Acetylation of H2AZ Lys 14 is associated with genome-wide gene activity in yeast

Abstract

Histone variants and their post-translational modifications help regulate chromosomal functions. Htz1 is an evolutionarily conserved H2A variant found at several promoters in the yeast Saccharomyces cerevisiae. In this study, we undertook a genome-wide analysis of Htz1 and its modifications in yeast. Using mass spectrometric analysis, we determined that Htz1 is acetylated at Lys 3, Lys 8, Lys 10, and Lys 14 within its N-terminal tail, with K14 being the most abundant acetylated site. ChIP and microarray analysis were then used to compare the location of Htz1-K14 acetylation to that of Htz1 genome-wide. The data presented here demonstrate that while Htz1 is associated preferentially with the promoters of repressed genes, K14 acetylation is enriched at the promoters of active genes, and requires two known histone acetyltransferases, Gcn5 and Esa1. In support of our genome-wide analysis, we found that the acetylatable lysines of Htz1 are required for its full deposition during nucleosome reassembly upon repression of PHO5. Since the majority of Htz1 acetylation is seen at active promoters, where nucleosomes are known to be disassembled, our data argue for a dynamic process in which reassembly of Htz1 is regulated by its acetylation at promoters during transcription.

Figure 1.

Mass spectrometric analysis of Htz1 identifies acetylation on four lysines in the N-terminal tail. (A) Histones isolated from asynchronously growing yeast cells were separated by RP-HPLC (C4 column). Htz1 is eluted after H2B, H4, and H2A but before H3. (B) The ESI-MS of peptides covering the sequence 1–20 from Htz1 treated with D₆-acetyl anhydride and digested with trypsin. Doubly charged ions at m/z 1047.9, 1049.4, 1050.9, 1052.4, and 1053.9 correspond to the peptide with sequence Ac¹SGKAHGGKGKSGAKDSGS LR that is un-, mono-, di-, tri-, and tetra-acetylated at K3, K8, K10, and K14, respectively. (C) ESI/MS/MS was performed on the ion at m/z 1019.6 (observed doubly charged ion at m/z 510.3) to verify its identity. The spectrum shows singly charged b-type and y-type ions corresponding to the sequence shown, in which Lys 14 is acetylated. (D) ESI/MS/MS was performed on the ion at m/z 1246.6 (observed doubly charged ion at m/z 623.8) to verify its identity. The spectrum shows singly charged b-type and y-type ions matching the sequence shown, in which both Lys 10 and Lys 14 are acetylated.

Figure 2.

Htz1 is depleted but hyperacetylated at active genes. (A,B) Moving averages (window size 100) of Htz1 binding and transcriptional frequencies at ORFs (A) and IGRs (B) are plotted relative to each other. The transcriptional frequencies for IGRs are the values for the cognate ORF. The Pearson correlation values (r) for the data sets are indicated. (C–F) Moving averages (window size 100) of Htz1-K14 acetylation at ORFs (C,E) or IGRs (D,F) are plotted relative to transcriptional frequency. The raw data are plotted in C and D; in E and F, the acetylation data have been normalized to bulk Htz1 occupancy. The Pearson correlations (r) are shown in each panel.

Figure 3.

Htz1-K14 acetylation correlates positively with other histone acetylation sites. Correlation values (r) of Htz1-K14 acetylation with other core histone acetylation sites are displayed graphically. Data for ORFs are shown in A and for IGRs in B.

Figure 4.

Htz1 and Htz1-K14 acetylation are enriched on promoters. (A) Htz1 is enriched on promoters relative to ORFs. Histogram plot of the ratios of Htz1 binding at promoter IGRs and their cognate ORFs (IGR:ORF ratios). Bars representing IGR:ORF ratios close to 1 are gray; bars representing ratios different from 1 are black. (B) Distribution of IGR classes within the total Htz1 data set (dark-gray bars) relative to the Htz1-enriched (black bars) and Htz1-depleted (light-gray bars) subsets. The abundance of each IGR subtype in each data set is expressed as a percentage of the total. (C) Histogram plot of the ratios of Htz1-K14 acetylation at promoter IGRs and their cognate ORFs (IGR:ORF ratios). Bars representing IGR:ORF ratios close to 1 are gray; bars representing ratios different from 1 are black. (D) Distribution of IGR classes within the total Htz1-K14 acetylation data set (dark-gray bars) relative to the Htz1-K14 hyperacetylated (black bars) and Htz1-K14 hypoacetylated (light-gray bars) subsets. The abundance of each IGR subtype in each data set is expressed as a percentage of the total.

