Thermodynamic analysis of acetylation-dependent Pb1 bromodomain-histone H3 interactions

An acetyl-histone peptide library was used to determine the thermodynamic parameters that define acetylation-dependent bromodomain-histone interactions. Bromodomains interact with histones by binding acetylated lysines. The bromodomain used in this study, BrD3, is derived from the polybromo-1 protein, which is a subunit of the PBAF chromatin remodeling complex. Steady-state fluorescence anisotropy was used to examine the variations in specificity and affinity that drive molecular recognition. Temperature and salt concentration dependence studies demonstrate that the hydrophobic effect is the primary driving force, consistent with lysine acetylation being required for binding. An electrostatic effect was observed in only two complexes where the acetyl-lysine was adjacent to an arginine. The large change in heat capacity determined for the specific complex suggests that the dehydrated BrD3-histone interface forms a tightly bound, high-affinity complex with the target site. These explorations into the thermodynamic driving forces that confer acetylation site-dependent BrD3-histone interactions improve our understanding of how individual bromodomains work in isolation. Furthermore, this work will permit the development of hypotheses regarding how the native Pb1, and the broader class of bromodomain proteins, directs multisubunit chromatin remodeling complexes to specific acetyl-nucleosome sites in vivo.     

Evolutionary Conservation of Structural and Functional Coupling between the BRM AT-Hook and Bromodomain

The BAF chromatin remodeling complex is critical for genome regulation. The central ATPase of BAF is either BRM or BRG1, both of which contain a C-terminal bromodomain, known to associate with acetylated lysines. We have recently demonstrated that in addition to acetyl-lysine binding, the BRG1/BRM bromodomain can associate with DNA through a lysine/arginine rich patch that is adjacent to the acetyl-lysine binding pocket. Flanking the bromodomain is an AT-hook separated by a short, proline-rich linker. We previously found that the AT-hook and bromodomain can associate with DNA in a multivalent manner. Here, we investigate the conservation of this composite module and find that the AT-hook, linker, and lysine/arginine rich bromodomain patch are ancient, conserved over ~1 billion years. We utilize extensive mutagenesis, NMR spectroscopy, and fluorescence anisotropy to dissect the contribution of each of these conserved elements in association of this module with DNA. Our results reveal a structural and functional coupling of the AT-hook and bromodomain mediated by the linker. The lysine/arginine rich patch on the bromodomain and the conserved elements of the AT-hook are critical for robust affinity for DNA, while the conserved elements of the linker are dispensable for overall DNA affinity but critical for maintaining the relative conformation of the AT-hook and bromodomain in binding to DNA. This supports that the coupled action of the AT-hook and bromodomain are important for BAF activity.     

Small-Molecule Inhibition of BRDT for Male Contraception

A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception. PAPERCLIP:     

Polybromo-1-bromodomains bind histone H3 at specific acetyl-lysine positions

The human polybromo-1 protein is thought to localize the Polybromo, BRG1-associated factors chromatin-remodeling complex to kinetochores during mitosis via direct interaction of its six tandem bromodomains with acetylated nucleosomes. Bromodomains are acetyl-lysine binding modules roughly 100 amino acids in length originally found in chromatin associated proteins. Previous studies verified acetyl-histone binding by each bromodomain, but site-specificity, a central tenet of the histone code hypothesis, was not examined. Here, the acetylation site-dependence of bromodomain-histone interactions was examined using steady-state fluorescence anisotropy. Results indicate that single bromodomains bind specific acetyl-lysine sites within the histone tail with sub-micromolar affinity. Identification of duplicate target sites suggests that native Pb1 interacts with both copies of histone H3 upon nucleosome assembly. Quantitative analysis of single bromodomain-histone interactions can be used to develop hypotheses regarding the histone acetylation pattern that acts as the binding target of the native polybromo-1 protein.     

Ligand-induced transrepressive function of VDR requires a chromatin remodeling complex, WINAC

We have previously shown that the novel ATP-dependent chromatin remodeling complex WINAC is required for the ligand-bound Vitamin D receptor (VDR)-mediated transrepression of the 25(OH)D₃ 1-hydroxylase [1(OH)ase] gene. However, the molecular basis for VDR promoter association, which does not involve its binding to specific DNA sequences, remains unclear. To address this issue, we investigated the function of WSTF in terms of the association between WINAC and chromatin for ligand-induced transrepression by VDR. Results of in vitro experiments using chromatin templates showed that the association of unliganded VDR with the promoter required physical interactions between WSTF and both VDR and acetylated histones prior to VDR association with chromatin. The acetylated histone-interacting region of WSTF was mapped to the bromodomain, and a WSTF mutant lacking the bromodomain served as a dominant-negative mutant in terms of ligand-induced transrepression of the 1(OH)ase gene. Thus, our findings indicate that WINAC associates with chromatin through a physical interaction between the WSTF bromodomain and acetylated histones, that appears to be indispensable for VDR/promoter association for ligand-induced transrepression of 1(OH)ase gene expression.