Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family

Abstract

SummaryBromodomains (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.

The bromodomain interaction module

“…The solution structure of PCAF established in the late 90s the ability of BRDs to bind acetylayted lysine and supported the hypothesis that BRD-containing proteins contribute to highly specific histone acetylation by targeting HATs to specific chromosomal sites [24]. The BRD of PCAF has been shown to interact in vitro with acetylated histone H3 sites (K9 ac [41], K14 ac [33,41], K36 ac [41]) as well as histone H4 sites (K8 ac [24], K16 ac [33,41] and K20 ac [33,41]). It has also been shown to bind to the HIV-1 transcriptional trans-activator Tat (K50 ac [41]), an interaction required to stimulate transcription of the integrated HIV-1 genome.…”

“…A dissociation constant (K D ) of 360 lM for the di-acetylated substrate peptide H4K5 ac /K12 ac has been determined in vitro by SPR [36] and a K D for the mono-acetylated H4K12 ac peptide of 2.9 mM has been determined by NMR titrations [38]. Furthermore, closely spaced multiple acetylation sites have been shown to increase affinity of the H4 tail for the murine BET family member BRDT [39] and this observation has been studied in detail for the entire subfamily of BET proteins [33] suggesting a cooperative role of neighbouring sites for substrate binding. Although there have been reports linking BRDs to acetylation sites in proteins other than histones, current research has been focussed on histone proteins given the significance of BRDs in chromatin biology [34].…”

“…FALZ was shown to interact with histone H4 (K5 ac [33]) as well as a poly-arginine 11-mer peptide that carried a central acetylated lysine epitope, demonstrating how affinity for this class of modules is mainly driven by electrostatic attractions of the rim-regions that flank the central K ac docking site. CECR2 also has affinity for histone H3 peptides (K9 ac , K14 ac , [33]) and binds to single marks even on diacetylated peptides (K9 ac /K14 ac [33]). Interestingly binding to histone H3 K9 ac /K14 ac was found to be attenuated by phosphoryla-tion at serine 10 (pS10), a modification required for mitotic progression suggesting that this PTM can influence binding to the surrounding acetylation marks.…”

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states

“…The BET family shares a C-terminal extra-terminal domain and two tandem bromodomains, BD1 and BD2, that primarily bind to multi-acetylated histone H4 tail at K5, K8, K12, and K16 in vitro , but not to the monoacetylated histone H3/H4 peptides, including that of acetylated H3 K27 (H3K27ac) [9,10,12,13]. Since acetylation of histone H4 in the nucleus is proposed to occur at K16 first, then at K12, K8, and K5 [14], the simultaneous acetylation of K5 and K8 indicates a typical state of histone H4 hyperacetylation [14].…”

“…Since acetylation of histone H4 in the nucleus is proposed to occur at K16 first, then at K12, K8, and K5 [14], the simultaneous acetylation of K5 and K8 indicates a typical state of histone H4 hyperacetylation [14]. Indeed, BET proteins preferentially bind to the K5/K8-diacetylated H4 tail peptides mimicking hyperacetylated H4 in vitro [12,13]. Additionally, H4 K5 acetylation (H4K5ac) facilitated by histone acetyltransferase p300 (EP300) and disruptor of telomere silencing 1-like (DOT1L) might facilitate the binding of BRD4 to the chromatin [15].…”