Modulation of transcription via assembly of negative regulators

Project Overview

Transcriptional programs are often deregulated in disease, offering opportunities for therapeutic intervention. One of the most promising over recent years is through targeting epigenetic readers of the bromo and extra-terminal (BET) family (BRD2, BRD3, BRD4 and BRDT in human) which share common, modular domain architecture, including two N-terminal bromodomain (BRD) modules and an extra-terminal (ET) interaction domain (Filippakopoulos et al., 2012; Filippakopoulos et al., 2010; Wang and Filippakopoulos, 2015). This novel mechanism of targeting readers of acetylation resulted in intense clinical activity since 2010, with several active trials seeking to attenuate BET function in hematopoietic and solid tumours. However, despite the promising initial clinical reports, the ubiquitous expression of BETs, the existence of isoforms, their broad and central involvement in transcription as well as their overlapping functional roles, have raised concerns of toxicity upon targeting them, while indications of resistance have called for better mechanistic understanding of BET-controlled processes (Filippakopoulos and Knapp, 2014; Fujisawa and Filippakopoulos, 2017; Filippakopoulos, 2018).

BETs bind to acetylated proteins (including histones) via their BRD modules, facilitating the aggregation of transcriptional regulators to chromatin, leading to locus-specific transcriptional stimulation or repression. For example, BRD4 stimulates transcriptional elongation by tethering the positive transcription elongation factor b (P-TEFb) complex to chromatin. There is however evidence that BETs can stimulate transcription in a P-TEFb independent manner, via their ET domain which functions as a protein:protein interaction module, however the precise mode and biological implication of these interactions remain elusive. We have established a proteomic network of linkages initiated by BETs, identifying partners associated with negative regulation of transcription (Lambert et al., 2018). We seek to explore the role(s) of these interesting linkages in suppressing transcriptional programmes, employing cell, structural and chemical biology approaches, combined with high throughput genomic technologies. Understanding the molecular and structural basis of these interactions will help demystify the biological role(s) of these important complex assemblies, offering opportunities for alternative BET-targeting in cancer.

Training Opportunities

In this graduate studentship you would employ cell biology techniques (cell culture, western blotting, real time quantitative PCR, iRNA, CRISPR) to characterize interactions initiated by BET proteins with transcriptional attenuators previously identified in our lab. You would employ high throughput sequencing approaches (ChIP-sequencing, chromatin accessibility and conformation capture) to interrogate where, when and how BET proteins aggregate transcriptional complexes. You would express and purify fragments and full length versions of human BET proteins as well as their identified partners. You would employ biophysical techniques such as analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) to study these protein:protein interactions in solution. You would generate crystals of the purified complexes and solve their structures by X-ray crystallography.  This studentship would provide an excellent opportunity to develop skills in cell biology, recombinant protein biochemistry, biophysics and bioinformatics. The outcome of this project would contribute to a major area in biomedical sciences as BET proteins are implicated in cancer as parts of oncogenic fusions and structural insights into their involvement in transcriptional elongation will be of major impact for the development of specific inhibitors.


Protein Science & Structural Biology and Genetics & Genomics


Project reference number: 438

Funding and admissions information


Name Department Institution Country Email
Professor Panagis Filippakopoulos Structural Genomics Consortium Oxford University, Old Road Campus Research Building GBR
Professor Skirmantas Kriaucionis Oxford Ludwig Institute Oxford University, Old Road Campus Research Building GBR
Mr Steve Taylor Weatherall Institute of Molecular Medicine Oxford University, Weatherall Institute of Molecular Medicine GBR

Filippakopoulos P, Knapp S. 2012. The bromodomain interaction module. FEBS Lett., 586 (17), pp. 2692-704. Read abstract | Read more

ε-N-acetylation of lysine residues (K(ac)) is one of the most abundant post-translation modifications (PTMs) in the human proteome. In the nucleus, acetylation of histones has been linked to transcriptional activation of genes but the functional consequences of most acetylation events and proteins recruited to these sites remains largely unknown. Bromodomains (BRDs) are small helical interaction modules that specifically recognize acetylation sites in proteins. BRDs have recently emerged as interesting targets for the development of specific protein interaction inhibitors, enabling a novel exiting strategy for the development of new therapies. This review provides an overview over sequence requirements of BRDs, known substrates and the structural mechanisms of specific K(ac) recognition. Hide abstract

Filippakopoulos P, Picaud S, Mangos M, Keates T, Lambert JP, Barsyte-Lovejoy D, Felletar I, Volkmer R, Müller S, Pawson T, Gingras AC, Arrowsmith CH, Knapp S. 2012. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell, 149 (1), pp. 214-31. Read abstract | Read more

Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family. Hide abstract

Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, Morse EM, Keates T, Hickman TT, Felletar I, Philpott M, Munro S, McKeown MR, Wang Y, Christie AL, West N, Cameron MJ, Schwartz B, Heightman TD, La Thangue N, French CA, Wiest O, Kung AL, Knapp S, Bradner JE. 2010. Selective inhibition of BET bromodomains. Nature, 468 (7327), pp. 1067-73. Read abstract | Read more

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family. Hide abstract

Fujisawa T, Filippakopoulos P. 2017. Functions of bromodomain-containing proteins and their roles in homeostasis and cancer. Nat. Rev. Mol. Cell Biol., 18 (4), pp. 246-262. Read abstract | Read more

Bromodomains (BRDs) are evolutionarily conserved protein-protein interaction modules that are found in a wide range of proteins with diverse catalytic and scaffolding functions and are present in most tissues. BRDs selectively recognize and bind to acetylated Lys residues - particularly in histones - and thereby have important roles in the regulation of gene expression. BRD-containing proteins are frequently dysregulated in cancer, they participate in gene fusions that generate diverse, frequently oncogenic proteins, and many cancer-causing mutations have been mapped to the BRDs themselves. Importantly, BRDs can be targeted by small-molecule inhibitors, which has stimulated many translational research projects that seek to attenuate the aberrant functions of BRD-containing proteins in disease. Hide abstract

Wang CY, Filippakopoulos P. 2015. Beating the odds: BETs in disease. Trends Biochem. Sci., 40 (8), pp. 468-79. Read abstract | Read more

Bromodomains (BRDs) are evolutionarily conserved protein interaction modules that specifically recognise acetyl-lysine on histones and other proteins, facilitating roles in regulating gene transcription. BRD-containing proteins bound to chromatin loci such as enhancers are often deregulated in disease leading to aberrant expression of proinflammatory cytokines and growth-promoting genes. Recent developments targeting the bromo and extraterminal (BET) subset of BRD proteins demonstrated remarkable efficacy in murine models providing a compelling rationale for drug development and translation to the clinic. Here we summarise recent advances in our understanding of the roles of BETs in regulating gene transcription in normal and diseased tissue as well as the current status of their clinical translation. Hide abstract