Combined Interactions of Plant Homeodomain and Chromodomain Regulate NuA4 Activity at DNA Double-Strand Breaks

Abstract

DNA double-strand breaks (DSBs) represent one of the most threatening lesions to the integrity of genomes. In yeast Saccharomyces cerevisiae, NuA4, a histone acetylation complex, is recruited to DSBs, wherein it acetylates histones H2A and H4, presumably relaxing the chromatin and allowing access to repair proteins. Two subunits of NuA4, Yng2 and Eaf3, can interact in vitro with methylated H3K4 and H3K36 via their plant homeodomain (PHD) and chromodomain. However, the roles of the two domains and how they interact in a combinatorial fashion are still poorly characterized. In this study, we generated mutations in the PHD and chromodomain that disrupt their interaction with methylated H3K4 and H3K36. We demonstrate that the combined mutations in both the PHD and chromodomain impair the NuA4 recruitment, reduce H4K12 acetylation at the DSB site, and confer sensitivity to bleomycin that induces DSBs. In addition, the double mutant cells are defective in DSB repair as judged by Southern blot and exhibit prolonged activation of phospho-S129 of H2A. Cells harboring the H3K4R, H3K4R, K36R, or set1D set2D mutant that disrupts H3K4 and H3K36 methylation also show very similar phenotypes to the PHD and chromodomain double mutant. Our results suggest that multivalent interactions between the PHD, chromodomain, and methylated H3K4 and H3K36 act in a combinatorial manner to recruit NuA4 and regulate the NuA4 activity at the DSB site.

The Eaf3 chromodomain acts as a pH sensor for gene expression by altering its binding affinity for histone methylated-lysine residues

“…As described above and in previous studies [9,10,14,33], Eaf3 CD itself has high methylation specificity but low sequence specificity; therefore, combinatorial actions with the PHD of Yng2 in NuA4 and with the PHD of Rco1 in Rpd3S are necessary for the overall affinity and specificity of the H3K4me-and H3K36me-nucleosome interaction [12,16], and activation [15,38,39] of the complexes (Figure 7). A biochemical study previously demonstrated that Rpd3S preferentially binds di-nucleosomes by contacting two nucleosomes simultaneously via Eaf3 CD and Rco1 PHD [40].…”

“…In fact, Eaf3 CD can also bind weakly to H3K4me, facilitating recruitment of NuA4 to the promoter region. Similar to Rpd3S, the PHD of the Yng2 subunit of NuA4 plays an essential cooperative role in binding of Eaf3 CD to H3K4me [15,16]. Thus, Eaf3 CD seems to have no strong sequence specificity and can bind to both H3K4me and H3K36me, while the specific recruitment of NuA4 and Rpd3S to H3K4me and H3K36me, respectively, is cooperatively determined by the PHD of the respective Yng2 and Rco1 subunits.…”

Combined Action of Histone Reader Modules Regulates NuA4 Local Acetyltransferase Function but Not Its Recruitment on the Genome

“…During the last steps of preparation of the manuscript, a report implicating the combined action of Eaf3 CHD/H3K36me and Yng2 PHD/H3K4me in assisting NuA4 function during the repair of DNA double-strand breaks was published (67). While the concept is interesting, in our hands we could not detect any increased sensitivity to DNA-damaging agents for either our single or double mutant cells (20,54) (Fig.…”

YHMI: a web tool to identify histone modifications and histone/chromatin regulators from a gene list in yeast

“…Indeed, several studies have shown that the methylated H3K4 and H3K36 interact with PHD and chromodomain within the NuA4 complex by using the GST pull-down, immunoprecipitation and protein array assays ( 9 , 11 , 12 ). Furthermore, the set1Δ set2Δ mutants or mutants with combined mutations of the PHD and chromodomains show defective acetyltransferases activity of NuA4 ( 33 , 34 ). Additionally, an experiment has shown that the interaction of H3K4me3 with Tudor domain in Sgf29 within the SAGA complex is also essential for the acetyltransferase activity of SAGA ( 35 ).…”