register interest

Dr Annabelle Lewis

Research Area: Cell and Molecular Biology
Technology Exchange: SNP typing, Transcript profiling and Transgenesis
Scientific Themes: Cancer Biology and Genetics & Genomics
Keywords: Cancer, Gene Regulation, Colorectal cancer and Epigenetics

Many common genetic variants in the human genome have been associated with a small increase in the risk of cancer. However the majority of these variants lie in non-coding regions of DNA and their functions are currently unknown. It is likely that they affect gene regulatory elements such as promoters, enhancers and insulators, or epigenetic programmes, rather than altering the protein itself.

My group’s main interest lies in cancer gene regulation, with a particular focus on exploring the role of common genetic variants in cancer predisposition and progression. We are interested in identifying regulatory elements, particularly those that may be involved in colorectal cancer risk. We will investigate how they control the gene in normal and cancerous cells, the effect of SNP variants on their normal function and the role they play in cancer predisposition and progression.

A major focus of the lab is a cancer predisposition variant lying in the promoter of the mismatch repair pathway gene, MLH1. MLH1 is disrupted in about 15% of colorectal cancers, termed MSI+ cancers. These tumours generally show a good prognosis at early stages but have a differential response to chemotherapy. The common variant, rs1800734, near MLH1 is strongly linked to increased DNA methylation and MLH1 gene repression, but the mechanisms involved are poorly understood. Our aims are to investigate and correlate allele specific MLH1 expression, DNA methylation and protein binding in the region in patient samples. We will use cell lines and develop model systems to define the primary cause of repression (eg DNA methylation, transcription factor binding) and the critical effect that rs1800734 has in the pathway of cancer development.The knowledge from these studies will help us to understand the effects of MLH1 disruption on tumours and provide information to help with clinical diagnosis, prognosis and therapy.

We are also interested in SNP variants associated with colorectal cancer risk in the POLD3 locus. POLD3 is a component of the Pol δ polymerase which functions in both replication and repair. However, its role in colorectal cancer is largely unexplored. We are using in vitro and in vivo model systems to investigate the role of POLD3 in cancer pathways. In addition we are working to identify the causative SNP and regulatory element(s) and the mechanisms by which they influence cancer initiation or progression.

Name Department Institution Country
Professor Eric O'Neill Oxford Institute for Radiation Oncology United Kingdom
Professor Simon Leedham Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor Gareth Bond Oxford Ludwig Institute Oxford University, Old Road Campus Research Building United Kingdom
Dr Ben Davies Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Professor Ian Tomlinson Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine United Kingdom
Irshad S, Bansal M, Guarnieri P, Davis H, Zen AAH, Baran B, Pinna CMA, Rahman H, Biswas S, Bardella C et al. 2017. Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer JOURNAL OF PATHOLOGY, 242 (2), pp. 178-192. | Show Abstract | Read more

© 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context-dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ-secretase-independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co-localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal-subtype tumours, where it interacts with Notch to induce an epithelial–mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP–Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal-subtype tumours promotes a synergistic BMP–Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal-subtype CRC insensitive to γ-secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Irshad S, Bansal M, Guarnieri P, Davis H, Al Haj Zen A, Baran B, Pinna CMA, Rahman H, Biswas S, Bardella C et al. 2017. Bone morphogenetic protein and Notch signalling crosstalk in poor-prognosis, mesenchymal-subtype colorectal cancer. J Pathol, 242 (2), pp. 178-192. | Show Abstract | Read more

The functional role of bone morphogenetic protein (BMP) signalling in colorectal cancer (CRC) is poorly defined, with contradictory results in cancer cell line models reflecting the inherent difficulties of assessing a signalling pathway that is context-dependent and subject to genetic constraints. By assessing the transcriptional response of a diploid human colonic epithelial cell line to BMP ligand stimulation, we generated a prognostic BMP signalling signature, which was applied to multiple CRC datasets to investigate BMP heterogeneity across CRC molecular subtypes. We linked BMP and Notch signalling pathway activity and function in human colonic epithelial cells, and normal and neoplastic tissue. BMP induced Notch through a γ-secretase-independent interaction, regulated by the SMAD proteins. In homeostasis, BMP/Notch co-localization was restricted to cells at the top of the intestinal crypt, with more widespread interaction in some human CRC samples. BMP signalling was downregulated in the majority of CRCs, but was conserved specifically in mesenchymal-subtype tumours, where it interacts with Notch to induce an epithelial-mesenchymal transition (EMT) phenotype. In intestinal homeostasis, BMP-Notch pathway crosstalk is restricted to differentiating cells through stringent pathway segregation. Conserved BMP activity and loss of signalling stringency in mesenchymal-subtype tumours promotes a synergistic BMP-Notch interaction, and this correlates with poor patient prognosis. BMP signalling heterogeneity across CRC subtypes and cell lines can account for previous experimental contradictions. Crosstalk between the BMP and Notch pathways will render mesenchymal-subtype CRC insensitive to γ-secretase inhibition unless BMP activation is concomitantly addressed. © 2017 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cheng TH, Thompson DJ, O'Mara TA, Painter JN, Glubb DM, Flach S, Lewis A, French JD, Freeman-Mills L, Church D et al. 2016. Five endometrial cancer risk loci identified through genome-wide association analysis. Nat Genet, 48 (6), pp. 667-674. | Show Abstract | Read more

We conducted a meta-analysis of three endometrial cancer genome-wide association studies (GWAS) and two follow-up phases totaling 7,737 endometrial cancer cases and 37,144 controls of European ancestry. Genome-wide imputation and meta-analysis identified five new risk loci of genome-wide significance at likely regulatory regions on chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). We also found a second independent 8q24.21 signal (rs17232730). Functional studies of the 13q22.1 locus showed that rs9600103 (pairwise r(2) = 0.98 with rs11841589) is located in a region of active chromatin that interacts with the KLF5 promoter region. The rs9600103[T] allele that is protective in endometrial cancer suppressed gene expression in vitro, suggesting that regulation of the expression of KLF5, a gene linked to uterine development, is implicated in tumorigenesis. These findings provide enhanced insight into the genetic and biological basis of endometrial cancer.