Figure 5.

Htz1 distribution relative to chromosomal position. (A) Htz1 enrichment is plotted against distance to the nearest telomere (moving averages; window size 50). (B) Htz1-K14acetylation levels are plotted relative to distance from the nearest telomere (moving averages; window size 50).

Figure 6.

Gcn5 and Esa1 are redundantly required for Htz1-K14 acetylation. Levels of Htz1-K14 acetylation were determined by ChIP using α741 antibodies; α660 antibodies were used in ChIP to control for bulk Htz1 levels. Enrichments relative to a fragment 0.5 kb from the end of chromosome VIR (TelVIR) were calculated and normalized to input DNA. (A) Semiquantitative multiplex PCR analysis of Htz1-K14 acetylation (top), bulk Htz1 (middle), and input DNA (bottom) at SMX3. Inactivating mutations in either Esa1 or Gcn5 reduce the enrichment of Htz1-K14Ac at SMX3, but a double mutation reduces acetylation to background levels. Levels of Htz1 are shown for comparison. Increased levels of Htz1 in the HAT mutant strains correspond to reduced transcription (as measured by Pol II enrichment), but mutations in other HATs that caused reduced Pol II enrichment did not show loss of Htz1-K14 acetylation (data not shown). (B) Quantitation of ChIP data for four loci (two ORFs and two IGRs). K14 acetylation is displayed as a percentage of the wild-type level (WT) and has been normalized to total Htz1 occupancy. The mean values from three independent ChIPs with α741 (Htz1-K14Ac), divided by the mean values from ChIPs with α660 (bulk Htz1), are plotted. Error bars are not indicated because the data represent divided ratios, but variation between experiments was negligible.

Figure 7.

Acetylation regulates deposition of Htz1 at the PHO5 promoter. Levels of HA-Htz1, H3, and Htz1-K14 acetylation at the PHO5 promoter were determined by ChIP. Quantitations of enrichments at PHO5 relative to TelVIR are plotted. The schematic diagram (top) of the PHO5 promoter shows promoter nucleosomes as open circles and the 5′ ORF nucleosome as a filled circle, with UASp1 (black box), UASp2 (gray box), and the TATA box also shown. The positions of the primer sets used for PCR amplification are indicated by thick black lines above nucleosomes −1 and −2. An asterisk indicates the primer set for which the data are shown, although both primer sets gave identical results. (A) HA-Htz1 binding in cells that have wild-type (WT; black line) or mutated (4K-R; gray line) Htz1. At 0 min the cells were shifted from high- to low-phosphate media. (B) H3 binding in the Htz1-WT (black line) or Htz1-4K-R (gray line) strains. The cells were shifted from high- to low-phosphate media at 0 min. (C) HA-Htz1 binding in cells that have wild-type (WT; black line) or 4K-R (gray line) Htz1. At −60 min the cells were growing in low-phosphate medium; at 0 min phosphate was added. (D) H3 binding in wild-type (WT; black line) or 4K-R (gray line) strains. At −60 min the cells were growing in low-phosphate medium; at 0 min phosphate was added. (E) HA-Htz1 binding in cells that have wild-type (WT; black line) or 4K-R (gray line) Htz1. At −60 min the cells were growing in low-phosphate medium; at 0 min phosphate was added and samples were taken at 1, 5, 10, 30, and 60 min after phosphate addition. (F) Htz1-K14 acetylation at the PHO5 promoter under repressed (black bars) or active (gray bars) conditions. Raw data are plotted to the left; the values to the right have been normalized to bulk Htz1 levels to account for nucleosome loss during gene activity.