. 2006. The microphthalmia-associated transcription factor Mitf interacts with beta-catenin to determine target gene expression. Mol Cell Biol, 26 (23), pp. 8914-8927. | Show Abstract | Read more

Commitment to the melanocyte lineage is characterized by the onset of expression of the microphthalmia-associated transcription factor (Mitf). This transcription factor plays a fundamental role in melanocyte development and maintenance and seems to be crucial for the survival of malignant melanocytes. Furthermore, Mitf has been shown to be involved in cell cycle regulation and to play important functions in self-renewal and maintenance of melanocyte stem cells. Although little is known about how Mitf regulates these various processes, one possibility is that Mitf interacts with other regulators. Here we show that Mitf can interact directly with beta-catenin, the key mediator of the canonical Wnt signaling pathway. The Wnt signaling pathway plays a critical role in melanocyte development and is intimately involved in triggering melanocyte stem cell proliferation. Significantly, constitutive activation of this pathway is a feature of a number of cancers including malignant melanoma. Here we show that Mitf can redirect beta-catenin transcriptional activity away from canonical Wnt signaling-regulated genes toward Mitf-specific target promoters to activate transcription. Thus, by a feedback mechanism, Mitf can diversify the output of canonical Wnt signaling to enhance the repertoire of genes regulated by beta-catenin. Our results reveal a novel mechanism by which Wnt signaling and beta-catenin activate gene expression, with significant implications for our understanding of both melanocyte development and melanoma.

Goding C. 2006. Editorial: vigour, vitiligo and the RPE. Pigment Cell Res, 19 (5), pp. 379. | Read more

Goding C. 2006. Editorial: For support and illumination PIGMENT CELL RESEARCH, 19 (4), pp. 259-259. | Read more

Bilican B, Goding CR. 2006. Cell cycle regulation of the T-box transcription factor tbx2. Exp Cell Res, 312 (12), pp. 2358-2366. | Show Abstract | Read more

T-box transcription factors play key roles in development and in particular the determination or maintenance of cell fate. Tbx2 is a transcriptional repressor implicated in several developmental processes and which has also been implicated in cancer through its ability to suppress senescence via repression of the p19(ARF) and p21(Cip1) (CDKN1A) promoters. However, despite its importance, little is known about how Tbx2 may be regulated. Here, we show that Tbx2 protein expression is tightly regulated during cell cycle progression, with levels being low in G1, increasing in mid-S-phase and persisting at high levels though G2 until finally undergoing a dramatic reduction at the onset of mitosis. Moreover, in S-phase, Tbx2 is present at a subset of late, but not early, replication foci and a significant fraction of Tbx2 is tightly associated with the nucleus in small DNA-associated foci that do not correspond with telomeres, PML or cajal bodies. The results are consistent with Tbx2 playing a role in cell cycle progression and organization of subnuclear compartments.

Goding C. 2006. Editorial: history and all that. Pigment Cell Res, 19 (3), pp. 181-182. | Read more

Goding C. 2006. Editorial Pigment Cell Research, 19 (2), pp. 103-103. | Read more

Xella B, Goding C, Agricola E, Di Mauro E, Caserta M. 2006. The ISWI and CHD1 chromatin remodelling activities influence ADH2 expression and chromatin organization. Mol Microbiol, 59 (5), pp. 1531-1541. | Show Abstract | Read more

Nucleosome remodelling complexes play a key role in gene activation in response to environmental changes by driving promoter chromatin to reach an accessible configuration. They also mediate genome-wide chromatin organization, although their role in processes other than activation-related chromatin remodelling are poorly understood. The Saccharomyces cerevisiae ADH2 gene represents an excellent model for understanding the role of chromatin structure and remodelling in gene regulation. Following glucose depletion, highly positioned promoter nucleosomes are destabilized leading to strictly regulated kinetics of transcriptional activation. Nevertheless, no chromatin remodelling activities responsible for establishing or remodelling ADH2 chromatin structure have been identified to date. Here we show that the absence of the Isw1 and Chd1 ATP-dependent chromatin remodelling activities delays the maximal expression of ADH2 without impairing the chromatin remodelling that occurs upon activation. Instead, a destabilized chromatin structure on the ADH2 coding and termination region is observed in the absence of Isw1 or Chd1 in repressing conditions. The specific Isw1 complex involved in this nucleosome repositioning is Isw1b because the deletion of Ioc2 and Ioc4, but not of Ioc3, causes the same phenotype as the deletion of Isw1. Moreover, the lack of Chd1 combined with the absence of Isw1 and Isw2 impairs nucleosome spacing along the ADH2 gene, and genome-wide in S. cerevisiae. Thus, the ISWI and Chd1 remodelling factors are not only involved in transcription-related chromatin remodelling, but also are required to maintain a specific chromatin configuration across the yeast genome.

Goding C, Meyskens FL. 2006. Microphthalmic-associated transcription factor integrates melanocyte biology and melanoma progression. Clin Cancer Res, 12 (4), pp. 1069-1073. | Read more

Goding C. 2006. Editorial: Colour and form PIGMENT CELL RESEARCH, 19 (1), pp. 1-2. | Read more

Goding C. 2005. Melanosomes, light and transposable elements. Pigment Cell Res, 18 (5), pp. 321. | Read more

Goding C. 2005. Editorial: It's good to talk. Pigment Cell Res, 18 (6), pp. 391-392. | Read more

Crouch DH, Fisher F, La Rocca SA, Goding CR, Gillespie DAF. 2005. Viral mutations enhance the Max binding properties of the vMyc b-HLH-LZ domain. Nucleic Acids Res, 33 (16), pp. 5235-5242. | Show Abstract | Read more

Interaction with Max via the helix-loop-helix/leucine zipper (HLH-LZ) domain is essential for Myc to function as a transcription factor. Myc is commonly upregulated in tumours, however, its activity can also be potentiated by virally derived mutations. vMyc, derived from the virus, MC29 gag-Myc, differs from its cellular counterpart by five amino acids. The N-terminal mutation stabilizes the protein, however, the significance of the other mutations is not known. We now show that vMyc can sustain longer deletions in the LZ domain than cMyc before complete loss in transforming activity, implicating the viral mutations in contributing to Myc:Max complex formation. We confirmed this both in vitro and in vivo, with loss of Max binding correlating with a loss in the biological activity of Myc. A specific viral mutation, isoleucine383>leucine (I383>L) in helix 2 of the HLH domain, extends the LZ domain from four to five heptad repeats. Significantly, introduction of I383>L into a Myc mutant that is defective for Max binding substantially restored its ability to complex with Max in vitro and in vivo. We therefore propose that this virally derived mutation is functional by significantly contributing to establishing a more hydrophobic interface between the LZs of Myc and Max.

Kumasaka M, Sato S, Yajima I, Goding CR, Yamamoto H. 2005. Regulation of melanoblast and retinal pigment epithelium development by Xenopus laevis Mitf. Dev Dyn, 234 (3), pp. 523-534. | Show Abstract | Read more

Mitf is a central regulator of pigment cell development that is essential for the normal development of the melanocyte and retinal pigment epithelium (RPE) lineages. To understand better the role of Mitf, we have used the Xenopus laevis experimental system to allow a rapid examination of the role of Mitf in vivo. Here, we report the function of XlMitfalpha-M on melanophore development and melanization compared with that of Slug that is expressed in neural crest cells. Overexpression of XlMitfalpha-M led to an increase in melanophores that was partly contributed by an increase in Slug-positive cells, indicating that XlMitfalpha-M is a key regulator of melanocyte/melanophore development and melanization. Moreover, overexpression of a dominant-negative form of XlMitfalpha led to a decrease in the number of melanophores and induced abnormal melanoblast migration. We also observed an induction of ectopic RPE and extended RPE by overexpression of XlMitfalpha-M and possible interactions between XlMitfalpha and several eye-related genes essential for normal eye development.

Goding C. 2005. Editorial: Melanosomes, light and transposable elements PIGMENT CELL RESEARCH, 18 (5), pp. 321-321. | Read more

Goding C. 2005. Editorial: It's back - www.pigment.org PIGMENT CELL RESEARCH, 18 (4), pp. 225-226. | Read more

Goding C. 2005. Editorial: melanomas and Wnts. Pigment Cell Res, 18 (3), pp. 149. | Read more

Goding C. 2005. Editorial: a model, MIA and Sox. Pigment Cell Res, 18 (2), pp. 63. | Read more

Vance KW, Carreira S, Brosch G, Goding CR. 2005. Tbx2 is overexpressed and plays an important role in maintaining proliferation and suppression of senescence in melanomas. Cancer Res, 65 (6), pp. 2260-2268. | Show Abstract | Read more

The INK4a and ARF genes found at the CDKN2A locus are key effectors of cellular senescence that is believed to act as a powerful anticancer mechanism. Accordingly, mutations in these genes are present in a wide variety of spontaneous human cancers and CDKN2A germ line mutations are found in familial melanoma. The TBX2 gene encoding a key developmental transcription factor is amplified in pancreatic cancer cell lines and preferentially amplified and overexpressed in BRCA1 and BRCA2 mutated breast tumors. Overexpression of Tbx2 and the related factor Tbx3, which is also overexpressed in breast cancer and melanomas, can suppress senescence in defined experimental systems through repression of ARF expression. However, it is not known how Tbx2 mediates its repressive effect nor whether endogenous Tbx2 or Tbx3 perform a similar antisenescence function in transformed cells. This is a particularly important question because the loss of CDKN2A in many human cancers would, in principle, bypass the requirement for Tbx2/3-mediated repression of ARF in suppressing senescence. We show here that Tbx2 is overexpressed in melanoma cell lines and that Tbx2 targets histone deacetylase 1 to the p21Cip1 (CDKN1A) initiator. Strikingly, expression of an inducible dominant-negative Tbx2 (dnTbx2) leads to displacement of histone deacetylase 1, up-regulation of p21(Cip1) expression, and the induction of replicative senescence in CDKN2A-null B16 melanoma cells. In human melanoma cells, expression of dnTbx2 leads to severely reduced growth and induction of senescence-associated heterochromatin foci. The results suggest that the activity of endogenous Tbx2 is critically required to maintain proliferation and suppress senescence in melanomas.

Carreira S, Goodall J, Aksan I, La Rocca SA, Galibert M-D, Denat L, Larue L, Goding CR. 2005. Mitf cooperates with Rb1 and activates p21Cip1 expression to regulate cell cycle progression. Nature, 433 (7027), pp. 764-769. | Show Abstract | Read more

The controls that enable melanoblasts and melanoma cells to proliferate are likely to be related, but so far no key regulator of cell cycle progression specific to the melanocyte lineage has been identified. The microphthalmia-associated transcription factor Mitf has a crucial but poorly defined role in melanoblast and melanocyte survival and in differentiation. Here we show that Mitf can act as a novel anti-proliferative transcription factor able to induce a G1 cell-cycle arrest that is dependent on Mitf-mediated activation of the p21(Cip1) (CDKN1A) cyclin-dependent kinase inhibitor gene. Moreover, cooperation between Mitf and the retinoblastoma protein Rb1 potentiates the ability of Mitf to activate transcription. The results indicate that Mitf-mediated activation of p21Cip1 expression and consequent hypophosphorylation of Rb1 will contribute to cell cycle exit and activation of the differentiation programme. The mutation of genes associated with melanoma, such as INK4a or BRAF that would affect either Mitf cooperation with Rb1 or Mitf stability respectively, would impair Mitf-mediated cell cycle control.

Goding C. 2005. Editorial: Black to the future--the next 5 years. Pigment Cell Res, 18 (1), pp. 1. | Read more

Takahashi K, Kumasaka M, Sato S, Yajima I, Goding C, Yamamoto H. 2004. Mitt contributes to the dendricity of melanoblasts in Xenopus laevis ZOOLOGICAL SCIENCE, 21 (12), pp. 1301-1301.

Martinez-Campa C, Politis P, Moreau J-L, Kent N, Goodall J, Mellor J, Goding CR. 2004. Precise nucleosome positioning and the TATA box dictate requirements for the histone H4 tail and the bromodomain factor Bdf1. Mol Cell, 15 (1), pp. 69-81. | Show Abstract | Read more

Acetylation of histone tails plays a key role in chromatin dynamics and is associated with the potential for gene expression. We show here that a 2-3 bp mispositioning of the nucleosome covering the TATA box at PHO5 induces a dependency on the acetylatable lysine residues of the histone H4 N-terminal region and on the TFIID-associated bromodomain factor Bdf1. This dependency arises either through fusion of the PHO5 promoter to a lacZ reporter or by mutation of the TATA box in the natural gene. The results suggest that promoters in which the TATA box is either absent or poorly accessible on the surface of a nucleosome may compensate by using Bdf1 bromodomains and acetylated H4 tails to anchor TFIID to the promoter during the initial stages of transcription activation. We propose that nucleosome positioning at the nucleotide level provides a subtle, but highly effective, mechanism for gene regulation.