Davis H, Irshad S, Bansal M, Rafferty H, Boitsova T, Bardella C, Jaeger E, Lewis A, Freeman-Mills L, Giner FC et al. 2015. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche. Nat Med, 21 (1), pp. 62-70. | Show Abstract | Read more

Hereditary mixed polyposis syndrome (HMPS) is characterized by the development of mixed-morphology colorectal tumors and is caused by a 40-kb genetic duplication that results in aberrant epithelial expression of the gene encoding mesenchymal bone morphogenetic protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell fate that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem cell properties in Lgr5-negative progenitor cells that have exited the stem cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem cell is not the sole cell of origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic premalignant lesions with a hitherto unknown pathogenesis, and these lesions can be considered the sporadic equivalents of HMPS polyps.

Lewis A, Freeman-Mills L, de la Calle-Mustienes E, Giráldez-Pérez RM, Davis H, Jaeger E, Becker M, Hubner NC, Nguyen LN, Zeron-Medina J et al. 2014. A polymorphic enhancer near GREM1 influences bowel cancer risk through differential CDX2 and TCF7L2 binding. Cell Rep, 8 (4), pp. 983-990. | Show Abstract | Read more

A rare germline duplication upstream of the bone morphogenetic protein antagonist GREM1 causes a Mendelian-dominant predisposition to colorectal cancer (CRC). The underlying disease mechanism is strong, ectopic GREM1 overexpression in the intestinal epithelium. Here, we confirm that a common GREM1 polymorphism, rs16969681, is also associated with CRC susceptibility, conferring ∼20% differential risk in the general population. We hypothesized the underlying cause to be moderate differences in GREM1 expression. We showed that rs16969681 lies in a region of active chromatin with allele- and tissue-specific enhancer activity. The CRC high-risk allele was associated with stronger gene expression, and higher Grem1 mRNA levels increased the intestinal tumor burden in Apc(Min) mice. The intestine-specific transcription factor CDX2 and Wnt effector TCF7L2 bound near rs16969681, with significantly higher affinity for the risk allele, and CDX2 overexpression in CDX2/GREM1-negative cells caused re-expression of GREM1. rs16969681 influences CRC risk through effects on Wnt-driven GREM1 expression in colorectal tumors.

Davis H, Lewis A, Behrens A, Tomlinson I. 2014. Investigation of the atypical FBXW7 mutation spectrum in human tumours by conditional expression of a heterozygous propellor tip missense allele in the mouse intestines. Gut, 63 (5), pp. 792-799. | Show Abstract | Read more

OBJECTIVE: FBXW7 encodes the substrate recognition component of a ubiquitin ligase that degrades targets such as Notch1, c-Jun, c-Myc and cyclin E. FBXW7 mutations occur in several tumour types, including colorectal cancers. The FBXW7 mutation spectrum in cancers is unusual. Some tumours have biallelic loss of function mutations but most have monoallelic missense mutations involving specific arginine residues at β-propellor tips involved in substrate recognition. DESIGN: FBXW7 functional studies have generally used null systems. In order to analyse the most common mutations in human tumours, we created a Fbxw7(fl(R482Q))(/+) mouse and conditionally expressed this mutation in the intestines using Vill-Cre. We compared these mice with heterozygous null (Fbxw7(+/-)) mutants. RESULTS: A few sizeable intestinal adenomas occurred in approximately 30% of R482Q/+ and Fbxw7(+/-) mice at age >300 days. Breeding the R482Q allele onto Apc mutant backgrounds led to accelerated morbidity and increased polyp numbers and size. Within the small bowel, polyp distribution was shifted proximally. Elevated levels of two particular Fbxw7 substrates, Klf5 and Tgif1, were found in normal intestine and adenomas of R482Q/+, R482Q/R482Q and Fbxw7(-/-) mice, but not Fbxw7(+/-) animals. On the Apc mutant background, Fbxw7(+/-) mutants had a phenotype intermediate between Fbxw7 wild-type and R482Q/+ mice. CONCLUSIONS: Heterozygous Fbxw7 propellor tip (R482Q) mutations promote intestinal tumorigenesis on an Apc mutant background. Klf5 and Tgif1 are strong candidates for mediating this effect. Although heterozygous null Fbxw7 mutations also promote tumour growth, these have a weaker effect than R482Q. These findings explain the FBXW7 mutation spectrum found in human cancers, and emphasise the need for animal models faithfully to reflect human disease.

Lewis A, Tomlinson I. 2012. Cancer. The utility of mouse models in post-GWAS research. Science, 338 (6112), pp. 1301-1302. | Read more

Jaeger E, Leedham S, Lewis A, Segditsas S, Becker M, Cuadrado PR, Davis H, Kaur K, Heinimann K, Howarth K et al. 2012. Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1. Nat Genet, 44 (6), pp. 699-703. | Show Abstract | Read more

Hereditary mixed polyposis syndrome (HMPS) is characterized by apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma occurring in a high proportion of affected individuals. Here, we use genetic mapping, copy-number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a duplication spanning the 3' end of the SCG5 gene and a region upstream of the GREM1 locus. This unusual mutation is associated with increased allele-specific GREM1 expression. Whereas GREM1 is expressed in intestinal subepithelial myofibroblasts in controls, GREM1 is predominantly expressed in the epithelium of the large bowel in individuals with HMPS. The HMPS duplication contains predicted enhancer elements; some of these interact with the GREM1 promoter and can drive gene expression in vitro. Increased GREM1 expression is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel.

