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. 2006 Apr;26(7):2791-802.
doi: 10.1128/MCB.26.7.2791-2802.2006.

Genome-wide relationships between TAF1 and histone acetyltransferases in Saccharomyces cerevisiae

Melissa Durant  1 , B Franklin Pugh
Affiliations

Genome-wide relationships between TAF1 and histone acetyltransferases in Saccharomyces cerevisiae

Melissa Durant et al. Mol Cell Biol. 2006 Apr.

Abstract

Histone acetylation regulates gene expression, yet the functional contributions of the numerous histone acetyltransferases (HATs) to gene expression and their relationships with each other remain largely unexplored. The central role of the putative HAT-containing TAF1 subunit of TFIID in gene expression raises the fundamental question as to what extent, if any, TAF1 contributes to acetylation in vivo and to what extent it is redundant with other HATs. Our findings herein do not support the basic tenet that TAF1 is a major HAT in Saccharomyces cerevisiae, nor do we find that TAF1 is functionally redundant with other HATs, including Gcn5, Elp3, Hat1, Hpa2, Sas3, and Esa1, which is in contrast to previous conclusions regarding Gcn5. Our findings do reveal that of these HATs, only Gcn5 and Esa1 contribute substantially to gene expression genome wide. Interestingly, histone acetylation at promoter regions throughout the genome does not require TAF1 or RNA polymerase II, indicating that most acetylation is likely to precede transcription and not depend upon it. TAF1 function has been linked to Bdf1, which binds TFIID and acetylated histone H4 tails, but no linkage between TAF1 and the H4 HAT Esa1 has been established. Here, we present evidence for such a linkage through Bdf1.

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Figures

FIG. 1.
FIG. 1.
Gcn5, and not TAF1, is important for bulk H3 acetylation levels. All strains were grown in CSM-His medium at 25°C and then shifted to 37°C for 45 min to inactivate the taf1ts2 allele, when present. Crude whole-cell lysates were subjected SDS-polyacrylamide gel electrophoresis and immunoblot analysis using antibodies recognizing the indicated histone H3 modifications or total H3 (bottom immunoblot). Quantitation of three independent replicates is shown.
FIG. 2.
FIG. 2.
Gcn5, and not TAF1, is important for gene-specific H3 acetylation levels. (A) Frequency distribution of H3 Ac-K9,14 occupancy in intergenic regions containing (left panel) or lacking (right panel) a promoter, the latter being intergenic regions located between two convergently transcribed genes. chIP assays were performed on cross-linked, sheared chromatin from WT, taf1ts2, gcn5Δ, and taf1ts2 gcn5Δ strains that were grown at 25°C and then shifted to 37°C for 45 min to inactivate the taf1ts2 allele. Immunoprecipitated DNA from test strains was labeled and cohybridized to intergenic microarrays along with an independent wild-type sample. H3 Ac-K9,14 occupancy levels relative to the wild type were converted to a log2 scale and binned in 0.05 intervals, and the resulting frequency histogram was converted to an interpolated frequency distribution using Kaleidagraph software. (B) Cell growth and chIP were performed as described above (A) using a wild-type or rpb1-1 strain. H3 Ac-K9,14 occupancy data were normalized to a nonspecific immunoprecipitated chIP DNA data set (39), converted to a log2 scale, and centered to the median value for nonpromoter intergenic regions. Shown is a scatter plot comparison between H3 Ac-K9,14 in an rpb1-1 strain versus a wild-type strain at the nonpermissive temperature (37°C). (C) Immunoblots were performed on WT and rpb1-1 strains at the permissive (25°C) and nonpermissive (37°C) temperatures using antibodies against the indicated histone modifications or total H3. Quantitation of three independent H4 modification replicates is shown below the immunoblot.
FIG. 3.
FIG. 3.
Gcn5 and TAF1 make independent contributions to gene expression. (A) Genome-wide changes in gene expression in WT, taf1ts2, gcn5Δ, and taf1ts2 gcn5Δ strains relative to an independent wild-type strain. Expression changes were determined after cultures were shifted to 37°C for 45 min to inactivate the taf1ts2 allele. Frequency distributions are plotted as described in the legend of Fig. 2. This experiment is a repeat of an experiment described previously (18) but was performed in the context of the experiments described in the legend of Fig. 4. The dashed line represents the calculated distribution for the double mutant obtained by adding the log2 ratios of the single mutants. (B) Gene-by-gene scatter plot relating calculated and observed values for the taf1ts2 gcn5Δ strain.
FIG. 4.
FIG. 4.
TAF1 does not exhibit functional redundancy with a variety of yeast HATs, and many HATs do not make significant contributions to global gene expression or acetylation levels. (A to E) Changes in genome-wide mRNA expression levels were determined in mutant HAT strains in a wild-type or taf1ts2 background, as described in the legend of Fig. 3. Predicted values (dashed line) were calculated for the double mutants by adding the log2 ratios of the single mutants. (F) Changes in bulk H3 or H4 acetylation levels at specific lysine residues in each of the mutant HAT strains were assayed by immunoblotting, as described in the legend of Fig. 1.
FIG. 5.
FIG. 5.
Esa1, and not TAF1, is important for bulk H4 acetylation levels. Wild-type, taf1ts2, esa1-414, and taf1ts2 esa1-414 strains were grown and shifted to the nonpermissive temperature as described in the legend of Fig. 1. Blots were probed with the indicated antibody. Quantitation of three independent replicates is shown in the bar graph.
FIG. 6.
FIG. 6.
Esa1 and TAF1 are functionally linked. (A) Data from Fig. 4E (log2 changes in gene expression in wild-type, taf1ts2, esa1-414, and taf1ts2 esa1-414 strains) were filtered for a twofold cutoff and clustered into eight groups by K means (13). Average values for each group in the taf1ts2 and esa1-414 data sets are plotted against each other. (B) A general mechanism describing the potential interrelationship between Esa1 (NuA4) and TAF1 (TFIID) in gene activation. According to the model, recruitment of NuA4 to TATA-less promoters results in H4 tail acetylation, allowing Bdf1 to dock and escort in TFIID. Two potentially rate-limiting steps are shown. Below these steps are arbitrary rate constants in which either NuA4 or TFIID activity, as indicated, is limiting for transcription at five hypothetical genes. The graph represents simulated log2 changes in gene expression (RNA output) in a hypothetical esa1-414 mutant versus a hypothetical taf1ts2 mutant for each of the five genes. See Materials and Methods for a detailed description of the simulation using KinTekSim software.
FIG. 7.
FIG. 7.
H4 acetylation is linked to Bdf1 occupancy, which is linked to TAF1 occupancy. Bdf1-TAP, TAF1-TAP, and “null” (no tag) log2 occupancy levels at 25°C were derived from data reported previously (54). The data were centered to the median value for nonpromoter regions (intergenic regions located between two convergent genes). The H4 tetra-acetylated log2 occupancy levels at 30°C were derived from data reported previously (4) and centered to the genome-wide median. The x-axis data were sorted by value, and 100-gene sliding-window averages (step size = 1) were plotted against the corresponding y-axis sliding-window average.
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