The Swi2/Snf2 bromodomain is required for the displacement of SAGA and the octamer transfer of SAGA-acetylated nucleosomes

The SWI/SNF and SAGA chromatin-modifying complexes contain bromodomains that help anchor these complexes to acetylated promoter nucleosomes. To study the importance of bromodomains in these complexes, we have compared the chromatin-remodeling and octamer-transfer activity of the SWI/SNF complex to a mutant complex that lacks the Swi2/Snf2 bromodomain. Here we show that the SWI/SNF complex can remodel or transfer SAGA-acetylated nucleosomes more efficiently than the Swi2/Snf2 bromodomain-deleted complex. These results demonstrate that the Swi2/Snf2 bromodomain is important for the remodeling as well as for the octamer-transfer activity of the complex on H3-acetylated nucleosomes. Moreover, we show that, although the wild-type SWI/SNF complex displaces SAGA that is bound to acetylated nucleosomes, the bromodomain mutant SWI/SNF complex is less efficient in SAGA displacement. Thus, the Swi2/Snf2 bromodomain is required for the full functional activity of SWI/SNF on acetylated nucleosomes and is important for the displacement of SAGA from acetylated promoter nucleosomes.     

A cooperative activation loop among SWI/SNF, γ‐H2AX and H3 acetylation for DNA double‐strand break repair

Although recent studies highlight the importance of histone modifications and ATP‐dependent chromatin remodelling in DNA double‐strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser‐139 (S139ph) after DNA damage, binds to γ‐H2AX (the phosphorylated form of H2AX)‐containing nucleosomes in S139ph‐dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to γ‐H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with γ‐H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to γ‐H2AX nucleosomes. S139ph stimulates the H3 acetylation on γ‐H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on γ‐H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, γ‐H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.     

Solution structure of human Brg1 bromodomain and its specific binding to acetylated histone tails

Human brahma-related gene 1 (Brg1) is a core protein in human SWI/SNF chromatin-remodeling complex which regulates gene expression. Brg1 contains a bromodomain that has been shown to anchor the entire complex to promoter nucleosomes by interacting with histones that are acetylated at specific lysine residues. The Brg1 bromodomain belongs to an important subclass of the bromodomain family for which no structural information is known. Here we report the solution structure of the Brg1 bromodomain determined by NMR. The Brg1 bromodomain conserves the left-handed, four-helix bundle topology found in other bromodomain structures. However, the alphaZ helix of Brg1 bromodomain is about 4 residues shorter relative to previously published bromodomain structures. Using NMR perturbation studies, we demonstrate the Brg1 bromodomain binds acetyllysine in the context of histone tails, with no comparable affinity for unacetylated peptides. The estimated dissociation constants (KD) for acetylated histone peptides H4-AcK8 and H4-AcK12 are 4.0 and 3.6 mM, respectively. In this study the dominant substrate was H3-AcK14 (KD approximately 1.2 mM). Mutagenesis analysis reveals several residues important for the binding specificity. Using molecular dynamics simulations, we present a model of the Brg1 bromodomain in complex with H3-AcK14 and discuss the potential interactions which provide the selectivity of the Brg1 bromodomain for histone H3-AcK14.     

Acetylation-dependent chromatin reorganization by BRDT,a testis-specific bromodomain-containing protein

The association between histone acetylation and replacement observed during spermatogenesis prompted us to consider the testis as a source for potential factors capable of remodelling acetylated chromatin. A systematic search of data banks for open reading frames encoding testis-specific bromodomain-containing proteins focused our attention on BRDT, a testis-specific protein of unknown function containing two bromodomains. BRDT specifically binds hyperacetylated histone H4 tail depending on the integrity of both bromodomains. Moreover, in somatic cells, the ectopic expression of BRDT triggered a dramatic reorganization of the chromatin only after induction of histone hyperacetylation by trichostatin A (TSA). We then defined critical domains of BRDT involved in its activity. Both bromodomains of BRDT, as well as flanking regions, were found indispensable for its histone acetylation-dependent remodelling activity. Interestingly, we also observed that recombinant BRDT was capable of inducing reorganization of the chromatin of isolated nuclei in vitro only when the nuclei were from TSA-treated cells. This assay also allowed us to show that the action of BRDT was ATP independent, suggesting a structural role for the protein in the remodelling of acetylated chromatin. This is the first demonstration of a large-scale reorganization of acetylated chromatin induced by a specific factor.     

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.