Leedham SJ, Rodenas-Cuadrado P, Howarth K, Lewis A, Mallappa S, Segditsas S, Davis H, Jeffery R, Rodriguez-Justo M, Keshav S et al. 2013. A basal gradient of Wnt and stem-cell number influences regional tumour distribution in human and mouse intestinal tracts. Gut, 62 (1), pp. 83-93. | Show Abstract | Read more

OBJECTIVE: Wnt signalling is critical for normal intestinal development and homeostasis. Wnt dysregulation occurs in almost all human and murine intestinal tumours and an optimal but not excessive level of Wnt activation is considered favourable for tumourigenesis. The authors assessed effects of pan-intestinal Wnt activation on tissue homeostasis, taking into account underlying physiological Wnt activity and stem-cell number in each region of the bowel. DESIGN: The authors generated mice that expressed temporally controlled, stabilised β-catenin along the crypt-villus axis throughout the intestines. Physiological Wnt target gene activity was assessed in different regions of normal mouse and human tissue. Human intestinal tumour mutation spectra were analysed. RESULTS: In the mouse, β-catenin stabilisation resulted in a graduated neoplastic response, ranging from dysplastic transformation of the entire epithelium in the proximal small bowel to slightly enlarged crypts of non-dysplastic morphology in the colorectum. In contrast, stem and proliferating cell numbers were increased in all intestinal regions. In the normal mouse and human intestines, stem-cell and Wnt gradients were non-identical, but higher in the small bowel than large bowel in both species. There was also variation in the expression of some Wnt modulators. Human tumour analysis confirmed that different APC mutation spectra are selected in different regions of the bowel. CONCLUSIONS: There are variable gradients in stem-cell number, physiological Wnt activity and response to pathologically increased Wnt signalling along the crypt-villus axis and throughout the length of the intestinal tract. The authors propose that this variation influences regional mutation spectra, tumour susceptibility and lesion distribution in mice and humans.

Lewis A, Davis H, Deheragoda M, Pollard P, Nye E, Jeffery R, Segditsas S, East P, Poulsom R, Stamp G et al. 2012. The C-terminus of Apc does not influence intestinal adenoma development or progression. J Pathol, 226 (1), pp. 73-83. | Show Abstract | Read more

Adenomatous polyposis coli (APC ) mutations are found in most colorectal tumours. These mutations are almost always protein-truncating, deleting both central domains that regulate Wnt signalling and C-terminal domains that interact with the cytoskeleton. The importance of Wnt dysregulation for colorectal tumourigenesis is well characterized. It is, however, unclear whether loss of C-terminal functions contributes to tumourigenesis, although this protein region has been implicated in cellular processes--including polarity, migration, mitosis, and chromosomal instability (CIN)—that have been postulated as critical for the development and progression of intestinal tumours. Since almost all APC mutations in human patients disrupt both central and C-terminal regions, we created a mouse model to test the role of the C-terminus of APC in intestinal tumourigenesis. This mouse (Apc(ΔSAMP)) carries an internal deletion within Apc that dysregulates Wnt by removing the beta-catenin-binding and SAMP repeats, but leaves the C-terminus intact. We compared Apc(ΔSAMP) mice with Apc(1322T) animals. The latter allele represented the most commonly found human APC mutation and was identical to Apc(ΔSAMP) except for absence of the entire C-terminus. Apc(ΔSAMP) mice developed numerous intestinal adenomas indistinguishable in number, location, and dysplasia from those seen in Apc(1322T) mice. No carcinomas were found in Apc(ΔSAMP) or Apc(1322T) animals. While similar disruption of the Wnt signalling pathway was observed in tumours from both mice, no evidence of differential C-terminus functions (such as cell migration, CIN, or localization of APC and EB1) was seen. We conclude that the C-terminus of APC does not influence intestinal adenoma development or progression.

Tomlinson IPM, Carvajal-Carmona LG, Dobbins SE, Tenesa A, Jones AM, Howarth K, Palles C, Broderick P, Jaeger EEM, Farrington S et al. 2011. Multiple common susceptibility variants near BMP pathway loci GREM1, BMP4, and BMP2 explain part of the missing heritability of colorectal cancer. PLoS Genet, 7 (6), pp. e1002105. | Show Abstract | Read more

Genome-wide association studies (GWAS) have identified 14 tagging single nucleotide polymorphisms (tagSNPs) that are associated with the risk of colorectal cancer (CRC), and several of these tagSNPs are near bone morphogenetic protein (BMP) pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3), BMP4 (14q22.2), and BMP2 (20p12.3) using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10)) and BMP2 (rs4813802, P = 4.65×10(-11)). Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8)) and rs11632715 (P = 2.30×10(-10)). As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases.

Davis H, Lewis A, Spencer-Dene B, Tateossian H, Stamp G, Behrens A, Tomlinson I. 2011. FBXW7 mutations typically found in human cancers are distinct from null alleles and disrupt lung development. J Pathol, 224 (2), pp. 180-189. | Show Abstract | Read more

FBXW7 is the substrate recognition component of a SCF-type E3 ubiquitin ligase. It has multiple targets such as Notch1, c-Jun, and cyclin E that function in critical developmental and signalling pathways. Mutations in FBXW7 are often found in many types of cancer. In most cases, these mutations do not inactivate the protein, but are mono-allelic missense changes at specific arginine resides involved in substrate binding. We have hypothesized that FBXW7 mutations are selected in cancers for reasons other than haploinsufficiency or full loss-of-function. Given that the existing mutant Fbxw7 mice carry null alleles, we created a mouse model carrying one of the commonly occurring point mutations (Fbxw7(R482Q)) in the WD40 substrate recognition domain of Fbxw7. Mice heterozygous for this mutation apparently developed normally in utero, died perinatally due to a defect in lung development, and in some cases showed cleft palate and eyelid fusion defects. By comparison, Fbxw7(+/-) mice were viable and developed normally. Fbxw7(-/-) animals died of vascular abnormalities at E10.5. We screened known FBXW7 targets for changes in the lungs of the Fbxw7(R482Q/+) mice and found Tgif1 and Klf5 to be up-regulated. Fbxw7(R482Q) alleles are not functionally equivalent to heterozygous or homozygous null alleles, and we propose that they are selected in tumourigenesis because they cause a selective or partial loss of FBXW7 function.

