Metabolically Derived Lysine Acylations and Neighboring Modifications Tune the Binding of the BET Bromodomains to Histone H4

Abstract

Recent proteomic studies discovered histone lysines are modified by acylations beyond acetylation. These acylations derive from acyl-CoA metabolites, potentially linking metabolism to transcription. Bromodomains bind lysine acylation on histones and other nuclear proteins to influence transcription. However, the extent bromodomains bind non-acetyl acylations is largely unknown. Also unclear are the effects of neighboring post-translational modifications, especially within heavily modified histone tails. Using peptide arrays, binding assays, sucrose gradients, and computational methods, we quantified ten distinct acylations for binding to the bromodomain and extraterminal domain (BET) family. Four of these acylations – hydroxyisobutyrylation, malonylation, glutarylation, and homocitrullination – had never been tested for bromodomain binding. We found N-terminal BET bromodomains bound acetylated and propionylated peptides consistent with previous studies. Interestingly, all other acylations inhibited BET bromodomain binding to peptides and nucleosomes. To understand how context tunes bromodomain binding, effects of neighboring methylation, phosphorylation, and acylation within histone H4 tails were determined. Serine-1 phosphorylation inhibited BRD4 N-terminal bromodomain binding to polyacetylated H4 tails by >5-fold whereas methylation had no effect. Furthermore, BRDT and BRD4 N-terminal bromodomain binding to H4K5acetyl was enhanced 1.4- to 9.5-fold by any neighboring acylation of lysine-8, indicating a secondary H4K8acyl binding site that is more permissive of non-acetyl acylations than previously appreciated. In contrast, C-terminal BET bromodomains exhibited 9.9- to 13.5-fold weaker binding for polyacylated compared to monoacylated H4 tails indicating the C-terminal bromodomains do not cooperatively bind multiple acylations. These results suggest acyl-CoA levels tune or block BET bromodomain recruitment to histones, linking metabolism to bromodomain-mediated transcription.

Chemical Epigenetics: The Impact of Chemical and Chemical Biology Techniques on Bromodomain Target Validation

“…[35b] Interestingly,int he absence of K50 acetylation, PCAF binds to residues 20-40 within HIV-1T at. Acetylation of K28 in the model peptide HIV-1 Tat [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] K28Ac was reported to abrogate the interaction with PCAF. [36] Histones or histone-mimicking peptides with multiple KAc sites tend to show higher affinity for bromodomains in the BET family,c ompared to monoacetylated histones or peptides.…”

“…Scientists at GSK used aFBDD approach to develop the first potent and selective ATAD2-bromodomain ligands. [91a-c] While fragment hits for ATAD2 had previously been identified but not elaborated into ligands, [102] optimization of aquinolinone fragment hit (38)produced aseries of highaffinity ligands,exemplified by compounds 46-48 ( Figure 10). Compound 48 has an IC 50 value of 50 nm for ATAD2 and 150fold selectivity over other bromodomains,including the BET family (approximately 500-fold), facilitating selective studies of the function of the ATAD2 bromodomain ( Table 2).…”

Nucleosome scaffolding by Brd4 tandem bromodomains in acetylation-dependent chromatin compartmentalization

“…To measure the inter-bromodomain bindings site distance distribution experimentally, electron paramagnetic resonance (EPR) probe was designed in which a nitroxide spin label (TEMPO) was attached to an inhibitor (JQ1) (20) that binds all BET acetyl-lysine binding sites with nanomolar affinity (‘JQ1-TEMPO’; Figure S4A). Although a BET bromodomain-targeted spin label had not been previously reported, we and others have shown that the location on JQ1 where TEMPO was attached tolerates modification in a manner that does not affect bromodomain binding (33, 34). JQ1-TEMPO binding to the Brd4 tandem bromodomains was shown by decreased amplitude (increased width) of the resonance lines in the continuous wave (cw) EPR spectrum (Figure S4B).…”

“…For instance, the N -terminal bromodomains (BD1) of Brd4 and BrdT cooperatively bind two adjacent acetyl-lysine residues ( i.e. lysines 5 and 8) on the histone H4 tail (13, 32) with 3-to 20-fold tighter affinity compared to either acetylation in isolation (33, 34) suggesting that multiple neighboring acetylation sites are necessary to recruit these bromodomains to chromatin.…”

“…The BET family member Brd4 is especially critical for initiation and elongation by RNA Polymerase II (RNAP II) (46) and enhancer-mediated transcription (47, 48), particularly in the contexts of inflammation (49, 50) and Myc-driven cancer (51, 52). While the specificities of individual recombinant BET bromodomains toward histone lysine acetylation have been explored (13, 32–34), it was unknown if the tandem BET bromodomains are capable of simulataneously binding and scaffolding two acetylated nucleosomes, which would have broad implications on how 3D chromatin organization is established in an acetylation-dependent manner. Here, we directly test different possible mechanisms of multivalent BET tandem bromodomain engagement of nucleosomes using biochemical, structural, biophysical, and bioinformatic techniques.…”