NuA4-directed chromatin transactions throughout the Saccharomyces cerevisiae genome

Abstract

Two of the major histone acetyltransferases in Saccharomyces cerevisiae are NuA4 and SAGA, which acetylate histones H4 and H3, respectively. Acetylated H3 and H4 tails have been implicated in binding bromodomain proteins, including Bdf1. Bdf1 interacts with the general transcription factor TFIID, which might promote preinitiation complex (PIC) assembly. Bdf1 also interacts with the SWR complex (SWR-C). SWR-C is responsible for the deposition of the histone H2A variant H2A.Z. The placement of these interactions into a connected pathway of PIC assembly has not been fully established. Moreover, it is not known how widespread and how variable such a pathway might be on a genomic scale. Here we provide genomic evidence for S. cerevisiae that PIC assembly (TFIID occupancy) and chromatin remodeling (SWR-C and H2A.Z occupancy) are linked in large part to NuA4-directed H4 acetylation and subsequent Bdf1 binding, rather than through SAGA-directed H3 acetylation. Bdf1 and its homolog Bdf2 tend to have distinct locations in the genome. However, the deletion of BDF1 leads to the accumulation of Bdf2 at Bdf1-vacated sites. Thus, while Bdf1 and Bdf2 are at least partially redundant in function, their functions in the genome are geographically distinct.

FIG. 1.

Model for PIC assembly via nucleosome acetylation. Shown are a series of events starting with a typical promoter and its nucleosomal architecture. NuA4 and/or SAGA associate with promoter regions via targeting mechanisms that are not shown. These HAT complexes acetylate histone H4 and H3, respectively (and other sites as well). Bdf1 is preferentially recruited to acetylated nucleosomes, which could be replaced by Bdf2 at some promoters. It is not known to which nucleosomes Bdf1 binds. Bdf1 recruits TFIID and SWR-C, which leads to PIC assembly, the release of H2A.Z, and transcription by pol II (RNA polymerase II).

FIG. 2.

Cluster plot of genome-wide changes in factor occupancy in mutant versus wild-type strains. (A) Changes in factor occupancy in the indicated mutants. ChIP-chip was performed on the indicated factors in the indicated strains. Assays were run in parallel with mutant and wild-type (WT) strains, which were labeled separately with Cy3 and Cy5 fluorescent dyes and cohybridized to spotted microarrays containing ∼6,000 intergenic-region-length PCR-generated probes. Data are presented as a cluster diagram (14) in which each row is an intergenic promoter region and each column is an average of two dye-swapped replicates. Red, green, and black denote increases, decreases, and no change in binding, respectively. Data were filtered to include only those intergenic regions that contained a single promoter region and had the largest change in occupancy (i.e., <10th or >90th percentile in at least one column). Six hundred eleven intergenic regions (∼10% of the analyzed genome) met these criteria. These intergenic regions are intended to be the strongest representatives of the genome so as to generate the strongest patterns. Such patterns are nevertheless likely to be applicable, with lower intensity, to most other intergenic promoter regions. Rows were grouped according to k means into four clusters (with 283, 158, 108, and 62 intergenic regions) (14). Columns were clustered hierarchically (14). The table below the cluster plot provides the average log₂ ratio in each cluster for each experiment. “All” denotes all promoters, and “Non” denotes nonpromoter intergenic regions (i.e., regions between two convergently transcribed genes). (B) Average occupancy of the indicated factors for the promoter regions of clusters 1 to 4. “A” denotes all single-promoter intergenic regions (>3,000), and “N” denotes nonpromoter intergenic regions (>1,000). Data for sets designated “(1)” are the log₂ ratios of the wild-type reference data set presented in panel A (binding after a shift to 37°C for 45 min) divided by the ChIP result for a “Null” untagged control (58). Data for sets designated “(2)” are the log₂ ratios of the wild-type data set from reference (binding after a shift to 37°C for 15 min) divided by a “Null” result. The two data sets were collected from two different studies which differed only in the time at 37°C (15 versus 45 min) before binding was measured. Their trends were similar, thereby providing additional confidence in the conclusions drawn from the data. Log₂ values are relative to those for nonpromoter regions.

FIG. 3.

Intrinsic H4 acetylation levels at genes from clusters 1 to 4. (A) Relative occupancy level of acetylated H4 (K5,8,12,16) and H3 (K9,14) at clusters 1 to 4. Acetylation levels were obtained from reference and were normalized to the levels for nonpromoter intergenic regions. Data were transformed into log₂ ratios, and the average for each cluster was determined. “Non” denotes the average of >2,000; “All” denotes all promoter regions. (B) Changes (n-fold) in factor occupancy upon the loss of Esa1 correlate with the initial H4 acetylation levels. The H4 acetylation levels are from panel A. Changes in factor occupancy levels represent averages from Fig. 2A.

FIG. 4.

Changes in Bdf1 and Taf1 occupancy when Gcn5 is eliminated. ChIP-chip assays were performed with Bdf1-TAP and Taf1-TAP as described in the legend to Fig. 2A; the test and reference samples were derived from gcn5Δ and GCN5 strains, respectively. The average n-fold change in occupancy between the mutant and wild-type strains was determined for the set of genes in clusters 1 to 4 (Fig. 2A), log₂ transformed, and plotted. The data were centered such that there was, on average, no change in occupancy in nonpromoter intergenic regions. “All” denotes the value for all intergenic promoter regions for which there were valid data.

FIG. 5.

Regions that are depleted of NuA4-assembled transcription components tend to be enriched with repressor proteins. Average occupancy data (log₂ scale) for the promoter regions in the indicated clusters were obtained from reference , as described in the legend to Fig. 2B.