Corre S, Primot A, Sviderskaya E, Bennett DC, Vaulont S, Goding CR, Galibert M-D. 2004. UV-induced expression of key component of the tanning process, the POMC and MC1R genes, is dependent on the p-38-activated upstream stimulating factor-1 (USF-1). J Biol Chem, 279 (49), pp. 51226-51233. | Show Abstract | Read more

Protection against UV-mediated DNA damage and the onset of oncogenesis is afforded by the tanning response in which UV irradiation triggers melanocytes to increase production of melanin that is then transferred to keratinocytes. A key component of the tanning process is the UV-mediated induction of the pro-opiomelanocortin (POMC) and MC1R genes encoding the alpha-melanocyte-stimulating hormone and its receptor, respectively, which play a crucial role in pigmentation by regulating the intracellular levels of cAMP. How these genes are regulated in response to UV irradiation is not known. Here we have shown that UV-induced activation of the POMC and MC1R promoters is mediated by p38 stress-activated kinase signaling to the transcription factor, upstream stimulating factor-1 (USF-1). Importantly, melanocytes derived from USF-1 -/- mice exhibit a defective UV response and fail to activate POMC and MC1R expression in response to UV irradiation. The results define USF-1 as a critical UV-responsive activator of genes implicated in protection from solar radiation.

Vance KW, Goding CR. 2004. The transcription network regulating melanocyte development and melanoma. Pigment Cell Res, 17 (4), pp. 318-325. | Show Abstract | Read more

The enormous variety of pigmentation phenotypes in nature reflects a series of remarkable events that begin in the neural crest and end with the manufacture and distribution of pigment by mature melanocytes located in the epidermis and hair follicles. While the origins of melanoblasts from multipotent precursors in the neural crest is striking in itself, yet more so is the fact that these pioneer melanoblasts manage to undertake and survive their long migration, and in doing so proliferate and maintain their identity before ultimately arriving at their destination and undergoing differentiation. With the application of the powerful combination of genetics and molecular and cell biology the mystery surrounding the genesis of the melanocyte lineage is slowly being unravelled. At its heart is the powerful alliance between signal transduction and transcription that coordinates the program of gene expression that confers on a cell its identity, provides its passport for migration, and instructs it in the arts of survival and timely reproduction. The realization that the proliferation and migration of melanoblasts during development resembles closely the proliferation and metastasis of melanoma, a highly dangerous and increasingly common cancer, serves to highlight the value of the melanocyte system as a model for addressing key issues of general significance in both development and cancer.

Goodall J, Martinozzi S, Dexter TJ, Champeval D, Carreira S, Larue L, Goding CR. 2004. Brn-2 expression controls melanoma proliferation and is directly regulated by beta-catenin. Mol Cell Biol, 24 (7), pp. 2915-2922. | Show Abstract | Read more

Constitutive activation of the Wnt/beta-catenin signaling pathway is a notable feature of a large minority of cases of malignant melanoma, an aggressive and increasingly common cancer. The identification of target genes downstream from this pathway is therefore crucial to our understanding of the disease. The POU domain transcription factor Brn-2 has been implicated in control of proliferation and melanoma survival, and its expression is strongly upregulated in melanoma. We show here that in vivo Brn-2 is expressed in melanocytes but not in embryonic day 11.5 melanoblasts and that its expression is directly controlled by the Wnt/beta-catenin signaling pathway in melanoma cell lines and in transgenic mice. Moreover, silent interfering RNA-mediated inhibition of Brn-2 expression in melanoma cells overexpressing beta-catenin results in significantly decreased proliferation. These results, together with the observation that BRAF signaling also induces Brn-2 expression, reveal that Brn-2 is a focus for the convergence of two key melanoma-associated signaling pathways that are linked to cell proliferation.

Goodall J, Wellbrock C, Dexter TJ, Roberts K, Marais R, Goding CR. 2004. The Brn-2 transcription factor links activated BRAF to melanoma proliferation. Mol Cell Biol, 24 (7), pp. 2923-2931. | Show Abstract | Read more

Malignant melanoma, an aggressive and increasingly common cancer, is characterized by a strikingly high rate (70%) of mutations in BRAF, a key component of the mitogen-activated protein (MAP) kinase signaling pathway. How signaling events downstream from BRAF affect the underlying program of gene expression is poorly understood. We show that the Brn-2 POU domain transcription factor is highly expressed in melanoma cell lines but not in melanocytes or melanoblasts and that overexpression of Brn-2 in melanocytes results in increased proliferation. Expression of Brn-2 is strongly upregulated by Ras and MAP kinase signaling. Importantly, the Brn-2 promoter is stimulated by kinase-activating BRAF mutants and endogenous Brn-2 expression is inhibited by RNA interference-mediated downregulation of BRAF. Moreover, silent interfering RNA-mediated depletion of Brn-2 in melanoma cells expressing activated BRAF leads to decreased proliferation. The results suggest that the high levels of Brn-2 expression observed in melanomas link BRAF signaling to increased proliferation.

Prince S, Carreira S, Vance KW, Abrahams A, Goding CR. 2004. Tbx2 directly represses the expression of the p21(WAF1) cyclin-dependent kinase inhibitor. Cancer Res, 64 (5), pp. 1669-1674. | Show Abstract | Read more

T-box factors play a crucial role in the development of many tissues, and mutations in T-box factor genes have been implicated in multiple human disorders. Some T-box factors have been implicated in cancer; for example, Tbx2 and Tbx3 can suppress replicative senescence, whereas Tbx3 can cooperate with Myc and Ras in cellular transformation. The p21(WAF1) cyclin-dependent kinase inhibitor plays a key role in senescence and in cell cycle arrest after DNA damage. Here, using a combination of in vitro DNA-binding, transfection, and chromatin immunoprecipitation assays, we show that Tbx2 can bind and repress the p21 promoter in vitro and in vivo. Moreover, small interfering RNA-mediated down-regulation of Tbx2 expression results in a robust activation of p21 expression. Taken together, these results implicate Tbx2 as a novel direct regulator of p21 expression and have implications for our understanding of the role of T-box factors in the regulation of senescence and oncogenesis, as well as in development.

Moreau J-L, Lee M, Mahachi N, Vary J, Mellor J, Tsukiyama T, Goding CR. 2003. Regulated displacement of TBP from the PHO8 promoter in vivo requires Cbf1 and the Isw1 chromatin remodeling complex. Mol Cell, 11 (6), pp. 1609-1620. | Show Abstract | Read more

Regulated binding of TBP to a promoter is a key event in transcriptional regulation. We show here that on glucose depletion, the S. cerevisiae Isw1 chromatin remodeling complex is required for the displacement of TBP from the PHO8 promoter. Displacement of TBP also requires the sequence-specific bHLH-LZ factor Cbf1p that targets Isw1p to the PHO8 UAS. Cbf1p- and Isw1p-dependent displacement of TBP is also observed at the PHO84 promoter, but not at the ADH1 promoter, where loss of TBP is Cbf1p- and Isw1p independent. The results point to a promoter-specific Isw1p-dependent mechanism for targeted regulation of basal transcription by displacement of TBP from a promoter.

Yajima I, Endo K, Sato S, Toyoda R, Wada H, Shibahara S, Numakunai T, Ikeo K, Gojobori T, Goding CR, Yamamoto H. 2003. Cloning and functional analysis of ascidian Mitf in vivo: insights into the origin of vertebrate pigment cells. Mech Dev, 120 (12), pp. 1489-1504. | Show Abstract | Read more

The microphthalmia-associated transcription factor (Mitf) is a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) transcription factor essential for the development and function of all melanin-producing pigment cells in vertebrates. To elucidate the evolutionary history of Mitf and the antiquity of its association with pigment cells, we have isolated and characterized HrMitf, a sole member of the Mitf-TFE bHLH-ZIP subfamily in the ascidian Halocynthia roretzi. Maternal HrMitf mRNA is detected in the fertilized egg and in the animal hemisphere from 4-cell stage through the gastrula stage. From the neurula through the early tailbud stage, HrMitf is preferentially expressed in the pigment-lineage cells that express the lineage-specific melanogenesis genes tyrosinase (HrTyr) and Tyrp. Overexpression of HrMitf induced ectopic expression of HrTyr enzyme activity in mesenchymal cells where the same enzyme activity was induced by overexpression of HrPax3/7, suggesting that a part(s) of the Pax3-Mitf-tyrosinase gene regulatory cascade seen in vertebrate melanocytes is operative during ascidian embryogenesis. We also show HrMitf and mouse Mitf-A, a Mitf isoform abundantly expressed in pigmented epithelial cells, share similar functional characteristics. These results suggest antiquity of the association of the Mitf-TFE subfamily with pigment cells and may support the idea that acquisition of multiple promoters (isoforms) by an ancestral Mitf gene has allowed the evolution of multiple pigment cell types.

Li KKC, Goodall J, Goding CR, Liao S-K, Wang C-H, Lin Y-C, Hiraga H, Nojima T, Nagashima K, Schaefer K-L, Lee KAW. 2003. The melanocyte inducing factor MITF is stably expressed in cell lines from human clear cell sarcoma. Br J Cancer, 89 (6), pp. 1072-1078. | Show Abstract | Read more

Clear cell sarcoma (CCS) is associated with the EWS/ATF1 oncogene that is created by chromosomal fusion of the Ewings Sarcoma oncogene (EWS) and the cellular transcription factor ATF1. The melanocytic character of CCS suggests that the microphthalmia-associated transcription factor (Mitf), a major inducer of melanocytic differentiation, may be miss-expressed in CCS. Accordingly, we show that the mRNA and protein of the melanocyte-specific isoform of Mitf (Mitf-M) are present in several cultured CCS cell lines (Su-ccs-1, DTC1, Kao, MST-1, MST-2 and MST-3). The above cell lines thus provide a valuable experimental resource for examining the role of Mitf-M in both CCS and melanocyte differentiation. Melanocyte-specific expression of Mitf-M is achieved via an ATF-dependent melanocyte-specific cAMP-response element in the Mitf-M promoter, and expression of Mitf-M in CCS cells suggests that EWS/ATF1 (a potent and promiscuous activator of cAMP-inducible promoters) may activate the Mitf-M promoter. Surprisingly, however, the Mitf-M promoter is not activated by EWS/ATF1 in transient assays employing CCS cells, melanocytes or nonmelanocytic cells. Thus, our results indicate that Mitf-M promoter activation may require an appropriate chromosomal context in CCS cells or alternatively that the Mitf-M promoter is not directly activated by EWS/ATF1.

Larue L, Kumasaka M, Goding CR. 2003. Beta-catenin in the melanocyte lineage. Pigment Cell Res, 16 (3), pp. 312-317. | Show Abstract | Read more

beta-Catenin is a multifunctional protein involved in cell-cell adhesion, intracellular signalling and gene transcription. It has been implicated in the development of various lineages, including neural crest derivatives. Melanocytes are derived from neural crest cells and beta-catenin is expressed throughout the development of this cell lineage. The multifunctional activity of beta-catenin is directly associated with its participation in multi protein-protein interactions. The cell-cell adhesion function of beta-catenin is mediated by the large cadherin cell adhesion molecule family, the intracellular signalling function by its interaction with GSK3beta, and the gene transcription activity by the four known LEF/TCF DNA binding-proteins. Here, we review the known beta-catenin interacting factors and targets involved in the development and transformation of melanocytes and in particular its role in the expression of the crucial gene of melanocyte development, Mitf.

Erdemir T, Bilican B, Cagatay T, Goding CR, Yavuzer U. 2002. Saccharomyces cerevisiae C1D is implicated in both non-homologous DNA end joining and homologous recombination. Mol Microbiol, 46 (4), pp. 947-957. | Show Abstract | Read more

C1D is a gamma-irradiation inducible nuclear matrix protein that interacts with and activates the DNA-dependent protein kinase (DNA-PK) that is essential for the repair of the DNA double-strand breaks and V(D)J recombination. Recently, it was demonstrated that C1D can also interact with TRAX and prevent the association of TRAX with Translin, a factor known to bind DNA break-point junctions, and that over expression of C1D can induce p53-dependent apoptosis. Taken together, these findings suggest that mammalian C1D could be involved in maintenance of genome integrity by regulating the activity of proteins involved in DNA repair and recombination. To obtain direct evidence for the biological function of C1D that we show is highly conserved between diverse species, we have analysed the Saccharomyces cerevisiae C1D homologue. We report that the disruption of the YC1D gene results in a temperature sensitivity and that yc1d mutant strains exhibit defects in non-homologous DNA end joining (NHEJ) and accurate DNA repair. In addition, using a novel plasmid-based in vivo recombination assay, we show that yc1d mutant strains are also defective in homologous recombination. These results indicate that YC1D is implicated in both homologous recombination and NHEJ pathways for the repair of DNA double-strand breaks.

