Structural Role of BET proteins in Transcription Initiation

Project Overview

High resolution crystal structure of a di-acetylated histone 4 peptide (H4K5acK8ac) bound to the ...

Isothermal titration calorimetry (ITC) of the binding of a di-acetylated (H4K5acK8ac) and a ...

High resolution crystal structure of a histone 4 di-acetylated peptide (H4K8acK12ac) bridging two ...

Proteins of the bromo and extra terminal (BET) family are key mediators for the assembly of the positive transcription elongation factor b complex (P-TEFb), an event required for the initiation of transcription elongation (Bres et al., 2008). The P-TEFb core complex is composed of cyclin-dependent kinase-9 (CDK9) and its activator cyclin T, and it functions to activate RNA polymerase II (RNAPol-II). BET proteins (BRD2, BRD3, BRD4 and BRDT in human) contain two conserved N-terminal bromodomains (BRDs), small helical modules that specifically recognize acetylated lysine sites in proteins (Muller et al., 2011), an extra terminal domain (ET) and a more divergent C-terminal recruitment domain (CT motif or CTM). They bind to P-TEFb via their CT motif, tethering the complex to acetylated histone tails via their two N-terminal BRDs, resulting in assembly of the transcriptional machinery. Importantly, BET proteins have been shown to be implicated in cancer, where they can be expressed as malignant oncogenic fusions together with NUT (nuclear protein in testis), a protein of unknown function, resulting in very aggressive and poorly differentiated carcinomas that originate mainly from midline locations such as the head, neck or mediastinum. We have demonstrated that it is possible to target the BET/histone interaction and directly inhibit it using the small molecule acetyl-lysine competitive inhibitor JQ1, resulting in epithelial differentiation, tumour shrinkage and survival in BRD4-NUT xenograft mice (Filippakopoulos et al., 2010).

The spatial requirements for the recruitment of P-TEFb onto chromatin by BET proteins remain to date elusive and it is unclear which components of these proteins are directly implicated. Understanding such interactions will shed light onto their role in transcription providing vital information for the deciphering of disease and the design of therapeutics. This project aims to characterize the specific interactions between fragments or full length of BET proteins with P-TEFb and explore the dynamics of these interactions. Recombinant expression systems for both components of P-TEFb have been established (Baumli et al., 2008) and are available in our laboratory. We have also structurally characterized seven of the eight human BET BRDs and have established recombinant expression systems yielding highly pure material for larger parts of BET proteins. We have characterized the interactions with histone tails in vitro and in vivo and have unravelled a structural mode of interaction whereby two acetyl-lysine epitopes bind to one BRD module of each BET BRD (Filippakopoulos et al., 2012). We have also investigated new classes of small molecule inhibitors which can be used as tool compounds to further study the biological role of BET proteins (Filippakopoulos et al., 2011). Using this rich toolset we aim to explore the association of different fragments of BET proteins to P-TEFb and to structurally characterize these interactions.

Training Opportunities

In this graduate studentship you would express and purify large fragments and full length versions of human BET proteins as well as P-TEFb (CDK9 and cyclin T). You would study the interaction of the P-TEFb complex with BET proteins in solution using biophysical techniques such as analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). 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 protein expression and purification as well as in biophysical methods for the characterization of protein:protein interactions and structure determination. 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 Cancer Biology


Project reference number: 374

Funding and admissions information


Name Department Institution Country Email
Professor Panagis Filippakopoulos Structural Genomics Consortium University of Oxford GBR
Professor Colin R Goding Oxford Ludwig Institute University of Oxford GBR

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

Brès V, Yoh SM, Jones KA. 2008. The multi-tasking P-TEFb complex. Curr. Opin. Cell Biol., 20 (3), pp. 334-40. Read abstract | Read more

P-TEFb (CycT1:Cdk9), the metazoan RNA polymerase II Ser2 C-terminal domain (CTD) kinase, regulates transcription elongation at many genes and integrates mRNA synthesis with histone modification, pre-mRNA processing, and mRNA export. Recruitment of P-TEFb to target genes requires deubiquitination of H2Bub, phosphorylation of H3S10, and the bromodomain protein, Brd4. Brd4 activates growth-related genes in the G1 phase of the cell cycle and can also tether P-TEFb to mitotic chromosomes, possibly to mark sites of active transcription throughout cell division. P-TEFb co-operates with c-Myc during transactivation and cell transformation, and also requires SKIP (c-Ski-interacting protein), an mRNA elongation and splicing factor. Some functions of the P-TEFb/Ser2P CTD are executed by the Spt6 transcription elongation factor, which binds directly to the phosphorylated CTD and recruits the Iws1 ('interacts with Spt6') protein. Iws1, in turn, interacts with the REF1/Aly nuclear export adaptor and stimulates the kinetics of mRNA export. Given the prominent role of Spt6 in regulating chromatin structure, the CTD-bound Spt6:Iws1 complex may also control histone modifications during elongation. Following transcription, P-TEFb accompanies the mature mRNA to the cytoplasm to promote translation elongation. Hide abstract