Babaei-Jadidi R, Li N, Saadeddin A, Spencer-Dene B, Jandke A, Muhammad B, Ibrahim EE, Muraleedharan R, Abuzinadah M, Davis H et al. 2011. FBXW7 influences murine intestinal homeostasis and cancer, targeting Notch, Jun, and DEK for degradation. J Exp Med, 208 (2), pp. 295-312. | Show Abstract | Read more

The Fbxw7 (F-box/WD repeat-containing protein 7; also called CDC4, Sel10, Ago, and Fbw7) component of the SCF (Skp1/Cullin/F-box protein) E3 ubiquitin ligase complex acts as a tumor suppressor in several tissues and targets multiple transcriptional activators and protooncogenes for ubiquitin-mediated degradation. To understand Fbxw7 function in the murine intestine, in this study, we specifically deleted Fbxw7 in the murine gut using Villin-Cre (Fbxw7(ΔG)). In wild-type mice, loss of Fbxw7 in the gut altered homeostasis of the intestinal epithelium, resulted in elevated Notch and c-Jun expression, and induced development of adenomas at 9-10 mo of age. In the context of APC (adenomatous polyposis coli) deficiency (Apc(Min/+) mice), loss of Fbxw7 accelerated intestinal tumorigenesis and death and promoted accumulation of β-catenin in adenomas at late but not early time points. At early time points, Fbxw7 mutant tumors showed accumulation of the DEK protooncogene. DEK expression promoted cell division and altered splicing of tropomyosin (TPM) RNA, which may also influence cell proliferation. DEK accumulation and altered TPM RNA splicing were also detected in FBXW7 mutant human colorectal tumor tissues. Given their reduced lifespan and increased incidence of intestinal tumors, Apc(Min/+)Fbxw7(ΔG) mice may be used for testing carcinogenicity and drug screening.

Lewis A, Segditsas S, Deheragoda M, Pollard P, Jeffery R, Nye E, Lockstone H, Davis H, Clark S, Stamp G et al. 2010. Severe polyposis in Apc(1322T) mice is associated with submaximal Wnt signalling and increased expression of the stem cell marker Lgr5. Gut, 59 (12), pp. 1680-1686. | Show Abstract | Read more

BACKGROUND AND AIMS: Adenomatous polyposis coli (APC) is a tumour suppressor gene mutated in the germline of patients with familial adenomatous polyposis (FAP) and somatically in most colorectal cancers. APC mutations impair β-catenin degradation, resulting in increased Wnt signalling. The most frequent APC mutation is a codon 1309 truncation that is associated with severe FAP. A previous study compared two mouse models of intestinal tumorigenesis, Apc(R850X) (Min) and Apc(1322T) (1322T), the latter a model of human codon 1309 changes. 1322T mice had more severe polyposis but, surprisingly, these tumours had lower levels of nuclear β-catenin than Min tumours. The consequences of these different β-catenin levels were investigated. METHODS: Enterocytes were isolated from 1322T and Min tumours by microdissection and gene expression profiling was performed. Differentially expressed Wnt targets and other stem cell markers were validated using quantitative PCR, in situ hybridisation and immunohistochemistry. RESULTS: As expected, lower nuclear β-catenin levels in 1322T lesions were associated with generally lower levels of Wnt target expression. However, expression of the Wnt target and stem cell marker Lgr5 was significantly higher in 1322T tumours than in Min tumours. Other stem cell markers (Musashi1, Bmi1 and the Wnt target Cd44) were also at higher levels in 1322T tumours. In addition, expression of the Bmp antagonist Gremlin1 was higher in 1322T tumours, together with lower Bmp2 and Bmp4 expression. CONCLUSIONS: The severe phenotype caused by truncation of Apc at codon 1322 is associated with an increased number of stem cells. Thus, a submaximal level of Wnt signalling favours the stem cell phenotype and this may promote tumorigenesis. A level of Wnt signalling exists that is too high for optimal tumour growth.

Pollard P, Deheragoda M, Segditsas S, Lewis A, Rowan A, Howarth K, Willis L, Nye E, McCart A, Mandir N et al. 2009. The Apc 1322T mouse develops severe polyposis associated with submaximal nuclear beta-catenin expression. Gastroenterology, 136 (7), pp. 2204-2213.e1-13. | Show Abstract | Read more

BACKGROUND & AIMS: We previously demonstrated that the 2 APC mutations in human colorectal tumors are coselected, because tumorigenesis requires an optimal level of Wnt signaling. We and others subsequently showed that the truncated APC proteins in colorectal tumors usually retain a total of 1-2 beta-catenin binding/degradation repeats (20AARs); very few intestinal tumors have proteins with no 20AARs. The coselection of the "2 hits" at APC makes it difficult to undertake further mechanistic studies in this area in humans. In mice, however, second hits appear to vary with the strain or genetic background used. This suggested the possibility of creating suboptimal Apc genotypes in the mouse. METHODS: We have constructed a mouse, Apc(1322T), with a mutant protein retaining one 20AAR. After repeated backcrossing to the C57BL/6J background, we compared the 1322T animals with the widely used Min mouse in which the mutant Apc protein has zero 20AARs. RESULTS: In both mice, intestinal adenomas showed copy-neutral loss of heterozygosity, making them homozygous for the mutant Apc allele. 1322T animals had markedly more severe polyposis, with earlier-onset, larger, more numerous, and more severely dysplastic adenomas. 1322T tumors also had more marked Paneth cell differentiation and higher frequencies of crypt fission. Somewhat surprisingly, nuclear beta-catenin expression was lower in 1322T than Min tumors. CONCLUSIONS: We propose that the Apc(1322T) mutation produces submaximal beta-catenin levels that promote early tumor growth more effectively than the Apc(Min) mutation.