Erdemir T, Bilican B, Oncel D, Goding CR, Yavuzer U. 2002. DNA damage-dependent interaction of the nuclear matrix protein C1D with Translin-associated factor X (TRAX). J Cell Sci, 115 (Pt 1), pp. 207-216. | Show Abstract

The nuclear matrix protein C1D is an activator of the DNA-dependent protein kinase (DNA-PK), which is essential for the repair of DNA double-strand breaks (DSBs) and V(D)J recombination. C1D is phosphorylated very efficiently by DNA-PK, and its mRNA and protein levels are induced upon gamma-irradiation, suggesting that C1D may play a role in repair of DSBs in vivo. In an attempt to identify the biological function of C1D, we have employed the yeast two-hybrid system and found that C1D interacts specifically with Translin-associated factor X, TRAX. Although the biological function of TRAX remains unknown, its bipartite nuclear targeting sequences suggest a role for TRAX in the movement of associated proteins, including Translin, into the nucleus. We show that C1D and TRAX interact specifically in both yeast and mammalian cells. Interestingly, however, interaction of these two proteins in mammalian cells only occur following gamma-irradiation, raising the possibility of involvement of TRAX in DNA double-strand break repair and providing evidence for biological functions of the nuclear matrix protein C1D and TRAX. Moreover, we show, using fluorescently tagged proteins, that the relative expression levels of TRAX and Translin affect their subcellular localization. These results suggest that one role for C1D may be to regulate TRAX/Translin complex formation.

Galibert MD, Carreira S, Goding CR. 2001. The Usf-1 transcription factor is a novel target for the stress-responsive p38 kinase and mediates UV-induced Tyrosinase expression. EMBO J, 20 (17), pp. 5022-5031. | Show Abstract | Read more

The stress-activated signalling cascade leading to phosphorylation of the p38 family of kinases plays a crucial role during development and in the cellular response to a wide variety of stress-inducing agents. Although alterations in gene expression characteristic of the stress response require the regulation of key transcription factors by the p38 family, few downstream targets for this signalling pathway have been identified. By examining the ability of pigment cells to respond to UV irradiation as part of the UV-induced tanning response, we show that while the microphthalmia-associated transcription factor Mitf regulates basal Tyrosinase expression, it is the ubiquitous basic helix-loop-helix-leucine zipper transcription factor Usf-1 that is required for the UV activation of the Tyrosinase promoter. Consistent with this we demonstrate that Usf-1 is phosphorylated and activated by the stress-responsive p38 kinase. The results suggest that activation of Usf-1 by p38 at a wide variety of viral and cellular promoters will provide a link between stimuli as diverse as UV irradiation, glucose, viral infection and pro-inflammatory cytokines, and the changes in gene expression associated with the stress response.

Toyoda R, Sato S, Ikeo K, Gojobori T, Numakunai T, Goding CR, Yamamoto H. 2000. Pigment cell-specific expression of the tyrosinase gene in ascidians has a different regulatory mechanism from vertebrates. Gene, 259 (1-2), pp. 159-170. | Show Abstract | Read more

Tyrosinase is the key enzyme required for the synthesis of melanin pigments. Sequence comparison and functional analysis of the 5' upstream regions of vertebrate tyrosinase genes have revealed the importance of conserved E-box motifs in regulating their specific expression in pigment cells, optic cup-derived retinal pigment epithelium (RPE) and neural crest-derived melanocytes. In ascidians (more basal protochordates), two pigment cells that resemble vertebrate RPE cells are formed and specifically express the orthologous tyrosinase gene (HrTyr) in the cerebral vesicle located at the anterior end of the neural tube. To define regulatory sequences required for pigment cell-lineage-specific expression of HrTyr during embryogenesis, a series of mutations of the 5' upstream region of HrTyr were fused to the lacZ reporter gene and were microinjected into fertilized eggs. We found that the -152bp upstream of the translational start site is essential for expression in pigment cell precursors of tailbud-stage embryos. Further, additional positive and unique restriction elements were identified in the region up to -1.8kb. Surprisingly, in the -152bp minimal promoter or in other regions with regulatory activities, there are no E-box motifs or sequences correlating with other conserved elements regulating vertebrate tyrosinase promoters. The possibility that Pax proteins regulate HrTyr expression is also discussed.

Lee M, Goodall J, Verastegui C, Ballotti R, Goding CR. 2000. Direct regulation of the Microphthalmia promoter by Sox10 links Waardenburg-Shah syndrome (WS4)-associated hypopigmentation and deafness to WS2. J Biol Chem, 275 (48), pp. 37978-37983. | Show Abstract | Read more

The transcription factor Sox10 is genetically linked with Waardenburg syndrome 4 (WS4) in humans and the Dominant megacolon (Dom) mouse model for this disease. The pigmentary defects observed in the Dom mouse and WS4 are reminiscent of those associated with mutations in the microphthalmia (Mitf) gene, which encodes a transcription factor essential for the development of the melanocyte lineage. We demonstrate here that wild type Sox10 directly binds and activates transcription of the MITF promoter, whereas a mutant form of the Sox10 protein genetically linked with WS4 acts as a dominant-negative repressor of MITF expression and can reduce endogenous MITF protein levels. The ability of Sox10 to activate transcription of the MITF promoter implicates Sox10 in the regulation of melanocyte development and provides a molecular basis for the hypopigmentation and deafness associated with WS4.

Goding CR. 2000. Melanocyte development and malignant melanoma. Forum (Genova), 10 (3), pp. 176-187. | Show Abstract

Malignant melanoma is a notoriously aggressive disease that can affect relatively young individuals and whose incidence is rising at an alarming rate. Unlike many cancers, metastatic melanoma is poorly responsive to current therapies and mutations affecting p53, the retinoblastoma gene product or Ras which occur frequently in many other cancer types, appear to be rare or at least relatively late events in the progression of the disease. Recent advances in our understanding of the disease at the molecular level have indicated that in addition to the loss of cell cycle checkpoints which may be common to all cancers, malignant melanoma shares many characteristics in common with developmental precursors to melanocytes, the mature pigment producing cells of the skin and hair follicles which are responsible for skin and hair colour. This review therefore focuses on the signalling pathways that play a crucial role in the development of the melanocyte lineage which are subject to deregulation in malignant melanoma namely signalling by receptor tyrosine kinases, the Wnt signalling pathway, as well as loss of the p16INK4a cyclin-dependent kinase inhibitor. Intriguingly all three pathways impact on the expression or function of the microphthalmia-associated transcription factor which plays an essential role in melanocyte development.

Lee M, O'Regan S, Moreau JL, Johnson AL, Johnston LH, Goding CR. 2000. Regulation of the Pcl7-Pho85 cyclin-cdk complex by Pho81. Mol Microbiol, 38 (2), pp. 411-422. | Show Abstract | Read more

Saccharomyces cerevisiae strains lacking a functional Pho85 cyclin-dependent kinase (cdk) exhibit a complex phenotype, including deregulation of phosphatase genes controlled by the transcription factor Pho4, slow growth on rich media, failure to grow using galactose, lactate or glycerol as a carbon source and hyperaccumulation of glycogen. The ability of Pho85 to regulate the transcription factor Pho4 is mediated by its association the Pho80 cyclin. Some other regulatory functions of the Pho85 cdk have been shown to be mediated via its interaction with a recently identified family of Pho80-related cyclins (Pcls). Here, we show that the poorly characterized Pho80-like protein Pcl7 forms a functional kinase complex with the Pho85 cdk, and that the activity of this complex is inhibited in response to phosphate starvation. Additionally, we show that Pcl7 interacts with the phosphate-regulated cyclin-cdk inhibitor Pho81, and that the regulation of the Pcl7-Pho85 complex in response to changes in phosphate levels is dependent on Pho81. Thus, we demonstrate for the first time that the Pho81 regulator is not dedicated to regulating Pho80, but may act to co-ordinate the activity of both the Pho80-Pho85 and Pcl7-Pho85 cyclin-cdk complexes in response to phosphate levels. We also demonstrate that expression of Pcl7 is cell cycle regulated, with maximal activity occurring in mid to late S-phase, perhaps suggesting a role for Pcl7 in cell cycle progression. Finally, we describe the phenotype of pcl7Delta and pcl6Delta yeast strains that have defects in carbon source utilization.

Carreira S, Liu B, Goding CR. 2000. The gene encoding the T-box factor Tbx2 is a target for the microphthalmia-associated transcription factor in melanocytes. J Biol Chem, 275 (29), pp. 21920-21927. | Show Abstract | Read more

Commitment to the melanocyte lineage is characterized by the onset of microphthalmia-associated transcription factor (Mitf) expression. Mitf plays a fundamental role in melanocyte development, with mice lacking Mitf being entirely devoid of pigment cells. In the absence of functional Mitf protein, melanoblasts expressing Mitf mRNA disappear around 2 days after their first appearance either by apoptosis or by losing their identity and adopting an alternative cell fate. The role of Mitf must therefore be to regulate genes required for melanoblast survival, proliferation, or the maintenance of melanoblast identity. Yet to date, Mitf has been shown to regulate genes such as Tyrosinase, Tyrp-1, and Dct, which are required for pigmentation, a differentiation-specific process. Because expression of these genes cannot account for the complete absence of pigment cells in Mitf-negative mice, Mitf must regulate the expression of other as yet uncharacterized genes. Here we provide several lines of evidence to suggest that Mitf may regulate the expression of the Tbx2 transcription factor, a member of the T-box family of proteins implicated in the maintenance of cell identity. First, isolation and sequencing of the entire murine Tbx2 gene revealed that the Tbx2 promoter contains a full consensus Mitf recognition element; second, Mitf could bind the promoter in vitro and activate Tbx2 expression in vivo in an E box-dependent fashion; and third, Tbx2 is expressed in melanoma cell lines expressing Mitf, but not in a line in which Mitf expression was not detectable. Taken together, with the fact that Tbx2 is expressed in Mitf-positive melanoblasts and melanocytes, but not in Mitf-negative melanoblast precursor cells, the evidence suggests that the Tbx2 gene may represent one of the first known targets for Mitf that is not a gene involved directly in the manufacture of pigment.

Goding CR. 2000. Mitf from neural crest to melanoma: signal transduction and transcription in the melanocyte lineage. Genes Dev, 14 (14), pp. 1712-1728.

Galibert MD, Yavuzer U, Dexter TJ, Goding CR. 1999. Pax3 and regulation of the melanocyte-specific tyrosinase-related protein-1 promoter. J Biol Chem, 274 (38), pp. 26894-26900. | Show Abstract | Read more

Previous work has established that the melanocyte-specific tyrosinase-related protein-1 (TRP-1) promoter is regulated positively by the microphthalmia-associated transcription factor Mitf, acting through the conserved M box and negatively by the T-box factor Tbx2, which can bind two "melanocyte-specific elements" termed the MSEu and MSEi. Both the MSEu and MSEi, which share a 6-base pair GTGTGA consensus, are also recognized by a previously unidentified melanocyte-specific factor, MSF. Here we show using a combination of DNA binding assays, proteolytic clipping, and anti-Pax3 antibodies that MSF is indistinguishable from Pax3, a paired homeodomain transcription factor implicated genetically in melanocyte development and the regulation of the Mitf promoter. Consistent with Pax3 being able to bind the TRP-1 promoter, Pax3 is expressed in melanocytes and melanomas, and TRP-1 promoter activity is up-regulated by Pax3. The results identify a novel role for Pax3 in the expression of TRP-1, and the potential role of Pax3 in the melanocyte lineage is discussed.

Yajima I, Sato S, Kimura T, Yasumoto K, Shibahara S, Goding CR, Yamamoto H. 1999. An L1 element intronic insertion in the black-eyed white (Mitf[mi-bw]) gene: the loss of a single Mitf isoform responsible for the pigmentary defect and inner ear deafness. Hum Mol Genet, 8 (8), pp. 1431-1441. | Show Abstract | Read more

Waardenburg syndrome type 2 (WS2) is an autosomal dominant disorder characterized by a combination of pigmentary and auditory abnormalities. Approximately 20% of WS2 cases are associated with mutations in the gene encoding microphthalmia-associated transcription factor (MITF). MITF plays a critical role in the development of both neural-crest-derived melanocytes and optic cup-derived retinal pigmented epithelium (RPE); the loss of a functional Mitf in mice results in complete absence of all pigment cells, which in turn induces microphthalmia and inner ear deafness. The black-eyed white Mitf mi-bw homozygous mouse normally has a pigmented RPE but lacks melanocytes essential for the pigmentation of the body and hearing. We show here that Mitf mi-bw is caused by an insertion into intron 3 of a 7.2 kb novel L1 element, L1bw, which belongs to an actively retrotransposing TF subfamily. The L1bw insertion reduces the amount of mRNAs for two Mitf isoforms, Mitf-A and Mitf-H, by affecting their overall expression levels and pre-mRNA splicing patterns, while it abolishes mRNA expression of another isoform, Mitf-M, which is specifically expressed in neural-crest-derived melanocytes. The consequence of the L1 insertion in the black-eyed white Mitf mi-bw mouse is that the developmental programme for RPE cells proceeds normally, most likely because of the presence of residual, full-length Mitf-A and Mitf-H proteins, whereas the lack of Mitf-M results in loss of the melanocyte population. The results suggest that melanocyte development depends critically on a single Mitf isoform, Mitf-M, and raise the possibility that specific mutations affecting MITF-M, the human equivalent of Mitf-M, may be responsible for a subset of WS2 conditions.