Muller S, Filippakopoulos P, Knapp S. 2011. Bromodomains as therapeutic targets. Expert Rev Mol Med, 13 pp. e29. Read abstract | Read more

Acetylation of lysine residues is a post-translational modification with broad relevance to cellular signalling and disease biology. Enzymes that 'write' (histone acetyltransferases, HATs) and 'erase' (histone deacetylases, HDACs) acetylation sites are an area of extensive research in current drug development, but very few potent inhibitors that modulate the 'reading process' mediated by acetyl lysines have been described. The principal readers of ɛ-N-acetyl lysine (K(ac)) marks are bromodomains (BRDs), which are a diverse family of evolutionary conserved protein-interaction modules. The conserved BRD fold contains a deep, largely hydrophobic acetyl lysine binding site, which represents an attractive pocket for the development of small, pharmaceutically active molecules. Proteins that contain BRDs have been implicated in the development of a large variety of diseases. Recently, two highly potent and selective inhibitors that target BRDs of the BET (bromodomains and extra-terminal) family provided compelling data supporting targeting of these BRDs in inflammation and in an aggressive type of squamous cell carcinoma. It is likely that BRDs will emerge alongside HATs and HDACs as interesting targets for drug development for the large number of diseases that are caused by aberrant acetylation of lysine residues. Hide abstract

Baumli S, Lolli G, Lowe ED, Troiani S, Rusconi L, Bullock AN, Debreczeni JE, Knapp S, Johnson LN. 2008. The structure of P-TEFb (CDK9/cyclin T1), its complex with flavopiridol and regulation by phosphorylation. EMBO J., 27 (13), pp. 1907-18. Read abstract | Read more

The positive transcription elongation factor b (P-TEFb) (CDK9/cyclin T (CycT)) promotes mRNA transcriptional elongation through phosphorylation of elongation repressors and RNA polymerase II. To understand the regulation of a transcriptional CDK by its cognate cyclin, we have determined the structures of the CDK9/CycT1 and free cyclin T2. There are distinct differences between CDK9/CycT1 and the cell cycle CDK CDK2/CycA manifested by a relative rotation of 26 degrees of CycT1 with respect to the CDK, showing for the first time plasticity in CDK cyclin interactions. The CDK9/CycT1 interface is relatively sparse but retains some core CDK-cyclin interactions. The CycT1 C-terminal helix shows flexibility that may be important for the interaction of this region with HIV TAT and HEXIM. Flavopiridol, an anticancer drug in phase II clinical trials, binds to the ATP site of CDK9 inducing unanticipated structural changes that bury the inhibitor. CDK9 activity and recognition of regulatory proteins are governed by autophosphorylation. We show that CDK9/CycT1 autophosphorylates on Thr186 in the activation segment and three C-terminal phosphorylation sites. Autophosphorylation on all sites occurs in cis. 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

Filippakopoulos P, Picaud S, Fedorov O, Keller M, Wrobel M, Morgenstern O, Bracher F, Knapp S. 2012. Benzodiazepines and benzotriazepines as protein interaction inhibitors targeting bromodomains of the BET family. Bioorg. Med. Chem., 20 (6), pp. 1878-86. Read abstract | Read more

Benzodiazepines are psychoactive drugs with anxiolytic, sedative, skeletal muscle relaxant and amnestic properties. Recently triazolo-benzodiazepines have been also described as potent and highly selective protein interaction inhibitors of bromodomain and extra-terminal (BET) proteins, a family of transcriptional co-regulators that play a key role in cancer cell survival and proliferation, but the requirements for high affinity interaction of this compound class with bromodomains has not been described. Here we provide insight into the structure-activity relationship (SAR) and selectivity of this versatile scaffold. In addition, using high resolution crystal structures we compared the binding mode of a series of benzodiazepine (BzD) and related triazolo-benzotriazepines (BzT) derivatives including clinically approved drugs such as alprazolam and midazolam. Our analysis revealed the importance of the 1-methyl triazolo ring system for BET binding and suggests modifications for the development of further high affinity bromodomain inhibitors. Hide abstract