Redrup L, Branco MR, Perdeaux ER, Krueger C, Lewis A, Santos F, Nagano T, Cobb BS, Fraser P, Reik W. 2009. The long noncoding RNA Kcnq1ot1 organises a lineage-specific nuclear domain for epigenetic gene silencing. Development, 136 (4), pp. 525-530. | Show Abstract | Read more

Long noncoding RNAs are implicated in a number of regulatory functions in eukaryotic genomes. The paternally expressed long noncoding RNA (ncRNA) Kcnq1ot1 regulates epigenetic gene silencing in an imprinted gene cluster in cis over a distance of 400 kb in the mouse embryo, whereas the silenced region extends over 780 kb in the placenta. Gene silencing by the Kcnq1ot1 RNA involves repressive histone modifications, including H3K9me2 and H3K27me3, which are partly brought about by the G9a and Ezh2 histone methyltransferases. Here, we show that Kcnq1ot1 is transcribed by RNA polymerase II, is unspliced, is relatively stable and is localised in the nucleus. Analysis of conditional Dicer mutants reveals that the RNAi pathway is not involved in gene silencing in the Kcnq1ot1 cluster. Instead, using RNA/DNA FISH we show that the Kcnq1ot1 RNA establishes a nuclear domain within which the genes that are epigenetically inactivated in cis are frequently found, whereas nearby genes that are not regulated by Kcnq1ot1 are localised outside of the domain. The Kcnq1ot1 RNA domain is larger in the placenta than in the embryo, consistent with more genes in the cluster being silenced in the placenta. Our results show for the first time that autosomal long ncRNAs can establish nuclear domains, which might create a repressive environment for epigenetic silencing of adjacent genes. Long ncRNAs in imprinting clusters and the Xist RNA on the inactive X chromosome thus appear to regulate epigenetic gene silencing by similar mechanisms.

Green K, Lewis A, Dawson C, Dean W, Reinhart B, Chaillet JR, Reik W. 2007. A developmental window of opportunity for imprinted gene silencing mediated by DNA methylation and the Kcnq1ot1 noncoding RNA. Mamm Genome, 18 (1), pp. 32-42. | Show Abstract | Read more

The Kcnq1 imprinted domain encodes a paternally expressed noncoding RNA Kcnq1ot1 and several paternally repressed protein-coding genes. Transcriptional regulation is controlled by the Kcnq1ot1 gene whose maternal germline methylation imprint overlaps with the Kcnq1ot1 promoter. The domain can be divided into two groups of genes. One group is imprinted in all lineages and is reliant on DNA methylation for its imprinting. The other group contains genes that are imprinted specifically in the placenta and retain their imprinting in the absence of Dnmt1, the primary DNA maintenance methylase. In the placenta paternal Kcnq1ot1 expression is associated with the acquisition of repressive histone modifications throughout the domain. Using the Dnmt1o knockout, we have analyzed the effect of removing DNA maintenance methylation at the eight-cell stage on the Kcnq1 imprinted domain. In the placenta the expression of the normally silent maternal Kcnq1ot1 allele leads to reduced expression of the surrounding maternally expressed genes. This repression is seen in both the placental-specific imprinted genes and the ubiquitously imprinted genes. Conversely, reduction of functional Dnmt1 results solely in reduced expression of the ubiquitously imprinted genes in the placenta. This suggests that Kcnq1ot1 expression can epigenetically silence placentally imprinted genes in the cluster only during a specific developmental window. This highlights the possibility that Kcnq1ot1-mediated repression is temporally regulated leading to epigenetic silencing of placental-specific genes. We show that allele-specific histone modifications are still present in the Dnmt1 ( -/- ) trophoblast at placental-specific imprinted loci and are likely responsible for maintaining the imprinting of these genes in the absence of DNA methylation.

Lewis A, Green K, Dawson C, Redrup L, Huynh KD, Lee JT, Hemberger M, Reik W. 2006. Epigenetic dynamics of the Kcnq1 imprinted domain in the early embryo. Development, 133 (21), pp. 4203-4210. | Show Abstract | Read more

The mouse Kcnq1 imprinted domain is located on distal chromosome 7 and contains several imprinted genes that are paternally repressed. Repression of these genes is regulated by a non-coding antisense transcript, Kcnq1ot1, which is paternally expressed. Maternal repression of Kcnq1ot1 is controlled by DNA methylation originating in the oocyte. Some genes in the region are imprinted only in the placenta, whereas others are imprinted in both extra-embryonic and embryonic lineages. Here, we show that Kcnq1ot1 is paternally expressed in preimplantation embryos from the two-cell stage, and that ubiquitously imprinted genes proximal to Kcnq1ot1 are already repressed in blastocysts, ES cells and TS cells. Repressive histone marks such as H3K27me3 are present on the paternal allele of these genes in both ES and TS cells. Placentally imprinted genes that are distal to Kcnq1ot1, by contrast, are not imprinted in blastocysts, ES or TS cells. In these genes, paternal silencing and differential histone marks arise during differentiation of the trophoblast lineage between E4.5 and E7.5. Our findings show that the dynamics during preimplantation development of gene inactivation and acquisition of repressive histone marks in ubiquitously imprinted genes of the Kcnq1 domain are very similar to those of imprinted X inactivation. By contrast, genes that are only imprinted in the placenta, while regulated by the same non-coding RNA transcript Kcnq1ot1, undergo epigenetic inactivation during differentiation of the trophoblast lineage. Our findings establish a model for how epigenetic gene silencing by non-coding RNA may depend on distance from the non-coding RNA and on lineage and differentiation specific factors.

Lewis A, Reik W. 2006. How imprinting centres work. Cytogenet Genome Res, 113 (1-4), pp. 81-89. | Show Abstract | Read more

Imprinted genes tend to be clustered in the genome. Most of these clusters have been found to be under the control of discrete DNA elements called imprinting centres (ICs) which are normally differentially methylated in the germline. ICs can regulate imprinted expression and epigenetic marks at many genes in the region, even those which lie several megabases away. Some of the molecular and cellular mechanisms by which ICs control other genes and regulatory regions in the cluster are becoming clear. One involves the insulation of genes on one side of the IC from enhancers on the other, mediated by the insulator protein CTCF and higher-order chromatin interactions. Another mechanism may involve non-coding RNAs that originate from the IC, targeting histone modifications to the surrounding genes. Given that several imprinting clusters contain CTCF dependent insulators and/or non-coding RNAs, it is likely that one or both of these two mechanisms regulate imprinting at many loci. Both mechanisms involve a variety of epigenetic marks including DNA methylation and histone modifications but the hierarchy of and interactions between these modifications are not yet understood. The challenge now is to establish a chain of developmental events beginning with differential methylation of an IC in the germline and ending with imprinting of many genes, often in a lineage dependent manner.

