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 sas3⌬ 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.In eukaryotes, DNA is packaged into chromatin, a nucleoprotein structure consisting of DNA, histones, and nonhistone proteins. The assembly of DNA into chromatin modulates the access of cellular machinery to DNA and thus regulates transcription, replication, repair, and recombination. Chromatin structure can be regulated by the addition of posttranslational modifications to histones, including acetylation, methylation, phosphorylation, and ubiquitination. Consistent with this, several histone posttranslational modifications have been linked to the regulation of gene expression.Numerous multiprotein complexes are involved in the posttranslational modification of histones. These complexes vary in both their protein components and the modifications they effect. The most well-studied group of modifying complexes is the histone acetyltransferases (HATs), which use acetyl coenzyme A as a substrate for the acetylation of lysine residues within both the tail and globular domains of histones (39,43,45,73). In the budding yeast Saccharomyces cerevisiae, there are at least eight proteins that have been identified as having HAT activity in vitro, including Gcn5p, Hat1p, Esa1p, Elp3p, Nut1p, Hpa2p, Sas2p, and Sas3p (45,73), although for many of these proteins, whether histones represent their true substrates in vivo is not known. Three of these proteins are found in complexes that specifically acetylate the lysines within the tail of histone H3, including the GCN5-dependent SAGA, SLIK/ SALSA, ADA, and HAT-A2 complexes (12,16,49,58,60), the ELP3-dependent elongator complex (70), and the SAS3-dependent NuA3 complex (25). The acetylation of histone tails is associated with regions of transcriptional activity, and histone acetylation is thought to modulate chromatin structure through two different mechanisms. First, the neutralization of charge associated with histone acetylation is believed to re...
Rsc4p, a subunit of the RSC chromatin-remodeling complex, is acetylated at lysine 25 by Gcn5p, a wellcharacterized histone acetyltransferase (HAT). Mutation of lysine 25 does not result in a significant growth defect, and therefore whether this modification is important for the function of the essential RSC complex was unknown. In a search to uncover the molecular basis for the lethality resulting from loss of multiple histone H3-specific HATs, we determined that loss of Rsc4p acetylation is lethal in strains lacking histone H3 acetylation. Phenotype comparison of mutants with arginine and glutamine substitutions of acetylatable lysines within the histone H3 tail suggests that it is a failure to neutralize the charge of the H3 tail that is lethal in strains lacking Rsc4p acetylation. We also demonstrate that Rsc4p acetylation does not require any of the known Gcn5p-dependent HAT complexes and thus represents a truly novel function for Gcn5p. These results demonstrate for the first time the vital and yet redundant functions of histone H3 and Rsc4p acetylation in maintaining cell viability.Posttranslational modifications can augment protein function extending the diversity of proteins produced by the cell. For example, many thousands of proteins in a typical eukaryotic cell are modified by the covalent addition of a phosphate group (22), which can serve to either directly alter protein structure or mediate protein-protein interactions. Another well-studied modification is protein acetylation. Amino-terminal acetylation is one of the most common protein modifications in eukaryotes, occurring on ca. 85% of proteins. In addition, the acetylation of the epsilon-amino group of internal lysines occurs on ␣-tubulin, high-mobility group proteins, transcription factors, nuclear import factors, and histones (28).Histones H2A, H2B, H3, and H4 are the best-characterized substrates for posttranslational acetylation of internal lysines, with the majority of histone acetylation occurring on the unstructured amino-terminal "tails" of these proteins. These modifications are proposed to have two functions: to directly alter chromatin structure by weakening histone-DNA, as well as internucleosome interactions (1,2,11,33,34), and to act as a "molecular dock" for recruitment of factors that modify chromatin structure (42). Histone acetylation is catalyzed by histone acetyltransferases (HATs), which are comprised of a catalytic subunit complexed with accessory proteins that serve to either target or potentiate HAT activity. The best-studied catalytic subunit is Gcn5p, a component of multiple histone H3-specific HAT complexes in Saccharomyces cerevisiae. These complexes are responsible for acetylation of lysines 9, 14, 18, 23, 27, and 36 of histone H3 (14, 26, 37). All Gcn5p-dependent HAT complexes share the accessory proteins Ada2p and Ada3p, and several studies have demonstrated that ADA2 and ADA3 are essential for both the nucleosomal HAT activity of Gcn5p and its incorporation into HAT complexes (5, 7, 13). Indeed, the majority of p...
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