Barbaric S, Münsterkötter M, Goding C, Hörz W. 1998. Cooperative Pho2-Pho4 interactions at the PHO5 promoter are critical for binding of Pho4 to UASp1 and for efficient transactivation by Pho4 at UASp2. Mol Cell Biol, 18 (5), pp. 2629-2639. | Show Abstract | Read more

The activation of the PHO5 gene in Saccharomyces cerevisiae in response to phosphate starvation critically depends on two transcriptional activators, the basic helix-loop-helix protein Pho4 and the homeodomain protein Pho2. Pho4 acts through two essential binding sites corresponding to the regulatory elements UASp1 and UASp2. Mutation of either of them results in a 10-fold decrease in promoter activity, and mutation of both sites renders the promoter totally uninducible. The role of Pho4 appears relatively straightforward, but the mechanism of action of Pho2 had remained elusive. By in vitro footprinting, we have recently mapped multiple Pho2 binding sites adjacent to the Pho4 sites, and by mutating them individually or in combination, we now show that each of them contributes to PHO5 promoter activity. Their function is not only to recruit Pho2 to the promoter but to allow cooperative binding of Pho4 together with Pho2. Cooperativity requires DNA binding of Pho2 to its target sites and Pho2-Pho4 interactions. A Pho4 derivative lacking the Pho2 interaction domain is unable to activate the promoter, but testing of UASp1 and UASp2 individually in a minimal CYC1 promoter reveals a striking difference between the two UAS elements. UASp1 is fully inactive, presumably because the Pho4 derivative is not recruited to its binding site. In contrast, UASp2 activates strongly in a Pho2-independent manner. From in vivo footprinting experiments and activity measurements with a promoter variant containing two UASp2 elements, we conclude that at UASp2, Pho2 is mainly required for the ability of Pho4 to transactivate.

Aksan I, Goding CR. 1998. Targeting the microphthalmia basic helix-loop-helix-leucine zipper transcription factor to a subset of E-box elements in vitro and in vivo. Mol Cell Biol, 18 (12), pp. 6930-6938. | Show Abstract | Read more

The development of melanocytes, which are pigment-producing cells responsible for skin, hair, and eye color, is absolutely dependent on the action of the microphthalmia basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor (Mi); mice lacking a functional Mi protein are entirely devoid of pigment cells. Mi has been shown to activate transcription of the tyrosinase, TRP-1, TRP-2, and QNR-71 genes through specific E-box elements, most notably the highly conserved M box. We investigated the mechanism which enables Mi to be recruited specifically to a restricted subset of E boxes in target promoters while being prevented from binding E-box elements in other promoters. We show both in vitro and in vivo that the presence of a T residue flanking a CATGTG E box is an essential determinant of the ability of Mi to bind DNA, and we successfully predict that the CATGTG E box from the P gene would not bind Mi. In contrast, no specific requirement for the sequences flanking a CACGTG E box was observed, and no binding to an atypical E box in the c-Kit promoter was detected. The relevance of these observations to the control of melanocyte-specific gene expression was highlighted by the fact that the E-box elements located in the tyrosinase, TRP-1, TRP-2, and QNR-71 promoters without exception possess a 5' flanking T residue which is entirely conserved between species as diverse as man and turtle. The ability of Mi to discriminate between different E-box motifs provides a mechanism to restrict the repertoire of genes which are likely to be regulated by Mi and provides insight into the ability of bHLH-LZ transcription factors to achieve the specificity required for the precise coordination of transcription during development.

McAndrew PC, Svaren J, Martin SR, Hörz W, Goding CR. 1998. Requirements for chromatin modulation and transcription activation by the Pho4 acidic activation domain. Mol Cell Biol, 18 (10), pp. 5818-5827. | Show Abstract | Read more

Perhaps the best characterized example of an activator-induced chromatin transition is found in the activation of the Saccharomyces cerevisiae acid phosphatase gene PHO5 by the basic helix-loop-helix (bHLH) transcription factor Pho4. Transcription activation of the PHO5 promoter by Pho4 is accompanied by the remodeling of four positioned nucleosomes which is dependent on the Pho4 activation domain but independent of transcription initiation. Whether the requirements for transcription activation through the TATA sequence are different from those necessary for the chromatin transition remains a major outstanding question. In an attempt to understand better the ability of Pho4 to activate transcription and to remodel chromatin, we have initiated a detailed characterization of the Pho4 activation domain. Using both deletion and point mutational analysis, we have defined residues between positions 75 and 99 as being both essential and sufficient to mediate transcription activation. Significantly, there is a marked concordance between the ability of mutations in the Pho4 activation domain to induce chromatin opening and transcription activation. Interestingly, the requirements for transcription activation within the Pho4 activation domain differ significantly if fused to a heterologous bHLH-leucine zipper DNA-binding domain. The implications for transcription activation by Pho4 are discussed.

Carreira S, Dexter TJ, Yavuzer U, Easty DJ, Goding CR. 1998. Brachyury-related transcription factor Tbx2 and repression of the melanocyte-specific TRP-1 promoter. Mol Cell Biol, 18 (9), pp. 5099-5108. | Show Abstract | Read more

Previous work has demonstrated that two key melanocyte-specific elements termed the MSEu and MSEi play critical roles in the expression of the melanocyte-specific tyrosinase-related protein 1 (TRP-1) promoter. Both the MSEu and MSEi, located at position -237 and at the initiator, respectively, bind a melanocyte-specific factor termed MSF but are also recognized by a previously uncharacterized repressor, since mutations affecting either of these elements result in strong up-regulation of TRP-1 promoter activity in melanoma cells. Here we demonstrate that repression mediated by the MSEu and MSEi also operates in melanocytes. We also report that both the MSEu and MSEi are recognized by the brachyury-related transcription factor Tbx2, a member of the recently described T-box family, and that Tbx2 is expressed in melanocyte and melanoblast cell lines but not in melanoblast precursor cells. Although Tbx2 and MSF each recognize the TRP-1 MSEu and MSEi motifs, it is binding by Tbx-2, not binding by MSF, that correlates with repression. Several lines of evidence tend to point to the brachyury-related transcription factor Tbx2 as being the repressor of TRP-1 expression: both the MSEu and MSEi bind Tbx2, and mutations in either element that result in derepression of the TRP-1 promoter diminish binding by Tbx2; the TRP-1 promoter, but not the tyrosinase, microphthalmia, or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) promoter, is repressed by Tbx2 in cotransfection assays; a high-affinity consensus brachyury/Tbx2-binding site is able to constitutively repress expression of the heterologous IE110 promoter; and a low-affinity brachyury/Tbx2 binding site is able to mediate Tbx2-dependent repression of the G3PDH promoter. Although we cannot rule out the presence of an additional, as yet unidentified factor playing a role in the negative regulation of TRP-1 in vivo, the evidence presented here suggests that Tbx2 most likely is the previously unidentified repressor of TRP-1 expression and as such is likely to represent the first example of transcriptional repression by a T-box family member.

Sato S, Roberts K, Gambino G, Cook A, Kouzarides T, Goding CR. 1997. CBP/p300 as a co-factor for the Microphthalmia transcription factor. Oncogene, 14 (25), pp. 3083-3092. | Show Abstract | Read more

The Microphthalmia basic-Helix-Loop-Helix-Leucine Zipper (bHLH-LZ) transcription factor (Mi) plays a crucial role in the genesis of melanocytes; mice deficient for a functional (Microphthalmia) gene product lack all pigment cells. We show here that the Mi activation domain resides N-terminal to the DNA-binding domain and that as little as 18 amino acids are sufficient to mediate transcription activation. The minimal activation region of Mi is highly conserved in the related transcription factor TFE3 and is predicted to adopt an amphipathic alpha-helical conformation. This region of Mi is also highly conserved with a region of E1A known to be essential for binding the CBP/p300 transcription cofactor. Consistent with these observations, the Mi activation domain can interact in vitro with CBP specifically through a region of CBP required for complex formation with E1A, P/CAF and c-Fos, and anti p300 antibodies can co-immunoprecipitate Mi from both melanocyte and melanoma cell lines. In addition, co-transfection of a vector expressing CBP2 (aas 1621-1891) fused to the VP16 activation domain potentiated the ability of Mi to activate transcription, confirming the significance of the CBP-Mi interaction observed in vitro. These data suggest that transcription activation by Mi is achieved at least in part by recruitment of CBP. The parallels between transcription regulation by Microphthalmia in melanocytes and MyoD in muscle cells are discussed.

Galibert MD, Boucontet L, Goding CR, Meo T. 1997. Recognition of the E-C4 element from the C4 complement gene promoter by the upstream stimulatory factor-1 transcription factor. J Immunol, 159 (12), pp. 6176-6183. | Show Abstract

Activation of complement gene expression plays a major role in the response to antigenic challenge. The induction of complement synthesis occurs primarily in liver and in macrophages and is mediated, at least in part, by increased transcription of the complement genes. For example, transcription of the C4 complement gene, which plays a crucial role in the complement pathway, is induced in response to acute inflammation or tissue injury. Previous work has defined the elements present in the C4 complement gene promoter that are required for its expression. Particularly important is an E-box motif, E-C4, that is conserved between the mouse, human, and rat promoters and that directed up to 90% of transcription from the mouse C4 promoter. Here we have purified the E-C4-binding factor to homogeneity using a novel and rapid affinity purification procedure. Following N-terminal microsequencing and subsequent isolation of the corresponding cDNA, the factor binding the E-C4 element was identified as upstream stimulatory factor-1 (USF-1), a basic helix-loop-helix-leucine zipper transcription factor. We also show for the first time that in vivo USF-1 is a phosphoprotein, but that phosphorylation of USF-1 is severely reduced in cells in culture. Moreover, the phosphorylated form of USF-1 binds DNA preferentially, indicating that phosphorylation may enhance the ability of USF-1 to bind DNA. The implications of USF-1 phosphorylation for C4 complement gene expression and transcription regulation are discussed.

Sviderskaya EV, Easty DJ, Hill SP, Goding CR, Bennett DC. 1997. Immortal cell lines representing precursors of melanoblasts, and their molecular markers Journal of Anatomy, 191 (1), pp. 134.

Goding CR, Fisher DE. 1997. Regulation of melanocyte differentiation and growth. Cell Growth Differ, 8 (9), pp. 935-940.

Crouch DH, Gallagher R, Goding CR, Neil JC, Fulton R. 1996. Multiple phenotypes associated with Myc-induced transformation of chick embryo fibroblasts can be dissociated by a basic region mutation. Nucleic Acids Res, 24 (16), pp. 3216-3221. | Show Abstract | Read more

Chimaeric alleles were constructed to assay the biological functions of an N-terminal deletion and C-terminal mutations which were found in a naturally occurring mutant of feline vMyc, T17. The mutant alleles were assayed for their ability to transform chick embryo fibroblasts in vitro by a number of criteria, namely the ability to induce morphological transformation, an accelerated growth rate and growth in soft agar. Feline cMyc could transform the avian cells, whilst T17 vMyc could not, and the N-terminal deletion was responsible for conferring the primary transformation defect on the mutant protein. The C-terminal mutations which consist of a point mutation adjacent to the nuclear localisation signal and a point mutation/amino acid insertion within the basic region (BR) could, however, dissociate the Myc-induced parameters of transformation. This effect was a specific function of the BR mutation alone, and the mutation could be transferred into avian cMyc with comparable biological consequences. The BR mutation did not disrupt the sequence specific DNA binding activity of the protein in vivo, despite exerting a biological effect. These data suggest a novel phenotype where the mutation may affect a subset of Myc-regulated genes through altered DNA binding specificity or protein-protein interactions.

