Promoter-specific recruitment of histone acetyltransferase activity is often critical for transcriptional activation. We present a detailed study of the interaction between the histone acetyltransferase complexes SAGA and NuA4, and transcription activators. We demonstrate by affinity chromatography and photo-cross-linking label transfer that acidic activators directly interact with Tra1p, a shared subunit of SAGA and NuA4. Mutations within the COOH-terminus of Tra1p disrupted its interaction with activators and resulted in gene-specific transcriptional defects that correlated with lowered promoter-specific histone acetylation. These data demonstrate that the essential Tra1 protein serves as a common target for activators in both SAGA and NuA4 acetyltransferases.

The histone methyltransferase Set2, which specifically methylates lysine 36 of histone H3, has been shown to repress transcription upon tethering to a heterologous promoter. However, the mechanism of targeting and the consequence of Set2-dependent methylation have yet to be demonstrated. We sought to identify the protein components associated with Set2 to gain some insights into the in vivo function of this protein. Mass spectrometry analysis of the Set2 complex, purified using a tandem affinity method, revealed that RNA polymerase II (pol II) is associated with Set2. Immunoblotting and immunoprecipitation using antibodies against subunits of pol II confirmed that the phosphorylated form of pol II is indeed an integral part of the Set2 complex. Gst-Set2 preferentially binds to CTD synthetic peptides phosphorylated at serine 2, and to a lesser extent, serine 5 phosphorylated peptides, but has no affinity for unphosphorylated CTD, suggesting that Set2 associates with the elongating form of the pol II. Furthermore, we show that set2⌬ ppr2⌬ double mutants (PPR2 encodes TFIIS, a transcription elongation factor) are synthetically hypersensitive to 6-azauracil, and that deletions in the CTD reduce in vivo levels of H3 lysine 36 methylation. Collectively, these results suggest that Set2 is involved in regulating transcription elongation through its direct contact with pol II.Nucleosomal structure, once thought to be quite static, turns out to be extremely dynamic and tightly regulated by various nonhistone proteins (1). These protein modulators can be generally categorized into two groups: those that utilize the energy released from ATP hydrolysis to alter histone-DNA contacts (2), and those that are capable of modifying histones covalently. To date, the known histone modifications include acetylation, methylation, phosphorylation, ubquitinylation, and ADP-ribosylation (3, 4). The consequence of complex histone modification patterns has yet to be fully understood. However, based on a growing body of evidence, a "histone code" hypothesis has been proposed suggesting that these modifications either directly alter chromatin structure (5) or create a series of molecular "bar codes" at the nucleosome surface for other proteins to recognize (3, 6).Most, if not all, chromatin modulators have a somewhat weak intrinsic affinity for their nucleosomal substrates in vitro. Nevertheless, the specific pattern of histone modifications and the particular loci where chromatin remodeling occurs throughout the genome strongly suggest that these activities are primarily targeted to certain regions by either DNA bound activators or repressors (7, 8). Acidic activators have been shown to be able to target SWI/SNF or Spt-Ada-Gcn5-acetyltranferase to local promoters, thereby facilitating transcription from nucleosomal templates (9 -12). Likewise, the repressor Ume6 or co-repressor Ssn6/Tup1 can recruit histone deacetylase complexes to remove the acetylation marks from histone tails, facilitating transcriptional repression (13-17). In addition ...

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Longstanding observations suggest that acetylation and/or amino-terminal tail structure of histones H3 and H4 are critical for eukaryotic cells. For Saccharomyces cerevisiae, loss of a single H4-specific histone acetyltransferase (HAT), Esa1p, results in cell cycle defects and death. In contrast, although several yeast HAT complexes preferentially acetylate histone H3, the catalytic subunits of these complexes are not essential for viability. To resolve the apparent paradox between the significance of H3 versus H4 acetylation, we tested the hypothesis that H3 modification is essential, but is accomplished through combined activities of two enzymes. We observed that Sas3p and Gcn5p HAT complexes have overlapping patterns of acetylation. Simultaneous disruption of SAS3, the homolog of the MOZ leukemia gene, and GCN5, the hGCN5/PCAF homolog, is synthetically lethal due to loss of acetyltransferase activity. This key combination of activities is specific for these two HATs because neither is synthetically lethal with mutations of other MYST family or H3-specific acetyltransferases. Further, the combined loss of GCN5 and SAS3 functions results in an extensive, global loss of H3 acetylation and arrest in the G 2 /M phase of the cell cycle. The strikingly similar effect of loss of combined essential H3 HAT activities and the loss of a single essential H4 HAT underscores the fundamental biological significance of each of these chromatin-modifying activities.