Reik W, Lewis A. 2005. Co-evolution of X-chromosome inactivation and imprinting in mammals. Nat Rev Genet, 6 (5), pp. 403-410. | Show Abstract | Read more

Recent studies have revealed mechanistic parallels between imprinted X-chromosome inactivation and autosomal imprinting. We suggest that neither mechanism was present in ancestral egg-laying mammals, and that both arose when the evolution of the placenta exerted selective pressure to imprint growth-related genes. We also propose that non-coding RNAs and histone modifications were adopted for the imprinting of growth suppressors on the X chromosome and on autosomes. This provides a unified hypothesis for the evolution of X-chromosome inactivation and imprinting.

Lewis A, Mitsuya K, Umlauf D, Smith P, Dean W, Walter J, Higgins M, Feil R, Reik W. 2004. Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation. Nat Genet, 36 (12), pp. 1291-1295. | Show Abstract | Read more

Imprinted genes are expressed from only one of the parental chromosomes and are marked epigenetically by DNA methylation and histone modifications. The imprinting center 2 (IC2) on mouse distal chromosome 7 is flanked by several paternally repressed genes, with the more distant ones imprinted exclusively in the placenta. We found that most of these genes lack parent-specific DNA methylation, and genetic ablation of methylation does not lead to loss of their imprinting in the trophoblast (placenta). The silent paternal alleles of the genes are marked in the trophoblast by repressive histone modifications (dimethylation at Lys9 of histone H3 and trimethylation at Lys27 of histone H3), which are disrupted when IC2 is deleted, leading to reactivation of the paternal alleles. Thus, repressive histone methylation is recruited by IC2 (potentially through a noncoding antisense RNA) to the paternal chromosome in a region of at least 700 kb and maintains imprinting in this cluster in the placenta, independently of DNA methylation. We propose that an evolutionarily older imprinting mechanism limited to extraembryonic tissues was based on histone modifications, and that this mechanism was subsequently made more stable for use in embryonic lineages by the recruitment of DNA methylation.

Lewis A, Mitsuya K, Constancia M, Reik W. 2004. Tandem repeat hypothesis in imprinting: deletion of a conserved direct repeat element upstream of H19 has no effect on imprinting in the Igf2-H19 region. Mol Cell Biol, 24 (13), pp. 5650-5656. | Show Abstract | Read more

Igf2 and H19 are reciprocally imprinted genes on mouse distal chromosome 7. They share several regulatory elements, including a differentially methylated region (DMR) upstream of H19 that is paternally methylated throughout development. The cis-acting sequence requirements for targeting DNA methylation to the DMR remain unknown; however, it has been suggested that direct tandem repeats near DMRs could be involved. Previous studies of the imprinted Rasgrf1 locus demonstrate indeed that a direct repeat element adjacent to a DMR is responsible for establishing paternal allele-specific methylation at the DMR and therefore allelic expression of the Rasgrf1 transcript. We identified a prominent and conserved direct tandem repeat 1 kb upstream of the H19 DMR and proposed that it played a similar role in imprinted regulation of H19. To test our hypothesis, we generated mice harboring a 1.7-kb targeted deletion of the direct repeat element and analyzed fetal growth, allelic expression, and methylation within the Igf2-H19 region. Surprisingly the deletion had no effect on imprinting. These results together with deletions of other repeats close to imprinted genes suggest that direct repeats may not be important for the targeting of methylation at the majority of imprinted loci and that the Rasgrf1 locus may be an exception to this rule.

Lewis A, Murrell A. 2004. Genomic imprinting: CTCF protects the boundaries. Curr Biol, 14 (7), pp. R284-R286. | Show Abstract | Read more

The DNA-binding protein CTCF, which acts as a chromatin 'insulator', regulates imprinting of the mammalian Igf2 and H19 genes in a methylation-sensitive manner. It has now been shown that CTCF is also required for protection against de novo methylation of the differentially methylated domain of H19 in the female germline.

Reik W, Murrell A, Lewis A, Mitsuya K, Umlauf D, Dean W, Higgins M, Feil R. 2004. Chromosome loops, insulators, and histone methylation: new insights into regulation of imprinting in clusters. Cold Spring Harb Symp Quant Biol, 69 pp. 29-37. | Read more

Lopes S, Lewis A, Hajkova P, Dean W, Oswald J, Forné T, Murrell A, Constância M, Bartolomei M, Walter J, Reik W. 2003. Epigenetic modifications in an imprinting cluster are controlled by a hierarchy of DMRs suggesting long-range chromatin interactions. Hum Mol Genet, 12 (3), pp. 295-305. | Show Abstract | Read more

Imprinted genes and their control elements occur in clusters in the mammalian genome and carry epigenetic modifications. Observations from imprinting disorders suggest that epigenetic modifications throughout the clusters could be under regional control. However, neither the elements that are responsible for regional control, nor its developmental timing, particularly whether it occurs in the germline or postzygotically, are known. Here we examine regional control of DNA methylation in the imprinted Igf2-H19 region in the mouse. Paternal germline specific methylation was reprogrammed after fertilization in two differentially methylated regions (DMRs) in Igf2, and was reestablished after implantation. Using a number of knockout strains in the region, we found that the DMRs themselves are involved in regional coordination in a hierarchical fashion. Thus the H19 DMR was needed on the maternal allele to protect the Igf2 DMRs 1 and 2 from methylation, and Igf2 DMR1 was needed to protect DMR2 from methylation. This regional coordination occurred exclusively after fertilization during somatic development, and did not involve linear spreading of DNA methylation, suggesting a model in which long-range chromatin interactions are involved in regional epigenetic coordination. These observations are likely to be relevant to other gene clusters in which epigenetic regulation plays a role, and in pathological situations in which epigenetic regulation is disrupted.