Yavuzer U, Goding CR. 1995. pWITCH: a versatile two-hybrid assay vector for the production of epitope/activation domain-tagged proteins both in vitro and in yeast. Gene, 165 (1), pp. 93-96. | Show Abstract | Read more

We describe the construction of a new vector, pWITCH, designed to facilitate the characterisation of proteins encoded by novel cDNAs isolated using either a one- or two-hybrid assay. Expression of directionally cloned cDNAs is directed in vivo in Saccharomyces cerevisiae from the inducible GAL10 promoter and in vitro from the T7 promoter, while translation of the expressed cDNAs results in proteins which are tagged in vitro with a specific epitope and in vivo with both the epitope and the VP16 transcription activation domain. The principle of using multiple promoters each able to operate under different conditions to express different combinations of protein domains without the need for subcloning should be generally applicable.

Yavuzer U, Keenan E, Lowings P, Vachtenheim J, Currie G, Goding CR. 1995. The Microphthalmia gene product interacts with the retinoblastoma protein in vitro and is a target for deregulation of melanocyte-specific transcription. Oncogene, 10 (1), pp. 123-134. | Show Abstract

Little is known of the molecular mechanisms underlying the differentiation of the melanocyte from the melanoblast or the progression from the melanocyte to a malignant melanoma. Since the adenovirus E1A products have proved a useful tool for understanding control of differentiation in other systems, we explored the possibility of using E1A as a probe for factors controlling melanocyte-specific gene expression and differentiation. The results obtained show that the adenovirus E1A 13S, but not the 12S, product can transform the highly pigmented and TPA-dependent melanocyte cell line melan-a. Transformation is characterised by a morphological change, loss of TPA-dependence, the ability to grow in soft agar and strikingly, loss of pigmentation which correlates with loss of expression of the melanocyte-specific TRP-1 and tyrosinase genes. Cotransfection assays demonstrated that repression of TRP-1 by E1A correlated with E1A binding to p105Rb and p300, with the target in the TRP-1 promoter being the M-box, and 11 bp basic-Helix-loop-Helix (bHLH) factor-binding motif conserved between melanocyte-specific promoters. Consistent with the M-box acting as a target for E1a-mediated transcription repression, we also show that the basic-helix-loop-helix-leucine zipper (bHLH-LZ) protein (Mi) encoded by the microphthalmia gene (mi), which is required for pigment cell differentiation, is a positive acting transcription factor which can interact with the retinoblastoma product in vitro and activate the TRP-1 promoter. Moreover, expression of the mi gene was reduced around 50-fold in the non-pigmented E1a-transformed melan-a cells compared to the nontransformed melan-a cell line, with ectopic expression of Mi able to prevent repression of the tyrosinase and TRP-1 promoters in the presence of E1A. Mi therefore appears to play a crucial role in melanocyte-specific gene expression. The parallels between repression of myogenesis and muscle cell bHLH factors, and Mi and melanocyte differentiation are discussed.

Eisen T, Easty DJ, Bennett DC, Goding CR. 1995. The POU domain transcription factor Brn-2: elevated expression in malignant melanoma and regulation of melanocyte-specific gene expression. Oncogene, 11 (10), pp. 2157-2164. | Show Abstract

Previous work has shown that melanoma cell lines express a distinct octamer binding protein. Given the role of octamer-binding proteins in cell differentiation and development, the role this factor is a key issue in understanding melanocyte differentiation and transformation. Using a proteolytic clipping assay, we show that the melanoma-specific octamer factor is Brn-2/N-Oct3, a POU domain protein previously known to be expressed in adult brain and in the developing nervous system. N-Oct3 mRNA was detected in a range of human melanoma cell lines and was around 10-fold elevated compared to normal human melanocytes while mRNA for Brn-2 was also detected in a mouse melanoblast cell line. Expression of Brn-2/N-Oct3, in melanoma cells in cotransfection assays activated the expression of the MHC class II DR alpha promoter but repressed the activity of the melanocyte-specific tyrosinase promoter. Repression correlated with Brn-2/N-Oct3 binding in a mutually exclusive fashion with basic-helix-loop-helix-leucine-zipper (bHLH-LZ) transcription factor USF in vitro and with Brn-2 expression preventing activation of the tyrosinase promoter by the bHLH-LZ factor Microphthalmia in vivo. The potential role of Brn-2/N-Oct3 in melanocyte differentiation and gene expression is discussed.

Bentley NJ, Eisen T, Goding CR. 1994. Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator. Mol Cell Biol, 14 (12), pp. 7996-8006. | Show Abstract | Read more

The tyrosinase gene is expressed specifically in melanocytes and the cells of the retinal pigment epithelium, which together are responsible for skin, hair, and eye color. By using a combination of DNase I footprinting and band shift assays coupled with mutagenesis of specific DNA elements, we examined the requirements for melanocyte-specific expression of the human tyrosinase promoter. We found that as little as 115 bp of the upstream sequence was sufficient to direct tissue-specific expression. This 115-bp stretch contains three positive elements: the M box, a conserved element found in other melanocyte-specific promoters; an Sp1 site; and a highly evolutionarily conserved element located between -14 and +1 comprising an E-box motif and an overlapping octamer element. In addition, two further elements, one positive and one negative, are located between positions -185 and -150 and positions -150 and -115, respectively. We also found that the basic helix-loop-helix factor encoded by the microphthalmia gene, which is essential for melanocyte differentiation, can transactivate the tyrosinase promoter via the M box and the conserved E box located close to the initiator. Since in vitro assays failed to identify any melanocyte-specific DNA-binding activity, the possibility that the specific arrangement of elements within the basal tyrosinase promoter determines melanocyte-specific expression is discussed.

Hirst K, Fisher F, McAndrew PC, Goding CR. 1994. The transcription factor, the Cdk, its cyclin and their regulator: directing the transcriptional response to a nutritional signal. EMBO J, 13 (22), pp. 5410-5420. | Show Abstract | Read more

The Pho80-Pho85 cyclin-cdk complex prevents transcription of PHO5 by inhibiting the ability of the basic-helix-loop-helix transcription factor Pho4 to activate transcription in response to high phosphate conditions. In low phosphate the Pho80-Pho85 complex is inactivated and Pho4 is then able to activate the acid phosphatase gene PHO5. We show here that Pho4 and the homeobox protein Pho2 interact in vivo and act cooperatively to activate the PHO5 UAS, with interaction being regulated by the phosphate switch. In addition, we also demonstrate that an additional factor, Pho81, interacts in high phosphate with both the Pho80 cyclin and with Pho4. In low phosphate, Pho80 and Pho81 dissociate from Pho4, but retain the ability to interact with each other. The evidence presented here supports the idea that Pho81 acts as a phosphate-sensitive trigger that regulates the ability of the Pho80-Pho85 cyclin-cdk complex to bind Pho4, while DNA binding by Pho4 is dependent on the phosphate-sensitive interaction with Pho2.

Yavuzer U, Goding CR. 1994. Melanocyte-specific gene expression: role of repression and identification of a melanocyte-specific factor, MSF. Mol Cell Biol, 14 (5), pp. 3494-3503. | Show Abstract | Read more

For a gene to be transcribed in a tissue-specific fashion, expression must be achieved in the appropriate cell type and also be prevented in other tissues. As an approach to understanding the regulation of tissue-specific gene expression, we have analyzed the requirements for melanocyte-specific expression of the tyrosinase-related protein 1 (TRP-1) promoter. Positive regulation of TRP-1 expression is mediated by both an octamer-binding motif and an 11-bp element, termed the M box, which is conserved between the TRP-1 and other melanocyte-specific promoters. We show here that, consistent with its ability to activate transcription in a non-tissue-specific fashion, the M box binds the basic-helix-loop-helix factor USF in vitro. With the use of a combination of site-directed mutagenesis and chimeric promoter constructs, additional elements involved in regulating TRP-1 expression were identified. These include the TATA region, which appears to contribute to the melanocyte specificity of the TRP-1 promoter. Mutational analysis also identified two repressor elements, one at the start site, the other located at -240, which function both in melanoma and nonmelanoma cells. In addition, a melanocyte-specific factor, MSF, binds to sites which overlap both repressor elements, with substitution mutations demonstrating that binding by MSF is not required for repression. Although a functional role for MSF has not been unequivocally determined, the location of its binding sites leads us to speculate that it may act as a melanocyte-specific antirepressor during transcription of the endogenous TRP-1 gene.

Jayaraman PS, Hirst K, Goding CR. 1994. The activation domain of a basic helix-loop-helix protein is masked by repressor interaction with domains distinct from that required for transcription regulation. EMBO J, 13 (9), pp. 2192-2199. | Show Abstract | Read more

While there are many examples of protein-protein interactions modulating the DNA-binding activity of transcription factors, little is known of the molecular mechanisms underlying the regulation of the transcription activation function. Using a two-hybrid system we show here that transcription repression of the basic domain/helix-loop-helix factor PHO4 is mediated by complex formation with the PHO80 repressor. In contrast to other systems, such as inhibition of GAL4 by GAL80 or of p53 by MDM2, where repression is mediated by direct interaction at regions overlapping the transcription activation domain, interaction with PHO80 involves two regions of PHO4 distinct from those involved in transcription activation or DNA-binding and dimerization. The possibility that repression of PHO4 by PHO80 may represent a general mechanism of transcription control, including regulation of the cell-type-specific transcription activation domain of c-Jun, is discussed.

FISHER F, CROUCH DH, GODING CR, JAYARAMAN PS, CLARK W, GILLESPIE DAF. 1993. MAX AND MYC/MAX COMPLEXES ACTIVATE TRANSCRIPTION INVIVO JOURNAL OF CELLULAR BIOCHEMISTRY, pp. 181-181.

Crouch DH, Fisher F, Clark W, Jayaraman PS, Goding CR, Gillespie DA. 1993. Gene-regulatory properties of Myc helix-loop-helix/leucine zipper mutants: Max-dependent DNA binding and transcriptional activation in yeast correlates with transforming capacity. Oncogene, 8 (7), pp. 1849-1855. | Show Abstract

Max is a basic helix-loop-helix/leucine zipper (bHLH/LZ) protein that forms sequence-specific DNA-binding complexes with the c-Myc oncoprotein (Myc). Using Saccharomyces cerevisiae, we have shown that the Max bHLH/LZ domain enables Myc to activate transcription through CACGTG and CACATG sequences in vivo, and that the number and context of such sites determines the level of activation. In addition, we have used yeast to investigate the role of the Myc helix-loop-helix (HLH) and leucine zipper (LZ) motifs in mediating Max-dependent DNA-binding and transcriptional activation in vivo using HLH/LZ mutants generated by site-directed mutagenesis. The results show that, while both motifs are essential for Myc to activate transcription, helix 2 of the HLH together with the contiguous LZ suffice to mediate complex formation with Max, whilst helix 1 is essential for sequence-specific DNA binding of Myc-Max complexes. Furthermore, the ability of Myc HLH/LZ mutants to bind DNA and activate transcription in collaboration with Max correlates closely with their neoplastic transforming activity in higher eukaryotic cells.

Fisher F, Crouch DH, Jayaraman PS, Clark W, Gillespie DA, Goding CR. 1993. Transcription activation by Myc and Max: flanking sequences target activation to a subset of CACGTG motifs in vivo. EMBO J, 12 (13), pp. 5075-5082. | Show Abstract | Read more

The Myc oncoprotein has been implicated in control of cell growth, division and differentiation. Although Myc contains a bHLH-LZ motif, it fails to bind DNA alone but can do so by forming heterodimers with an unrelated bHLH-LZ protein, Max. Max homodimers and Myc-Max heterodimers share the ability to bind CACGTG or CATGTG elements. Current models, based on experimentally induced overexpression of Myc and Max in mammalian cells, propose that Max-Max homodimers repress while Myc-Max heterodimers activate transcription through CACGTG binding sites. The interpretation of the results using mammalian cells is complicated by the presence of numerous unrelated CACGTG binding transcription activators and the existence of two alternative Max dimerization partners, Mad and Mxi-1. Thus, the mechanism whereby overexpression of Max leads to transcriptional repression remains to be established. Using a yeast system we show that Max homodimers have the potential to activate transcription through CACGTG motifs. Activation by Max requires DNA binding and amino acids outside the bHLH-LZ domain but is reduced compared with activation by Myc-Max heterodimers. Moreover, transcriptional activation by Myc-Max heterodimers, but not Max-Max homodimers, is strongly inhibited in vivo by specific sequences flanking the core CACGTG binding motif, presumably reflecting reduced DNA binding affinity. These results suggest a mechanism for directing the Myc-Max complex to a specific subset of CACGTG-containing target genes.

