Phospho-dependent recruitment of the yeast NuA4 acetyltransferase complex by MRX at DNA breaks regulates RPA dynamics during resection.

The KAT5 (Tip60/Esa1) histone acetyltransferase is part of NuA4, a large multifunctional complex highly conserved from yeast to mammals that targets lysines on H4 and H2A (X/Z) tails for acetylation. It is essential for cell viability, being a key regulator of gene expression, cell proliferation, and stem cell renewal and an important factor for genome stability. The NuA4 complex is directly recruited near DNA double-strand breaks (DSBs) to facilitate repair, in part through local chromatin modification and interplay with 53BP1 during the DNA damage response. While NuA4 is detected early after appearance of the lesion, its precise mechanism of recruitment remains to be defined. Here, we report a stepwise recruitment of yeast NuA4 to DSBs first by a DNA damage-induced phosphorylation-dependent interaction with the Xrs2 subunit of the Mre11-Rad50-Xrs2 (MRX) complex bound to DNA ends. This is followed by a DNA resection-dependent spreading of NuA4 on each side of the break along with the ssDNA-binding replication protein A (RPA). Finally, we show that NuA4 can acetylate RPA and regulate the dynamics of its binding to DNA, hence targeting locally both histone and nonhistone proteins for lysine acetylation to coordinate repair.

Structure/function analysis of the phosphatidylinositol-3-kinase domain of yeast tra1.

Tra1 is an essential component of the Saccharomyces cerevisiae SAGA and NuA4 complexes. Using targeted mutagenesis, we identified residues within its C-terminal phosphatidylinositol-3-kinase (PI3K) domain that are required for function. The phenotypes of tra1-P3408A, S3463A, and SRR3413-3415AAA included temperature sensitivity and reduced growth in media containing 6% ethanol or calcofluor white or depleted of phosphate. These alleles resulted in a twofold or greater change in expression of approximately 7% of yeast genes in rich media and reduced activation of PHO5 and ADH2 promoters. Tra1-SRR3413 associated with components of both the NuA4 and SAGA complexes and with the Gal4 transcriptional activation domain similar to wild-type protein. Tra1-SRR3413 was recruited to the PHO5 promoter in vivo but gave rise to decreased relative amounts of acetylated histone H3 and histone H4 at SAGA and NuA4 regulated promoters. Distinct from other components of these complexes, tra1-SRR3413 resulted in generation-dependent telomere shortening and synthetic slow growth in combination with deletions of a number of genes with roles in membrane-related processes. While the tra1 alleles have some phenotypic similarities with deletions of SAGA and NuA4 components, their distinct nature may arise from the simultaneous alteration of SAGA and NuA4 functions.

Regulation of NuA4 histone acetyltransferase activity in transcription and DNA repair by phosphorylation of histone H4.

The NuA4 complex is a histone H4/H2A acetyltransferase involved in transcription and DNA repair. While histone acetylation is important in many processes, it has become increasingly clear that additional histone modifications also play a crucial interrelated role. To understand how NuA4 action is regulated, we tested various H4 tail peptides harboring known modifications in HAT assays. While dimethylation at arginine 3 (R3M) had little effect on NuA4 activity, phosphorylation of serine 1 (S1P) strongly decreased the ability of the complex to acetylate H4 peptides. However, R3M in combination with S1P alleviates the repression of NuA4 activity. Chromatin from cells treated with DNA damage-inducing agents shows an increase in phosphorylation of serine 1 and a concomitant decrease in H4 acetylation. We found that casein kinase 2 phosphorylates histone H4 and associates with the Rpd3 deacetylase complex, demonstrating a physical connection between phosphorylation of serine 1 and unacetylated H4 tails. Chromatin immunoprecipitation experiments also link local phosphorylation of H4 with its deacetylation, during both transcription and DNA repair. Time course chromatin immunoprecipitation data support a model in which histone H4 phosphorylation occurs after NuA4 action during double-strand break repair at the step of chromatin restoration and deacetylation. These findings demonstrate that H4 phospho-serine 1 regulates chromatin acetylation by the NuA4 complex and that this process is important for normal gene expression and DNA repair.

Yeast G1 DNA damage checkpoint regulation by H2A phosphorylation is independent of chromatin remodeling.

Recent studies of yeast G1 DNA damage response have identified characteristic changes in chromatin adjacent to double-strand breaks (DSBs). Histone H2A (yeast H2AX) is rapidly phosphorylated on S129 by the kinase Tel1 (ATM) over a domain extending kilobases from the DSB. The adaptor protein Rad9 (53BP1) is recruited to this chromatin domain through binding of its tudor domains to histone H3 diMe-K79. Multisite phosphorylation of Rad9 by Mec1 (ATR) then activates the signaling kinase Rad53 (CHK2) to induce a delay in G1. Here, we report a previously undescribed role for Tel1 in G1 checkpoint response and show that H2A is the likely phosphorylation target, in a much as S129 mutation to Ala confers defects in G1 checkpoint arrest, Rad9 phosphorylation, and Rad53 activation. Importantly, Rad9 fails to bind chromatin adjacent to DSBs in H2A-S129A mutants. Previous work showed that H2A phosphorylation allows binding of NuA4, SWR, and INO80 chromatin remodeling complexes, perhaps exposing H3 diMe-K79. Yet, mutants lacking SWR or INO80 remain checkpoint competent, whereas loss of NuA4-dependent histone acetylation leads to G1 checkpoint persistence, suggesting that H2A phosphorylation promotes two independent events, rapid Rad9 recruitment to DSBs and subsequent remodeling by NuA4, SWR, and INO80.

Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites.

Yeast histone H2A is phosphorylated on Ser129 upon DNA damage, an event required for efficient repair. We show that phosphorylation occurs rapidly over a large region around DNA double-strand breaks (DSBs). Histone H4 acetylation is also important for DSB repair, and we found that the NuA4 HAT complex associates specifically with phospho-H2A peptides. A single NuA4 subunit, Arp4, is responsible for the interaction. The NuA4 complex is recruited to a DSB concomitantly with the appearance of H2A P-Ser129 and Arp4 is important for this binding. Arp4 is also a subunit of the Ino80 and Swr1 chromatin remodeling complexes, which also interact with H2A P-Ser129 and are recruited to DSBs. This association again requires Arp4 but also prior NuA4 recruitment and action. Thus, phosphorylation of H2A at DNA damage sites creates a mark recognized by different chromatin modifiers. This interaction leads to stepwise chromatin reconfiguration, allowing efficient DNA repair.