Cheng TH, Thompson DJ, O'Mara TA, Painter JN, Glubb DM, Flach S, Lewis A, French JD, Freeman-Mills L, Church D et al. 2016. Five endometrial cancer risk loci identified through genome-wide association analysis. Nat Genet, 48 (6), pp. 667-674. | Show Abstract | Read more

We conducted a meta-analysis of three endometrial cancer genome-wide association studies (GWAS) and two follow-up phases totaling 7,737 endometrial cancer cases and 37,144 controls of European ancestry. Genome-wide imputation and meta-analysis identified five new risk loci of genome-wide significance at likely regulatory regions on chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). We also found a second independent 8q24.21 signal (rs17232730). Functional studies of the 13q22.1 locus showed that rs9600103 (pairwise r(2) = 0.98 with rs11841589) is located in a region of active chromatin that interacts with the KLF5 promoter region. The rs9600103[T] allele that is protective in endometrial cancer suppressed gene expression in vitro, suggesting that regulation of the expression of KLF5, a gene linked to uterine development, is implicated in tumorigenesis. These findings provide enhanced insight into the genetic and biological basis of endometrial cancer.

Davis H, Irshad S, Bansal M, Rafferty H, Boitsova T, Bardella C, Jaeger E, Lewis A, Freeman-Mills L, Giner FC et al. 2015. Aberrant epithelial GREM1 expression initiates colonic tumorigenesis from cells outside the stem cell niche. Nat Med, 21 (1), pp. 62-70. | Show Abstract | Read more

Hereditary mixed polyposis syndrome (HMPS) is characterized by the development of mixed-morphology colorectal tumors and is caused by a 40-kb genetic duplication that results in aberrant epithelial expression of the gene encoding mesenchymal bone morphogenetic protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell fate that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem cell properties in Lgr5-negative progenitor cells that have exited the stem cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem cell is not the sole cell of origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic premalignant lesions with a hitherto unknown pathogenesis, and these lesions can be considered the sporadic equivalents of HMPS polyps.

Lewis A, Freeman-Mills L, de la Calle-Mustienes E, Giráldez-Pérez RM, Davis H, Jaeger E, Becker M, Hubner NC, Nguyen LN, Zeron-Medina J et al. 2014. A polymorphic enhancer near GREM1 influences bowel cancer risk through differential CDX2 and TCF7L2 binding. Cell Rep, 8 (4), pp. 983-990. | Show Abstract | Read more

A rare germline duplication upstream of the bone morphogenetic protein antagonist GREM1 causes a Mendelian-dominant predisposition to colorectal cancer (CRC). The underlying disease mechanism is strong, ectopic GREM1 overexpression in the intestinal epithelium. Here, we confirm that a common GREM1 polymorphism, rs16969681, is also associated with CRC susceptibility, conferring ∼20% differential risk in the general population. We hypothesized the underlying cause to be moderate differences in GREM1 expression. We showed that rs16969681 lies in a region of active chromatin with allele- and tissue-specific enhancer activity. The CRC high-risk allele was associated with stronger gene expression, and higher Grem1 mRNA levels increased the intestinal tumor burden in Apc(Min) mice. The intestine-specific transcription factor CDX2 and Wnt effector TCF7L2 bound near rs16969681, with significantly higher affinity for the risk allele, and CDX2 overexpression in CDX2/GREM1-negative cells caused re-expression of GREM1. rs16969681 influences CRC risk through effects on Wnt-driven GREM1 expression in colorectal tumors.

Lewis A, Tomlinson I. 2012. Cancer. The utility of mouse models in post-GWAS research. Science, 338 (6112), pp. 1301-1302. | Read more

Jaeger E, Leedham S, Lewis A, Segditsas S, Becker M, Cuadrado PR, Davis H, Kaur K, Heinimann K, Howarth K et al. 2012. Hereditary mixed polyposis syndrome is caused by a 40-kb upstream duplication that leads to increased and ectopic expression of the BMP antagonist GREM1. Nat Genet, 44 (6), pp. 699-703. | Show Abstract | Read more

Hereditary mixed polyposis syndrome (HMPS) is characterized by apparent autosomal dominant inheritance of multiple types of colorectal polyp, with colorectal carcinoma occurring in a high proportion of affected individuals. Here, we use genetic mapping, copy-number analysis, exclusion of mutations by high-throughput sequencing, gene expression analysis and functional assays to show that HMPS is caused by a duplication spanning the 3' end of the SCG5 gene and a region upstream of the GREM1 locus. This unusual mutation is associated with increased allele-specific GREM1 expression. Whereas GREM1 is expressed in intestinal subepithelial myofibroblasts in controls, GREM1 is predominantly expressed in the epithelium of the large bowel in individuals with HMPS. The HMPS duplication contains predicted enhancer elements; some of these interact with the GREM1 promoter and can drive gene expression in vitro. Increased GREM1 expression is predicted to cause reduced bone morphogenetic protein (BMP) pathway activity, a mechanism that also underlies tumorigenesis in juvenile polyposis of the large bowel.