Lowings P, Yavuzer U, Goding CR. 1992. Positive and negative elements regulate a melanocyte-specific promoter. Mol Cell Biol, 12 (8), pp. 3653-3662. | Show Abstract | Read more

Melanocytes are specialized cells residing in the hair follicles, the eye, and the basal layer of the human epidermis whose primary function is the production of the pigment melanin, giving rise to skin, hair, and eye color. Melanogenesis, a process unique to melanocytes that involves the processing of tyrosine by a number of melanocyte-specific enzymes, including tyrosinase and tyrosinase-related protein 1 (TRP-1), occurs only after differentiation from the melanocyte precursor, the melanoblast. In humans, melanogenesis is inducible by UV irradiation, with melanin being transferred from the melanocyte in the epidermis to the surrounding keratinocytes as protection from UV-induced damage. Excessive exposure to UV, however, is the primary cause of malignant melanoma, an increasingly common and highly aggressive disease. As an initial approach to understanding the regulation of melanocyte differentiation and melanocyte-specific transcription, we have isolated the gene encoding TRP-1 and examined the cis- and trans-acting factors required for cell-type-specific expression. We find that the TRP-1 promoter comprises both positive and negative regulatory elements which confer efficient expression in a TRP-1-expressing, pigmented melanoma cell line but not in NIH 3T3 or JEG3 cells and that a minimal promoter extending between -44 and +107 is sufficient for cell-type-specific expression. Assays for DNA-protein interactions coupled with extensive mutagenesis identified three factors, whose binding correlated with the function of two positive and one negative regulatory element. One of these factors, termed M-box-binding factor 1, binds to an 11-bp motif, the M box, which acts as a positive regulatory element both in TRP-1-expressing and -nonexpressing cell lines, despite being entirely conserved between the melanocyte-specific tyrosinase and TRP-1 promoters. The possible mechanisms underlying melanocyte-specific gene expression are discussed.

Fisher F, Goding CR. 1992. Single amino acid substitutions alter helix-loop-helix protein specificity for bases flanking the core CANNTG motif. EMBO J, 11 (11), pp. 4103-4109. | Show Abstract | Read more

While all basic region/helix-loop-helix (bHLH) proteins bind the consensus CANNTG motif, other factors must be involved in determining regulatory specificity. In this report we show that bases outside this core 6 bp are involved in determining the specificity of binding. Thus, binding of the yeast bHLH protein PHO4, but not CPF-1, is inhibited by the presence of a T residue immediately 5' to their common CACGTG recognition sequence. PHO4 binding specificity is altered by mutation at any of three different positions in the basic region, including a single Glu to Asp substitution. The significance of these data for DNA-binding and transcription regulation by the bHLH family of transcription factors is discussed.

Cox PM, Goding CR. 1992. An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II DR alpha promoter and activation by SV40 T-antigen. Nucleic Acids Res, 20 (18), pp. 4881-4887. | Show Abstract | Read more

Constitutive expression of major histocompatibility complex class II (MHC II) antigens normally occurs in B-lymphocytes and antigen presenting cells of the monocyte/macrophage lineage. However, many malignant tumours and transformed cells express these proteins aberrantly. We demonstrate here that the MHC II DR alpha promoter is constitutively active both in the SV40 large T antigen transformed cell line, COS, and in CV1 cells from which they are derived. As an approach to understanding the molecular mechanisms underlying aberrant DR alpha expression we have examined the cis- and trans-acting requirements for DR alpha transcription in these cell types. Electrophoretic mobility shift assays showed that the region immediately 3' to the X-box was bound by a member of the ATF/CREB family of transcription factors. Using deletions and point mutations in the DR alpha promoter we demonstrate that, in contrast to B-cells, the octamer motif and conserved X- and Y-boxes make only a minor contribution to promoter function while single point mutations in the ATF/CREB motif reduced transcription up to 20-fold. In addition, we show that the DR alpha promoter is activated by SV40 large T-antigen and that activation requires an intact ATF/CREB motif. Similar data were obtained using B16 melanoma cells. These results suggest that the ATF/CREB motif may be a target for transcription deregulation in several transformed cell types.

Fisher F, Jayaraman PS, Goding CR. 1991. C-myc and the yeast transcription factor PHO4 share a common CACGTG-binding motif. Oncogene, 6 (7), pp. 1099-1104. | Show Abstract

The basic-helix-loop-helix (b-HLH) motif is common to a number of proteins involved in transcriptional regulation and cell-type determination. The b-HLH motif is also present in the S. cerevisiae transcription factor PHO4 which positively regulates the acid phosphatase gene PHO5. In this report we show that the b-HLH region of PHO4 is sufficient to confer specific DNA-binding to the sequence CACGTG and, by comparison of the basic regions of PHO4 with those of other recently isolated CACGTG-binding proteins, we identify a specific subset of conserved amino acids in the basic region likely to confer DNA-binding specificity. On the basis of these observations we predict successfully the effect of substituting the PHO4 basic region with that from c-myc and show that the chimaeric protein activates transcription from the CACGTG elements present in the PHO5 UAS. From these data it is clear that the myc basic region confers specific binding to the sequence CACGTG.

Cox PM, Goding CR. 1991. Transcription and cancer. Br J Cancer, 63 (5), pp. 651-662. | Show Abstract | Read more

The normal growth, development and function of an organism requires precise and co-ordinated control of gene expression. A major part of this control is exerted by regulating messenger RNA (mRNA) production and involves complex interactions between an array of transcriptionally active proteins and specific regulatory DNA sequences. The combination of such proteins and DNA sequences is specific for given gene or group of genes in a particular cell type and the proteins regulating the same gene may vary between cell types. In addition the expression or activity of these regulatory proteins may be modified depending on the state of differentiation of a cell or in response to an external stimulus. Thus, the differentiation of embryonic cells into diverse tissues is achieved and the mature structure and function of the organism is maintained. This review focusses on the role of perturbations of these transcriptional controls in neoplasia. Deregulation of transcription may result in the failure to express genes responsible for cellular differentiation, or alternatively, in the transcription of genes involved in cell division, through the inappropriate expression or activation of positively acting transcription factors and nuclear oncogenes. Whether the biochemical abnormalities that lead to the disordered growth and differentiation of a malignant tumour affect cell surface receptors, membrane or cytoplasmic signalling proteins or nuclear transcription factors, the end result is the inappropriate expression of some genes and failure to express others. Current research is starting to elucidate which of the elements of this complicated system are important in neoplasia.

Goding CR, O'Hare P. 1989. Herpes simplex virus Vmw65-octamer binding protein interaction: a paradigm for combinatorial control of transcription. Virology, 173 (2), pp. 363-367. | Show Abstract | Read more

The transcriptional status of a given viral or cellular gene is determined both by the availability of functional transcription factors and by the combination and spatial arrangement of the cis-acting elements to which they bind. While differential gene expression can be achieved to some extent by the interaction of different factors with different genes it is clear that in some cases the same factor is required for the regulation of genes that are not coordinately expressed. How a transcription factor achieves selective function in the absence of selective binding is a question that remains largely unanswered. The recent advances in understanding how both protein-protein and protein-DNA interactions are required to mediate the induction of herpes simplex virus immediate early (HSV IE) gene expression have highlighted a novel mechanism for combinatorial control of transcription which has significant implications for the differential control of cellular gene expression.

Cousens DJ, Greaves R, Goding CR, O'Hare P. 1989. The C-terminal 79 amino acids of the herpes simplex virus regulatory protein, Vmw65, efficiently activate transcription in yeast and mammalian cells in chimeric DNA-binding proteins. EMBO J, 8 (8), pp. 2337-2342. | Show Abstract | Read more

Activation of herpes simplex virus immediate early gene expression normally requires the formation of a ternary complex between a virus trans-activator, Vmw65, a cellular octamer-binding protein, TRF and the cis-acting target sequence, the TAATGARAT motif. We report that the C-terminal 79 amino acids of Vmw65, which contain a potential acidic amphipathic helix, can activate transcription in both yeast and mammalian cells in the absence of TRF interaction when fused to the DNA-binding domain of the yeast transcription factor, GAL4. Together with our previous report which showed that the recruitment of TRF to the DNA by Vmw65 is insufficient for transcription activation, these results indicate that the octamer binding protein may not be directly involved in transcriptional induction mediated by Vmw65. The TRF-Vmw65 complex may therefore represent a novel class of transcription activator in which the protein domain responsible for sequence-specific DNA binding, present in TRF, and that necessary for induction of transcription, within Vmw65, are located on separate proteins. These results are discussed with reference to combinatorial transcriptional control and the role of octamer-binding proteins in other systems.

O'Hare P, Goding CR, Haigh A. 1988. Direct combinatorial interaction between a herpes simplex virus regulatory protein and a cellular octamer-binding factor mediates specific induction of virus immediate-early gene expression. EMBO J, 7 (13), pp. 4231-4238. | Show Abstract | Read more

We provide evidence for a novel mechanism of transcriptional regulation in which the immediate-early (IE) transactivating protein of herpes simplex virus, Vmw65, is assembled into a specific DNA-binding complex together with a cellular octamer-binding factor (TRF). The assembly of Vmw65/TRF complex requires not only the core TRF recognition site, but also flanking sequences which are dispensable for TRF binding alone. We show from functional analyses that TRF binding by a motif is required but not sufficient to confer induction on a heterologous promoter, and it is the ability of the motif to allow TRF/Vmw65 complex assembly which correlates with functional activity. Thus, for the induction of HSV IE expression, Vmw65 forms a complex with TRF by recognition of the specific subset of appropriately flanked TRF binding sites present in each of the IE genes. This mechanism may provide a paradigm for the selective utilization of the same transcription factor in differential gene expression.

Cox PM, Temperley SM, Kumar H, Goding CR. 1988. A distinct octamer-binding protein present in malignant melanoma cells. Nucleic Acids Res, 16 (23), pp. 11047-11056. | Show Abstract | Read more

The octamer-binding proteins present in HeLa cells, B-cells and malignant melanoma cells were compared by a gel-electrophoresis DNA-binding assay. Using an extract from the malignant melanoma cells a complex was formed using a variety of octamer containing probes that was distinct from those found using either a HeLa or B-cell extract. DNAase 1 footprints and methylation interference patterns of the melanoma-specific octamer-binding protein were indistinguishable from those obtained with the HeLa factor NF-A1, except for preferential binding of the melanoma-specific factor to DNA methylated at two G residues 16 base-pairs 3' to the octamer motif. Competition analyses using a variety of wild-type and mutant probes showed that mutations affecting binding of NF-A1 similarly affected binding of the melanoma octamer-binding factor. These data also revealed the extreme flexibility of the octamer-binding site, with one probe sharing only 4 bases with the 8 base consensus sequence binding efficiently.

O'Hare P, Goding CR. 1988. Herpes simplex virus regulatory elements and the immunoglobulin octamer domain bind a common factor and are both targets for virion transactivation. Cell, 52 (3), pp. 435-445. | Show Abstract | Read more

Functional upstream activator sequences (TAATGARAT motifs) of herpes simplex virus immediate-early genes were identified and shown both to bind a factor (TRF) present in uninfected HeLa cells and to confer inducibility by the virus regulatory protein, Vmw65, on a normally nonresponsive promoter. Point-mutation analyses demonstrated binding specificity and correlated binding with Vmw65 induction. Furthermore, the octamer domains of the adenovirus DNA replication origin, the histone H2B, and the immunoglobulin light chain genes bound and competed for TRF. The immunoglobulin octamer also conferred Vmw65 inducibility on the TK promoter. In addition, a modified form of TRF was specifically detected in infected cells. We conclude that TRF is similar or identical to the previously described octamer binding protein and is likely to be the target for coordinate induction of immediate-early gene expression by Vmw65.

Schneider JF, Fisher F, Goding CR, Jones NC. 1987. Mutational analysis of the adenovirus E1a gene: the role of transcriptional regulation in transformation. EMBO J, 6 (7), pp. 2053-2060. | Show Abstract | Read more

To determine whether the transcription regulatory activities of the adenoviral E1a gene play a role in its ability to transform primary cells we have constructed an extensive series of mutations within the E1a gene. The mutants have been characterized for their ability to transactivate the adenoviral early promoters, repress the transcriptional stimulation of the polyoma virus enhancer, establish primary baby rat kidney cells in culture and cooperate with the activated Ha-ras oncogene in morphologically transforming these cells. The mutant phenotypes reveal that: (i) the two transcription regulatory activities of E1a are separable since essential protein domains map within different regions of the protein; (ii) transactivation is unlikely to contribute significantly to E1a-mediated transformation since several isolated mutants lost the ability to transactivate but were nevertheless efficient at transformation; and (iii) both establishment and oncogene cooperation are linked to enhancer repression suggesting that E1a transforms cells by the repression of a cellular enhancer.

