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.     

Autoregulation of the rsc4 tandem bromodomain by gcn5 acetylation

An important issue for chromatin remodeling complexes is how their bromodomains recognize particular acetylated lysine residues in histones. The Rsc4 subunit of the yeast remodeler RSC contains an essential tandem bromodomain (TBD) that binds acetylated K14 of histone H3 (H3K14ac). We report a series of crystal structures that reveal a compact TBD that binds H3K14ac in the second bromodomain and, remarkably, binds acetylated K25 of Rsc4 itself in the first bromodomain. Endogenous Rsc4 is acetylated only at K25, and Gcn5 is identified as necessary and sufficient for Rsc4 K25 acetylation in vivo and in vitro. Rsc4 K25 acetylation inhibits binding to H3K14ac, and mutation of Rsc4 K25 results in altered growth rates. These data suggest an autoregulatory mechanism in which Gcn5 performs both the activating (H3K14ac) and inhibitory (Rsc4 K25ac) modifications, perhaps to provide temporal regulation. Additional regulatory mechanisms are indicated as H3S10 phosphorylation inhibits Rsc4 binding to H3K14ac peptides.     

Kinetic analysis of acetylation-dependent Pb1 bromodomain-histone interactions

Stopped-flow fluorescence anisotropy was used to determine the kinetic parameters that define acetylation-dependent bromodomain-histone interactions. Bromodomains are acetyllysine binding motifs found in many chromatin associated proteins. Individual bromodomains were derived from the polybromo-1 protein, which is a subunit of the PBAF chromatin-remodeling complex that has six tandem bromodomains in the amino-terminal region. The average k(on) and k(off) values for the formation of high-affinity complexes are 275 M(-1) s(-1) and 0.41 x 10(-3) s(-1), respectively. The average k(on) and k(off) values for the formation of low-affinity complexes are 119 M(-1) s(-1) and 1.42 x 10(-3) s(-1), respectively. Analysis of the on- and off-rates yields acetylation site-dependent equilibrium dissociation constants averaging 1.4 and 12.9 microM for high- and low-affinity complexes, respectively. This work represents the first examination of kinetic mechanisms of acetylation-dependent bromodomain-histone interactions.     

Acetylation increases access of remodelling complexes to their nucleosome targets to enhance initiation of V(D)J recombination

Targeted chromatin remodelling is essential for many nuclear processes, including the regulation of V(D)J recombination. ATP-dependent nucleosome remodelling complexes are important players in this process whose activity must be tightly regulated. We show here that histone acetylation regulates nucleosome remodelling complex activity to boost RAG cutting during the initiation of V(D)J recombination. RAG cutting requires nucleosome mobilization from recombination signal sequences. Histone acetylation does not stimulate nucleosome mobilization per se by CHRAC, ACF or their catalytic subunit, ISWI. Instead, we find the more open structure of acetylated chromatin regulates the ability of nucleosome remodelling complexes to access their nucleosome templates. We also find that bromodomain/acetylated histone tail interactions can contribute to this targeting at limited concentrations of remodelling complex. We therefore propose that the changes in higher order chromatin structure associated with histone acetylation contribute to the correct targeting of nucleosome remodelling complexes and this is a novel way in which histone acetylation can modulate remodelling complex activity.     

ATP依赖的染色质改构复合物及其作用机制

染色质高度紧密的折叠阻止了转录因子和辅因子与DNA的结合, 因而通过染色质重塑以解除这样的抑制环境, 对于转录活动的正常进行是至关重要的。目前认为, 染色质重塑至少是通过两种机制来完成的, 一种是通过ATP依赖的染色质改构复合物, 另一种是通过对组蛋白尾部进行共价修饰的组蛋白修饰酶复合物。文章结合近年来的研究进展, 对前者进行染色质重塑的机制及两者在基因转录调控过程中如何相互协作等进行了论述。     

Tandem bromodomains in the chromatin remodeler RSC recognize acetylated histone H3 Lys14

The coordination of chromatin remodeling with chromatin modification is a central topic in gene regulation. The yeast chromatin remodeling complex RSC bears multiple bromodomains, motifs for acetyl-lysine and histone tail interaction. Here, we identify and characterize Rsc4 and show that it bears tandem essential bromodomains. Conditional rsc4 bromodomain mutations were isolated, and were lethal in combination with gcn5Delta, whereas combinations with esa1 grew well. Replacements involving Lys14 of histone H3 (the main target of Gcn5), but not other H3 or H4 lysine residues, also conferred severe growth defects to rsc4 mutant strains. Importantly, wild-type Rsc4 bound an H3 tail peptide acetylated at Lys14, whereas a bromodomain mutant derivative did not. Loss of particular histone deacetylases suppressed rsc4 bromodomain mutations, suggesting that Rsc4 promotes gene activation. Furthermore, rsc4 mutants displayed defects in the activation of genes involved in nicotinic acid biosynthesis, cell wall integrity, and other pathways. Taken together, Rsc4 bears essential tandem bromodomains that rely on H3 Lys14 acetylation to assist RSC complex for gene activation.     

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.     

Histone H3 lysine 14 acetylation is required for activation of a DNA damage checkpoint in fission yeast

Histone lysine acetylation has emerged as a key regulator of genome organization. However, with a few exceptions, the contribution of each acetylated lysine to cellular functions is not well understood because of the limited specificity of most histone acetyltransferases and histone deacetylases. Here we show that the Mst2 complex in Schizosaccharomyces pombe is a highly specific H3 lysine 14 (H3K14) acetyltransferase that functions together with Gcn5 to regulate global levels of H3K14 acetylation (H3K14ac). By analyzing the effect of H3K14ac loss through both enzymatic inactivation and histone mutations, we found that H3K14ac is critical for DNA damage checkpoint activation by directly regulating the compaction of chromatin and by recruiting chromatin remodeling protein complex RSC.