Lewis A, Davis H, Deheragoda M, Pollard P, Nye E, Jeffery R, Segditsas S, East P, Poulsom R, Stamp G et al. 2012. The C-terminus of Apc does not influence intestinal adenoma development or progression. J Pathol, 226 (1), pp. 73-83. | Show Abstract | Read more

Adenomatous polyposis coli (APC ) mutations are found in most colorectal tumours. These mutations are almost always protein-truncating, deleting both central domains that regulate Wnt signalling and C-terminal domains that interact with the cytoskeleton. The importance of Wnt dysregulation for colorectal tumourigenesis is well characterized. It is, however, unclear whether loss of C-terminal functions contributes to tumourigenesis, although this protein region has been implicated in cellular processes--including polarity, migration, mitosis, and chromosomal instability (CIN)—that have been postulated as critical for the development and progression of intestinal tumours. Since almost all APC mutations in human patients disrupt both central and C-terminal regions, we created a mouse model to test the role of the C-terminus of APC in intestinal tumourigenesis. This mouse (Apc(ΔSAMP)) carries an internal deletion within Apc that dysregulates Wnt by removing the beta-catenin-binding and SAMP repeats, but leaves the C-terminus intact. We compared Apc(ΔSAMP) mice with Apc(1322T) animals. The latter allele represented the most commonly found human APC mutation and was identical to Apc(ΔSAMP) except for absence of the entire C-terminus. Apc(ΔSAMP) mice developed numerous intestinal adenomas indistinguishable in number, location, and dysplasia from those seen in Apc(1322T) mice. No carcinomas were found in Apc(ΔSAMP) or Apc(1322T) animals. While similar disruption of the Wnt signalling pathway was observed in tumours from both mice, no evidence of differential C-terminus functions (such as cell migration, CIN, or localization of APC and EB1) was seen. We conclude that the C-terminus of APC does not influence intestinal adenoma development or progression.

Tomlinson IPM, Carvajal-Carmona LG, Dobbins SE, Tenesa A, Jones AM, Howarth K, Palles C, Broderick P, Jaeger EEM, Farrington S et al. 2011. Multiple common susceptibility variants near BMP pathway loci GREM1, BMP4, and BMP2 explain part of the missing heritability of colorectal cancer. PLoS Genet, 7 (6), pp. e1002105. | Show Abstract | Read more

Genome-wide association studies (GWAS) have identified 14 tagging single nucleotide polymorphisms (tagSNPs) that are associated with the risk of colorectal cancer (CRC), and several of these tagSNPs are near bone morphogenetic protein (BMP) pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3), BMP4 (14q22.2), and BMP2 (20p12.3) using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10)) and BMP2 (rs4813802, P = 4.65×10(-11)). Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8)) and rs11632715 (P = 2.30×10(-10)). As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases.

Lewis A, Segditsas S, Deheragoda M, Pollard P, Jeffery R, Nye E, Lockstone H, Davis H, Clark S, Stamp G et al. 2010. Severe polyposis in Apc(1322T) mice is associated with submaximal Wnt signalling and increased expression of the stem cell marker Lgr5. Gut, 59 (12), pp. 1680-1686. | Show Abstract | Read more

BACKGROUND AND AIMS: Adenomatous polyposis coli (APC) is a tumour suppressor gene mutated in the germline of patients with familial adenomatous polyposis (FAP) and somatically in most colorectal cancers. APC mutations impair β-catenin degradation, resulting in increased Wnt signalling. The most frequent APC mutation is a codon 1309 truncation that is associated with severe FAP. A previous study compared two mouse models of intestinal tumorigenesis, Apc(R850X) (Min) and Apc(1322T) (1322T), the latter a model of human codon 1309 changes. 1322T mice had more severe polyposis but, surprisingly, these tumours had lower levels of nuclear β-catenin than Min tumours. The consequences of these different β-catenin levels were investigated. METHODS: Enterocytes were isolated from 1322T and Min tumours by microdissection and gene expression profiling was performed. Differentially expressed Wnt targets and other stem cell markers were validated using quantitative PCR, in situ hybridisation and immunohistochemistry. RESULTS: As expected, lower nuclear β-catenin levels in 1322T lesions were associated with generally lower levels of Wnt target expression. However, expression of the Wnt target and stem cell marker Lgr5 was significantly higher in 1322T tumours than in Min tumours. Other stem cell markers (Musashi1, Bmi1 and the Wnt target Cd44) were also at higher levels in 1322T tumours. In addition, expression of the Bmp antagonist Gremlin1 was higher in 1322T tumours, together with lower Bmp2 and Bmp4 expression. CONCLUSIONS: The severe phenotype caused by truncation of Apc at codon 1322 is associated with an increased number of stem cells. Thus, a submaximal level of Wnt signalling favours the stem cell phenotype and this may promote tumorigenesis. A level of Wnt signalling exists that is too high for optimal tumour growth.

Reik W, Lewis A. 2005. Co-evolution of X-chromosome inactivation and imprinting in mammals. Nat Rev Genet, 6 (5), pp. 403-410. | Show Abstract | Read more

Recent studies have revealed mechanistic parallels between imprinted X-chromosome inactivation and autosomal imprinting. We suggest that neither mechanism was present in ancestral egg-laying mammals, and that both arose when the evolution of the placenta exerted selective pressure to imprint growth-related genes. We also propose that non-coding RNAs and histone modifications were adopted for the imprinting of growth suppressors on the X chromosome and on autosomes. This provides a unified hypothesis for the evolution of X-chromosome inactivation and imprinting.

Lewis A, Mitsuya K, Umlauf D, Smith P, Dean W, Walter J, Higgins M, Feil R, Reik W. 2004. Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation. Nat Genet, 36 (12), pp. 1291-1295. | Show Abstract | Read more

Imprinted genes are expressed from only one of the parental chromosomes and are marked epigenetically by DNA methylation and histone modifications. The imprinting center 2 (IC2) on mouse distal chromosome 7 is flanked by several paternally repressed genes, with the more distant ones imprinted exclusively in the placenta. We found that most of these genes lack parent-specific DNA methylation, and genetic ablation of methylation does not lead to loss of their imprinting in the trophoblast (placenta). The silent paternal alleles of the genes are marked in the trophoblast by repressive histone modifications (dimethylation at Lys9 of histone H3 and trimethylation at Lys27 of histone H3), which are disrupted when IC2 is deleted, leading to reactivation of the paternal alleles. Thus, repressive histone methylation is recruited by IC2 (potentially through a noncoding antisense RNA) to the paternal chromosome in a region of at least 700 kb and maintains imprinting in this cluster in the placenta, independently of DNA methylation. We propose that an evolutionarily older imprinting mechanism limited to extraembryonic tissues was based on histone modifications, and that this mechanism was subsequently made more stable for use in embryonic lineages by the recruitment of DNA methylation.

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