Goding CR, Temperley SM, Fisher F. 1987. Multiple transcription factors interact with the adenovirus-2 EII-late promoter: evidence for a novel CCAAT recognition factor. Nucleic Acids Res, 15 (19), pp. 7761-7780. | Show Abstract | Read more

Multiple cellular transcription factors have been shown to interact with the upstream region of the adenovirus-2 EIIa-late promoter. One of these factors recognises each of the three CCAAT motifs present in the EIIL promoter at positions -72, -135 and -229, as well as the CCAAT elements in the rat albumin and herpes virus thymidine kinase promoters. A mutation known to reduce thymidine kinase promoter activity in vivo and in vitro abolishes binding of the factor, termed CCAAT recognition factor (CRF), which appears to be distinct from previously identified CCAAT factors. In addition, another protein, termed upstream factor II (USFII), shares binding sites at position -110 in the EIIL promoter and in the c-fos enhancer adjacent to the serum regulatable element. The recognition site for USFII is also found in the c-fos promoter and in the adenovirus early region EIV and EIIa-early promoters. An Sp1 recognition site has also been identified at position -41, and the binding sites for Sp1, USFII and CRF are all required for efficient EIIa-late promoter function. Finally, an additional factor recognising the consensus GGGGGGNT has been detected.

Goding C, Jalinot P, Zajchowski D, Boeuf H, Kédinger C. 1985. Sequence-specific trans-activation of the adenovirus EIIa early promoter by the viral EIV transcription unit. EMBO J, 4 (6), pp. 1523-1528. | Show Abstract | Read more

The contribution of adenovirus early genes, other than that of the well-documented EIa immediate early gene, to the transcriptional regulation of the viral EIIa early transcription unit was examined. HeLa cells were transfected with EIIa-containing plasmids and co-transfected with distinct plasmids bearing one of the viral regions EIa, EIII or EIV. Co-transfection with the EIV-recombinants, but not the EIII constructs, stimulated specific transcription from the major EIIaE start site (EIIaE1) by 5- to 15-fold, as concluded from quantitative S1 nuclease analysis of cytoplasmic RNA and in vitro nuclear 'run-on' transcription assays. The extent of the EIV-induced stimulation was similar to that achieved by EIa under identical conditions. However, in contrast to our observations for EIa-mediated stimulation, where no unique EIIaE1 promoter elements were implicated, maximal induction by EIV requires sequences between positions -48 and -19 (with respect to the EIIaE1 start site).

Leff T, Elkaim R, Goding CR, Jalinot P, Sassone-Corsi P, Perricaudet M, Kédinger C, Chambon P. 1984. Individual products of the adenovirus 12S and 13S EIa mRNAs stimulate viral EIIa and EIII expression at the transcriptional level. Proc Natl Acad Sci U S A, 81 (14), pp. 4381-4385. | Show Abstract | Read more

Recombinant plasmids containing mutant or wild-type adenovirus serotype 2 EIa genes that produce the 12S mRNA alone, the 13S mRNA alone, or both mRNAs were cotransfected into HeLa cells with plasmids containing the viral EIIa or EIII transcription units. The amount of RNA produced from the EIIa and EIII promoters was increased by the products of both the 13S and the 12S RNAs. By measuring the level of specific transcription in nuclei isolated from transfected cells we directly demonstrate that the increased amount of EIIa RNA is due to stimulation of the rate of transcription.

Goding CR, Russell WC. 1983. Adenovirus cores can function as templates in in vitro DNA replication. EMBO J, 2 (3), pp. 339-344. | Show Abstract | Read more

Adenovirus cores prepared by gentle disruption of virus by heating at 56 degrees C in the presence of deoxycholate were able to function as templates in an in vitro DNA replication system, allowing both initiation, indicated by the formation of terminal protein-dCMP complex, and elongation of > 300 nucleotides. Using both cores and DNA-protein complexes as templates, it was also demonstrated that novobiocin, an inhibitor of DNA gyrase, inhibited in vitro DNA replication by preventing formation of the initiation complex.

Elkaim R, Goding C, Kédinger C. 1983. The adenovirus-2 EIIa early gene promoter: sequences required for efficient in vitro and in vivo transcription. Nucleic Acids Res, 11 (20), pp. 7105-7117. | Show Abstract | Read more

A series of deletion mutants extending from -250 toward the capsite has been constructed in the early promoter region of the adenovirus 2 EIIa gene and tested both in vitro, and in vivo after transfection of HeLa cells, for the ability to act as a template for transcription. A region between positions -94 and -63 upstream from the major EIIa early cap site is essential both in vivo and in vitro for efficient promoter function. By cotransfection of the EIIa deletion mutants with the EIa transcription unit it has been possible to demonstrate that deletion to position -94 does not affect induction of transcription of the EIIa early gene by the EIa transcription unit, but deletion to position -63 results in loss of detectable levels of EIIa early specific RNA. Thus, sequences upstream from position -94 of the EIIa early gene are not involved in the induction of the EIIa early gene by the EIa transcription unit.

Goding CR, Russell WC. 1983. S1 sensitive sites in adenovirus DNA. Nucleic Acids Res, 11 (1), pp. 21-36. | Show Abstract | Read more

S1 nuclease has been used as a probe for regions of DNA secondary structure in supercoiled recombinant plasmids containing adenovirus (Ad) DNA sequences. In the sequences examined two S1 sensitive sites were identified in the left-terminal 16.5% of Ad 12 DNA, one of which aligned approximately with an inverted repeat region in the DNA sequence. In addition an S1 sensitive site was dictated by a potential cruciform structure in the region of the Ad 2 major late promoter. In contrast to the expected cleavage site at the loop of the cruciform, cleavage occurred at the base of the stem in the region of the TATA box. All three S1 sensitive sites identified were more sensitive to S1 than the endogenous sites in the parent plasmids.

Goding CR, Shaw CH, Blair GE, Russell WC. 1983. ADP-ribosylation in in vitro systems synthesizing adenovirus DNA. J Gen Virol, 64 (Pt 2) (FEB), pp. 477-483. | Show Abstract | Read more

Two systems utilizing extracts derived from nuclei of adenovirus-infected cells which synthesize adenovirus DNA in vitro were analysed for indications of ADP-ribosylation of virus proteins. On incubation with [32P]NAD or [14C]NAD, modification of the adenovirus T antigen could be demonstrated in one of these systems. ADP-ribosylation of adenovirus core proteins V and VII could also be demonstrated with both nuclear extracts. However, using 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase, there was no evidence either in vivo or in vitro that ADP-ribosylation played a critical role in the replication process.

Li Y, Tinoco R, Elmén L, Segota I, Xian Y, Fujita Y, Sahu A, Zarecki R, Marie K, Feng Y et al. 2019. Gut microbiota dependent anti-tumor immunity restricts melanoma growth in Rnf5-/- mice. Nat Commun, 10 (1), pp. 1492. | Show Abstract | Read more

Accumulating evidence points to an important role for the gut microbiome in anti-tumor immunity. Here, we show that altered intestinal microbiota contributes to anti-tumor immunity, limiting tumor expansion. Mice lacking the ubiquitin ligase RNF5 exhibit attenuated activation of the unfolded protein response (UPR) components, which coincides with increased expression of inflammasome components, recruitment and activation of dendritic cells and reduced expression of antimicrobial peptides in intestinal epithelial cells. Reduced UPR expression is also seen in murine and human melanoma tumor specimens that responded to immune checkpoint therapy. Co-housing of Rnf5-/- and WT mice abolishes the anti-tumor immunity and tumor inhibition phenotype, whereas transfer of 11 bacterial strains, including B. rodentium, enriched in Rnf5-/- mice, establishes anti-tumor immunity and restricts melanoma growth in germ-free WT mice. Altered UPR signaling, exemplified in Rnf5-/- mice, coincides with altered gut microbiota composition and anti-tumor immunity to control melanoma growth.

García-Jiménez C, Goding CR. 2019. Starvation and Pseudo-Starvation as Drivers of Cancer Metastasis through Translation Reprogramming. Cell Metab, 29 (2), pp. 254-267. | Show Abstract | Read more

Considerable progress has been made in identifying microenvironmental signals that effect the reversible phenotypic transitions underpinning the early steps in the metastatic cascade. However, although the general principles underlying metastatic dissemination have been broadly outlined, a common theme that unifies many of the triggers of invasive behavior in tumors has yet to emerge. Here we discuss how many diverse signals that induce invasion converge on the reprogramming of protein translation via phosphorylation of eIF2α, a hallmark of the starvation response. These include starvation as a consequence of nutrient or oxygen limitation, or pseudo-starvation imposed by cell-extrinsic microenvironmental signals or by cell-intrinsic events, including oncogene activation. Since in response to resource limitation single-cell organisms undergo phenotypic transitions remarkably similar to those observed within tumors, we propose that a starvation/pseudo-starvation model to explain cancer progression provides an integrated and evolutionarily conserved conceptual framework to understand the progression of this complex disease.

Ngeow KC, Friedrichsen HJ, Li L, Zeng Z, Andrews S, Volpon L, Brunsdon H, Berridge G, Picaud S, Fischer R et al. 2018. BRAF/MAPK and GSK3 signaling converges to control MITF nuclear export. Proc Natl Acad Sci U S A, 115 (37), pp. E8668-E8677. | Show Abstract | Read more

The close integration of the MAPK, PI3K, and WNT signaling pathways underpins much of development and is deregulated in cancer. In principle, combinatorial posttranslational modification of key lineage-specific transcription factors would be an effective means to integrate critical signaling events. Understanding how this might be achieved is central to deciphering the impact of microenvironmental cues in development and disease. The microphthalmia-associated transcription factor MITF plays a crucial role in the development of melanocytes, the retinal pigment epithelium, osteoclasts, and mast cells and acts as a lineage survival oncogene in melanoma. MITF coordinates survival, differentiation, cell-cycle progression, cell migration, metabolism, and lysosome biogenesis. However, how the activity of this key transcription factor is controlled remains poorly understood. Here, we show that GSK3, downstream from both the PI3K and Wnt pathways, and BRAF/MAPK signaling converges to control MITF nuclear export. Phosphorylation of the melanocyte MITF-M isoform in response to BRAF/MAPK signaling primes for phosphorylation by GSK3, a kinase inhibited by both PI3K and Wnt signaling. Dual phosphorylation, but not monophosphorylation, then promotes MITF nuclear export by activating a previously unrecognized hydrophobic export signal. Nonmelanocyte MITF isoforms exhibit poor regulation by MAPK signaling, but instead their export is controlled by mTOR. We uncover here an unanticipated mode of MITF regulation that integrates the output of key developmental and cancer-associated signaling pathways to gate MITF flux through the import-export cycle. The results have significant implications for our understanding of melanoma progression and stem cell renewal.

Li L, Friedrichsen HJ, Andrews S, Picaud S, Volpon L, Ngeow K, Berridge G, Fischer R, Borden KLB, Filippakopoulos P, Goding CR. 2018. A TFEB nuclear export signal integrates amino acid supply and glucose availability. Nat Commun, 9 (1), pp. 2685. | Show Abstract | Read more

How cells coordinate the response to fluctuating carbon and nitrogen availability required to maintain effective homeostasis is a key issue. Amino acid limitation that inactivates mTORC1 promotes de-phosphorylation and nuclear translocation of Transcription Factor EB (TFEB), a key transcriptional regulator of lysosome biogenesis and autophagy that is deregulated in cancer and neurodegeneration. Beyond its cytoplasmic sequestration, how TFEB phosphorylation regulates its nuclear-cytoplasmic shuttling, and whether TFEB can coordinate amino acid supply with glucose availability is poorly understood. Here we show that TFEB phosphorylation on S142 primes for GSK3β phosphorylation on S138, and that phosphorylation of both sites but not either alone activates a previously unrecognized nuclear export signal (NES). Importantly, GSK3β is inactivated by AKT in response to mTORC2 signaling triggered by glucose limitation. Remarkably therefore, the TFEB NES integrates carbon (glucose) and nitrogen (amino acid) availability by controlling TFEB flux through a nuclear import-export cycle.

Falletta P, Sanchez-Del-Campo L, Chauhan J, Effern M, Kenyon A, Kershaw CJ, Siddaway R, Lisle R, Freter R, Daniels MJ et al. 2017. Translation reprogramming is an evolutionarily conserved driver of phenotypic plasticity and therapeutic resistance in melanoma. Genes Dev, 31 (1), pp. 18-33. | Show Abstract | Read more

The intratumor microenvironment generates phenotypically distinct but interconvertible malignant cell subpopulations that fuel metastatic spread and therapeutic resistance. Whether different microenvironmental cues impose invasive or therapy-resistant phenotypes via a common mechanism is unknown. In melanoma, low expression of the lineage survival oncogene microphthalmia-associated transcription factor (MITF) correlates with invasion, senescence, and drug resistance. However, how MITF is suppressed in vivo and how MITF-low cells in tumors escape senescence are poorly understood. Here we show that microenvironmental cues, including inflammation-mediated resistance to adoptive T-cell immunotherapy, transcriptionally repress MITF via ATF4 in response to inhibition of translation initiation factor eIF2B. ATF4, a key transcription mediator of the integrated stress response, also activates AXL and suppresses senescence to impose the MITF-low/AXL-high drug-resistant phenotype observed in human tumors. However, unexpectedly, without translation reprogramming an ATF4-high/MITF-low state is insufficient to drive invasion. Importantly, translation reprogramming dramatically enhances tumorigenesis and is linked to a previously unexplained gene expression program associated with anti-PD-1 immunotherapy resistance. Since we show that inhibition of eIF2B also drives neural crest migration and yeast invasiveness, our results suggest that translation reprogramming, an evolutionarily conserved starvation response, has been hijacked by microenvironmental stress signals in melanoma to drive phenotypic plasticity and invasion and determine therapeutic outcome.

