Structural implications for K5/K12‐di‐acetylated histone H4 recognition by the second bromodomain of BRD2

Abstract

The BET family proteins recognize acetylated chromatin through their two bromodomains, acting as transcriptional activators or tethering viral genomes to the mitotic chromosomes of their host. The structural mechanism for how the N-terminal bromodomain of human BRD2 (BRD2-BD1) deciphers the mono-acetylated status of histone H4 tail was recently reported. Here we show the crystal structure of the second bromodomain of BRD2 (BRD2-BD2) in complex with the di-acetylated histone H4 tail (H4K5ac/K12ac). To our surprise, a single K5ac/K12ac peptide interacts with two BRD2-BD2 molecules simultaneously: the K5ac residue binds to one BRD2-BD2 molecule while the K12ac residue binds to another. These results provide a structural basis for the recognition of two different patterns of the histone acetylation status by a single bromodomain. Structured summary MINT-7989882, MINT-7989824, MINT-7989846, MINT-7989865: H4 (uniprotkb:P62805) binds (MI:0407) to BRD2 (uniprotkb:P25440) by surface plasmon resonance (MI:0107) MINT-7989539: H4 (uniprotkb:P62805) and BRD2 (uniprotkb:P25440) bind (MI:0407) by X-ray crystallography (MI:0114)

Association of bromodomain BET proteins with chromatin requires dimerization through the conserved motif B

“…While single bromodomains from Brd2 have been shown to attach to histone peptides in vitro (Huang et al, 2007;Nakamura et al, 2007;Umehara et al, 2010a;Umehara et al, 2010b), we have observed chromosome dissociation of a construct encompassing both intact bromodomains in the cell, highlighting the relevance of other structural characteristics in the context of the full-length protein for chromatin association in vivo. In this regard, motif B seems to be specifically required for chromosome association, since deletions of similar extent, N-terminal or Cterminal to motif B, do not affect Brd2 behavior or function.…”

“…However, additional acetylation marks have been reported to be recognized by Brd2, for instance acetyl-K5 and acetyl-K8 on histone H4 (Kanno et al, 2004;Umehara et al, 2010b). It is worth noting that these marks decrease during mitosis (Kruhlak et al, 2001;Sasaki et al, 2009).…”

“…In addition, here we show binding of Brd2 to a histone H3 peptide acetylated at K9/14. Although several reports suggest binding specificities of BET proteins for different acetylation marks, some discrepancies are observed in the literature, probably due to different experimental approaches (Dey et al, 2003;Ito et al, 2011;Kanno et al, 2004;LeRoy et al, 2008;Morinière et al, 2009;Sasaki et al, 2009;Umehara et al, 2010b). Thus, binding specificities have been determined either by in vitro pull-down experiments, SPR binding assays or real-time imaging in living cells, and using overexpressed proteins, either full-length fusion constructs, chimeric proteins or truncated fragments including just the bromodomains (Dey et al, 2003;Huang et al, 2007;Ito et al, 2011;Kanno et al, 2004;Morinière et al, 2009;Nakamura et al, 2007;Sasaki et al, 2009;Umehara et al, 2010a;Umehara et al, 2010b).…”

Bromo- and Extraterminal Domain Chromatin Regulators Serve as Cofactors for Murine Leukemia Virus Integration

“…BET proteins bind to acetylated histone tails via their bromodomains (44,45). The structures of BRD2 bromodomains BD1 and BD2 have been solved in association with H4 acetylated on Lys-5 and -12 (46,47). Recently, small-molecule inhibitors of BET proteins (I-BET and JQ1) have been developed that disrupt the binding interface between the bromodomain and the acetylated lysine groups on chromatin (48)(49)(50)(51).…”

Basic concepts of epigenetics

“…119 The complexity of BRDcontaining proteins role in the regulation of gene expression can be exemplified by the BRDs in human SWI/SNF BRG1 and hBRM ATPases, which have dynamic capacity to bind H3K14ac, H4K8ac, H4K5ac and H4K122ac, as well as DNA, via AT-hook or zinc finger binding motifs. [122][123][124][125] This is also exemplified by the Brg-1 component of remodeling complex BAF, which contains two bromodomains (BD 1 and BD 2 ) with capacity to trigger transcription through association with transcription factor E2F, transcription mediators such as CDK8 and TRAP220, RNAP II, 119 and with ability to dock to acetylated histone tails. 126 Other human BRD proteins, such as Brd4, are also recruited to acetylated H3/H4 tails or phosphorylated H3S10 and, in turn, recruit enzymes that phosphorylate RNAP II and recruit P-TEFb to transcriptional start sites.…”