Eaf1 is the platform for NuA4 molecular assembly that evolutionarily links chromatin acetylation to ATP-dependent exchange of histone H2A variants

Eaf1 (for Esa1-associated factor 1) and Eaf2 have been identified as stable subunits of NuA4, a yeast histone H4/H2A acetyltransferase complex implicated in gene regulation and DNA repair. While both SWI3-ADA2-N-CoR-TF IIIB domain-containing proteins are required for normal cell cycle progression, their depletion does not affect the global Esa1-dependent acetylation of histones. In contrast to all other subunits, Eaf1 is found exclusively associated with the NuA4 complex in vivo. It serves as a platform that coordinates the assembly of functional groups of subunits into the native NuA4 complex. Eaf1 shows structural similarities with human p400/Domino, a subunit of the NuA4-related TIP60 complex. On the other hand, p400 also possesses an SWI2/SNF2 family ATPase domain that is absent from the yeast NuA4 complex. This domain is highly related to the yeast Swr1 protein, which is responsible for the incorporation of histone variant H2AZ in chromatin. Since all of the components of the TIP60 complex are homologous to SWR1 or NuA4 subunits, we proposed that the human complex corresponds to a physical merge of two yeast complexes. p400 function in TIP60 then would be accomplished in yeast by cooperation between SWR1 and NuA4. In agreement with such a model, NuA4 and SWR1 mutants show strong genetic interactions, NuA4 affects histone H2AZ incorporation/acetylation in vivo, and both preset the PHO5 promoter for activation. Interestingly, the expression of a chimeric Eaf1-Swr1 protein recreates a single human-like complex in yeast cells. Our results identified the key central subunit for the structure and functions of the NuA4 histone acetyltransferase complex and functionally linked this activity with the histone variant H2AZ from yeast to human cells.     

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.     

Two distinct mechanisms of chromatin interaction by the Isw2 chromatin remodeling complex in vivo

We have previously shown that Saccharomyces cerevisiae Isw2 complex slides nucleosomes to remodel chromatin in vivo. Our data suggested a model in which Isw2 complex binds the histone octamer and DNA separately to generate the force necessary for nucleosome movement. Here we find that the histone H4 "basic patch" is the only portion of any amino-terminal histone tail required for both target-specific association of Isw2 complex with chromatin and chromatin remodeling in vivo, whereas it is dispensable for basal levels of chromatin binding. Similarly, we find that nonremodeled chromatin structure and integrity of Isw2 complex are required only for target-specific association of Isw2 with chromatin. These data demonstrate fundamental differences between the target-specific and basal modes of chromatin binding by Isw2 complex in vivo and suggest that only the former involves contributions from DNA, histone H4, and sequence-specific DNA binding proteins. We propose a model for target recognition and chromatin remodeling by Isw2 complex in vivo.     

The Schizosaccharomyces pombe JmjC-Protein,Msc1, Prevents H2A.Z Localization in Centromeric and Subtelomeric Chromatin Domains

Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation.     

The Yng1p plant homeodomain finger is a methyl-histone binding module that recognizes lysine 4-methylated histone H3

The ING (inhibitor of growth) protein family includes a group of homologous nuclear proteins that share a highly conserved plant homeodomain (PHD) finger domain at their carboxyl termini. Members of this family are found in multiprotein complexes that posttranslationally modify histones, suggesting that these proteins serve a general role in permitting various enzymatic activities to interact with nucleosomes. There are three members of the ING family in Saccharomyces cerevisiae: Yng1p, Yng2p, and Pho23p. Yng1p is a component of the NuA3 histone acetyltransferase complex and is required for the interaction of NuA3 with chromatin. To gain insight into the function of the ING proteins, we made use of a genetic strategy to identify genes required for the binding of Yng1p to histones. Using the toxicity of YNG1 overexpression as a tool, we showed that Yng1p interacts with the amino-terminal tail of histone H3 and that this interaction can be disrupted by loss of lysine 4 methylation within this tail. Additionally, we mapped the region of Yng1p required for overexpression of toxicity to the PHD finger, showed that this region capable of binding lysine 4-methylated histone H3 in vitro, and demonstrated that mutations of the PHD finger that abolish binding in vitro are no longer toxic in vivo. These results identify a novel function for the Yng1p PHD finger in promoting stabilization of the NuA3 complex at chromatin through recognition of histone H3 lysine 4 methylation.     

NuA4 acetyltransferase is required for efficient nucleotide excision repair in yeast

The nucleotide excision repair (NER) pathway is critical for removing damage induced by ultraviolet (UV) light and other helix-distorting lesions from cellular DNA. While efficient NER is critical to avoid cell death and mutagenesis, NER activity is inhibited in chromatin due to the association of lesion-containing DNA with histone proteins. Histone acetylation has emerged as an important mechanism for facilitating NER in chromatin, particularly acetylation catalyzed by the Spt-Ada-Gcn5 acetyltransferase (SAGA); however, it is not known if other histone acetyltransferases (HATs) promote NER activity in chromatin. Here, we report that the essential Nucleosome Acetyltransferase of histone H4 (NuA4) complex is required for efficient NER in Saccharomyces cerevisiae. Deletion of the non-essential Yng2 subunit of the NuA4 complex causes a general defect in repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in yeast; in contrast, deletion of the Sas3 catalytic subunit of the NuA3 complex does not affect repair. Rapid depletion of the essential NuA4 catalytic subunit Esa1 using the anchor-away method also causes a defect in NER, particularly at the heterochromatic HML locus. We show that disrupting the Sds3 subunit of the Rpd3L histone deacetylase (HDAC) complex rescued the repair defect associated with loss of Esa1 activity, suggesting that NuA4-catalyzed acetylation is important for efficient NER in heterochromatin.     

Methylation of histone H3 mediates the association of the NuA3 histone acetyltransferase with chromatin

The SAS3-dependent NuA3 histone acetyltransferase complex was originally identified on the basis of its ability to acetylate histone H3 in vitro. Whether NuA3 is capable of acetylating histones in vivo, or how the complex is targeted to the nucleosomes that it modifies, was unknown. To address this question, we asked whether NuA3 is associated with chromatin in vivo and how this association is regulated. With a chromatin pulldown assay, we found that NuA3 interacts with the histone H3 amino-terminal tail, and loss of the H3 tail recapitulates phenotypes associated with loss of SAS3. Moreover, mutation of histone H3 lysine 14, the preferred site of acetylation by NuA3 in vitro, phenocopies a unique sas3Delta phenotype, suggesting that modification of this residue is important for NuA3 function. The interaction of NuA3 with chromatin is dependent on the Set1p and Set2p histone methyltransferases, as well as their substrates, histone H3 lysines 4 and 36, respectively. These results confirm that NuA3 is functioning as a histone acetyltransferase in vivo and that histone H3 methylation provides a mark for the recruitment of NuA3 to nucleosomes.