Sáez-Ayala M, Montenegro MF, Sánchez-Del-Campo L, Fernández-Pérez MP, Chazarra S, Freter R, Middleton M, Piñero-Madrona A, Cabezas-Herrera J, Goding CR, Rodríguez-López JN. 2013. Directed phenotype switching as an effective antimelanoma strategy. Cancer Cell, 24 (1), pp. 105-119. | Show Abstract | Read more

Therapeutic resistance in melanoma and other cancers arises via irreversible genetic, and dynamic phenotypic, heterogeneity. Here, we use directed phenotype switching in melanoma to sensitize melanoma cells to lineage-specific therapy. We show that methotrexate (MTX) induces microphthalmia-associated transcription factor (MITF) expression to inhibit invasiveness and promote differentiation-associated expression of the melanocyte-specific Tyrosinase gene. Consequently, MTX sensitizes melanomas to a tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), that inhibits the essential enzyme DHFR with high affinity. The combination of MTX and TMECG leads to depletion of thymidine pools, double-strand DNA breaks, and highly efficient E2F1-mediated apoptosis in culture and in vivo. Importantly, this drug combination delivers an effective and tissue-restricted antimelanoma therapy in vitro and in vivo irrespective of BRAF, MEK, or p53 status.

Lu M, Breyssens H, Salter V, Zhong S, Hu Y, Baer C, Ratnayaka I, Sullivan A, Brown NR, Endicott J et al. 2013. Restoring p53 function in human melanoma cells by inhibiting MDM2 and cyclin B1/CDK1-phosphorylated nuclear iASPP. Cancer Cell, 23 (5), pp. 618-633. | Show Abstract | Read more

Nearly 90% of human melanomas contain inactivated wild-type p53, the underlying mechanisms for which are not fully understood. Here, we identify that cyclin B1/CDK1-phosphorylates iASPP, which leads to the inhibition of iASPP dimerization, promotion of iASPP monomer nuclear entry, and exposure of its p53 binding sites, leading to increased p53 inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most wild-type p53-expressing melanoma cell lines coexpress high levels of phosphorylated nuclear iASPP, MDM2, and cyclin B1. Inhibition of MDM2 and iASPP phosphorylation with small molecules induced p53-dependent apoptosis and growth suppression. Concurrent p53 reactivation and BRAFV600E inhibition achieved additive suppression in vivo, presenting an alternative for melanoma therapy.

Goodall J, Carreira S, Denat L, Kobi D, Davidson I, Nuciforo P, Sturm RA, Larue L, Goding CR. 2008. Brn-2 represses microphthalmia-associated transcription factor expression and marks a distinct subpopulation of microphthalmia-associated transcription factor-negative melanoma cells. Cancer Res, 68 (19), pp. 7788-7794. | Show Abstract | Read more

The origin of tumor heterogeneity is poorly understood, yet it represents a major barrier to effective therapy. In melanoma and in melanocyte development, the microphthalmia-associated transcription factor (Mitf) controls survival, differentiation, proliferation, and migration/metastasis. The Brn-2 (N-Oct-3, POU3F2) transcription factor also regulates melanoma proliferation and is up-regulated by BRAF and beta-catenin, two key melanoma-associated signaling molecules. Here, we show that Brn-2 also regulates invasiveness and directly represses Mitf expression. Remarkably, in melanoma biopsies, Mitf and Brn-2 each mark a distinct subpopulation of melanoma cells, providing a striking illustration of melanoma tumor heterogeneity with implications for melanoma therapy.

Rodriguez M, Aladowicz E, Lanfrancone L, Goding CR. 2008. Tbx3 represses E-cadherin expression and enhances melanoma invasiveness. Cancer Res, 68 (19), pp. 7872-7881. | Show Abstract | Read more

The T-box transcription factors Tbx2 and Tbx3 are overexpressed in many cancers and in melanoma promote proliferation by actively suppressing senescence. Whether they also contribute to tumor progression via other mechanisms is not known. Here, we identify a novel role for these factors, providing evidence that Tbx3, and potentially Tbx2, directly repress the expression of E-cadherin, a keratinocyte-melanoma adhesion molecule whose loss is required for the acquisition of an invasive phenotype. Overexpression of Tbx2 and Tbx3 in melanoma cells down-regulates endogenous E-cadherin expression, whereas depletion of Tbx3, but not Tbx2, increases E-cadherin mRNA and protein levels and decreases melanoma invasiveness in vitro. Consistent with these observations, in melanoma tissue, Tbx3 and E-cadherin expression are inversely correlated. Depletion of Tbx3 also leads to substantial up-regulation of Tbx2. The results suggest that Tbx2 and Tbx3 may play a dual role during the radial to vertical growth phase transition by both inhibiting senescence via repression of p21(CIP1) expression, and enhancing melanoma invasiveness by decreasing E-cadherin levels.

Delmas V, Beermann F, Martinozzi S, Carreira S, Ackermann J, Kumasaka M, Denat L, Goodall J, Luciani F, Viros A et al. 2007. Beta-catenin induces immortalization of melanocytes by suppressing p16INK4a expression and cooperates with N-Ras in melanoma development. Genes Dev, 21 (22), pp. 2923-2935. | Show Abstract | Read more

Tumor progression is a multistep process in which proproliferation mutations must be accompanied by suppression of senescence. In melanoma, proproliferative signals are provided by activating mutations in NRAS and BRAF, whereas senescence is bypassed by inactivation of the p16(Ink4a) gene. Melanomas also frequently exhibit constitutive activation of the Wnt/beta-catenin pathway that is presumed to induce proliferation, as it does in carcinomas. We show here that, contrary to expectations, stabilized beta-catenin reduces the number of melanoblasts in vivo and immortalizes primary skin melanocytes by silencing the p16(Ink4a) promoter. Significantly, in a novel mouse model for melanoma, stabilized beta-catenin bypasses the requirement for p16(Ink4a) mutations and, together with an activated N-Ras oncogene, leads to melanoma with high penetrance and short latency. The results reveal that synergy between the Wnt and mitogen-activated protein (MAP) kinase pathways may represent an important mechanism underpinning the genesis of melanoma, a highly aggressive and increasingly common disease.

Carreira S, Goodall J, Denat L, Rodriguez M, Nuciforo P, Hoek KS, Testori A, Larue L, Goding CR. 2006. Mitf regulation of Dia1 controls melanoma proliferation and invasiveness. Genes Dev, 20 (24), pp. 3426-3439. | Show Abstract | Read more

It is widely held that cells with metastatic properties such as invasiveness and expression of matrix metalloproteinases arise through the stepwise accumulation of genetic lesions arising from genetic instability and "clonal evolution." By contrast, we show here that in melanomas invasiveness can be regulated epigenetically by the microphthalmia-associated transcription factor, Mitf, via regulation of the DIAPH1 gene encoding the diaphanous-related formin Dia1 that promotes actin polymerization and coordinates the actin cytoskeleton and microtubule networks at the cell periphery. Low Mitf levels lead to down-regulation of Dia1, reorganization of the actin cytoskeleton, and increased ROCK-dependent invasiveness, whereas increased Mitf expression leads to decreased invasiveness. Significantly the regulation of Dia1 by Mitf also controls p27(Kip1)-degradation such that reduced Mitf levels lead to a p27(Kip1)-dependent G1 arrest. Thus Mitf, via regulation of Dia1, can both inhibit invasiveness and promote proliferation. The results imply variations in the repertoire of environmental cues that determine Mitf activity will dictate the differentiation, proliferative, and invasive/migratory potential of melanoma cells through a dynamic epigenetic mechanism.

Vance KW, Carreira S, Brosch G, Goding CR. 2005. Tbx2 is overexpressed and plays an important role in maintaining proliferation and suppression of senescence in melanomas. Cancer Res, 65 (6), pp. 2260-2268. | Show Abstract | Read more

The INK4a and ARF genes found at the CDKN2A locus are key effectors of cellular senescence that is believed to act as a powerful anticancer mechanism. Accordingly, mutations in these genes are present in a wide variety of spontaneous human cancers and CDKN2A germ line mutations are found in familial melanoma. The TBX2 gene encoding a key developmental transcription factor is amplified in pancreatic cancer cell lines and preferentially amplified and overexpressed in BRCA1 and BRCA2 mutated breast tumors. Overexpression of Tbx2 and the related factor Tbx3, which is also overexpressed in breast cancer and melanomas, can suppress senescence in defined experimental systems through repression of ARF expression. However, it is not known how Tbx2 mediates its repressive effect nor whether endogenous Tbx2 or Tbx3 perform a similar antisenescence function in transformed cells. This is a particularly important question because the loss of CDKN2A in many human cancers would, in principle, bypass the requirement for Tbx2/3-mediated repression of ARF in suppressing senescence. We show here that Tbx2 is overexpressed in melanoma cell lines and that Tbx2 targets histone deacetylase 1 to the p21Cip1 (CDKN1A) initiator. Strikingly, expression of an inducible dominant-negative Tbx2 (dnTbx2) leads to displacement of histone deacetylase 1, up-regulation of p21(Cip1) expression, and the induction of replicative senescence in CDKN2A-null B16 melanoma cells. In human melanoma cells, expression of dnTbx2 leads to severely reduced growth and induction of senescence-associated heterochromatin foci. The results suggest that the activity of endogenous Tbx2 is critically required to maintain proliferation and suppress senescence in melanomas.

Carreira S, Goodall J, Aksan I, La Rocca SA, Galibert M-D, Denat L, Larue L, Goding CR. 2005. Mitf cooperates with Rb1 and activates p21Cip1 expression to regulate cell cycle progression. Nature, 433 (7027), pp. 764-769. | Show Abstract | Read more

The controls that enable melanoblasts and melanoma cells to proliferate are likely to be related, but so far no key regulator of cell cycle progression specific to the melanocyte lineage has been identified. The microphthalmia-associated transcription factor Mitf has a crucial but poorly defined role in melanoblast and melanocyte survival and in differentiation. Here we show that Mitf can act as a novel anti-proliferative transcription factor able to induce a G1 cell-cycle arrest that is dependent on Mitf-mediated activation of the p21(Cip1) (CDKN1A) cyclin-dependent kinase inhibitor gene. Moreover, cooperation between Mitf and the retinoblastoma protein Rb1 potentiates the ability of Mitf to activate transcription. The results indicate that Mitf-mediated activation of p21Cip1 expression and consequent hypophosphorylation of Rb1 will contribute to cell cycle exit and activation of the differentiation programme. The mutation of genes associated with melanoma, such as INK4a or BRAF that would affect either Mitf cooperation with Rb1 or Mitf stability respectively, would impair Mitf-mediated cell cycle control.

Developing Treatment Paradigms for Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is one of the leading causes of blindness among the elderly affecting over 30 million individuals world-wide. AMD initiates in the back of the eye because of dysfunctions in the retinal pigment epithelium (RPE), a monolayer of cells that maintains vision through maintenance of photoreceptor healthy and integrity. AMD can lead to severe vision loss and blindness in advanced stages – “dry” and “wet” forms. In the dry stage, the death of RPE cells triggers ...

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Subversion of cellular defence mechanisms in H. pylori-induced stomach cancer

In nearly half of the human population, a Gram-negative bacterium called Helicobacter pylori colonises the gastric epithelium and is associated with an increased risk of developing gastric adenocarcinomas at the sites of infection. The epithelial depolarisation and hyperproliferation induced by H.pylori infection might be relevant events for cancer initiation (Bagnoli, Buti et al. 2005). However, the molecular mechanisms underlying H. Pylori-induced malignant transformation of gastric ...

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Identifying and characterising gastric stem cells and their role in H. pylori-induced stomach cancer

There is increasing evidence that bacterial infections are promoters or co-factors of human cancers. Strong epidemiological associations have been reported especially for  Helicobacter pylori that is considered to be responsible of 70% of the gastric malignancies (Boccellato and Meyer 2015). To figure causal relationships between an infection and cancer onset is difficult, mainly because of the long time span between the two events. Stronger connections to cancers might be delineated by ...

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