Histone variant H2A.Z marks the 5' ends of both active and inactive genes in euchromatin

Abstract

In S. cerevisiae, histone variant H2A.Z is deposited in euchromatin at the flanks of silent heterochromatin to prevent its ectopic spread. We show that H2A.Z nucleosomes are found at promoter regions of nearly all genes in euchromatin. They generally occur as two positioned nucleosomes that flank a nucleosome-free region (NFR) that contains the transcription start site. Astonishingly, enrichment at 5' ends is observed not only at actively transcribed genes but also at inactive loci. Mutagenesis of a typical promoter revealed a 22 bp segment of DNA sufficient to program formation of a NFR flanked by two H2A.Z nucleosomes. This segment contains a binding site of the Myb-related protein Reb1 and an adjacent dT:dA tract. Efficient deposition of H2A.Z is further promoted by a specific pattern of histone H3 and H4 tail acetylation and the bromodomain protein Bdf1, a component of the Swr1 remodeling complex that deposits H2A.Z.

Figure 1. H2A.Z Enrichment in Euchromatin

(A) Schematic of HA₃-Htz1 ChIP enrichment across chromosome III. Bars represent IP/WCE value as determined by QPCR for a single 300 bp segment. Each 5′ primer is separated by 1000 bp. (B) Log scale graph comparing H2A.Z enrichment values to distance to the nearest initiation codon. The correlation coefficient is 0.2662. (C and D) Diagram of BPH1-SNT1 interval (C) and the LEU2-YCL012c interval (D). Both regions were assayed by ChIP and QPCR for HA₃-Htz1 deposition. Enrichment values are average IP/WCE ratios from triplicate samples with standard error of the mean (SEM) error bars. Vertical dashed lines are drawn through each gene’s initiation codon. x axis values are the chromosomal coordinates of the 5′ primers of each pair used.

Figure 2. High-Resolution Mapping of H2A.Z Nucleosomes

Shown is a color depiction of ratio of the ChIP signal for mononucleosomal DNA immunoprecipitated using anti-Htz1 antibodies divided by those for DNA extracted from mononucleosomes for regions covered by a high-resolution oligonucleotide tiling microarray. (A) H2A.Z enrichment in representative euchromatic regions analyzed in Figures 1C and 1D. Shown are the data for five replicate microarray hybridizations. Yellow represents a positive relative enrichment for H2A.Z over the median enrichment versus blue for negative enrichment. (B) Clustered array dataset centered on nucleosome-free regions (NFRs) of gene promoters. Shown are data from probes from up to 1 kb upstream and 1 kb downstream of the position of the NFR estimated from previous studies (Yuan et al., 2005). Each row represents a single promoter region, and columns correspond to data from microarray oligonucleotides at a given position with respect to the NFR.

Figure 3. Comparison of H2A.Z Enrichment Normalized for Nucleosome Density with Transcription Rate and RNA Polymerase II Occupancy

Shown are plots of promoter H2A.Z enrichments shown in Figure 2 versus calculated transcription rates and RNA polymerase II occupancy as determined by ChIP. (A) and (B) show plots for H2A.Z nucleosomes 3′ of the whereas (C) and (D) show plots for the nucleosomes 5′ to the NFR. R² values are shown.

Figure 4. H2A.Z Enrichment at Meiosis-Specific and a-Specific Genes

All enrichment values are triplicate averages of HA₃-Htz1 or Ac₄H4 ChIP DNA amounts normalized to the BUD3 ORF region with SEM error bars. Sidebars show BUD3 and SGF29 (positive control) loci. (A and B) HA₃-Htz1 enrichment at the DIT1/DIT2 (A) and HOP1/SPO22 (B) promoter and ORF regions. QPCR fragments are for consecutive 200 bp segments; dashed lines are drawn through gene initiation codons to their approximate relative position. (C) HA₃-Htz1 and (D) Ac₄H4 normalized enrichment at AGA2 for MATa and MATα strains. QPCR probes correspond to consecutive 100 bp segments with the position of the 5′ primer relative to the initiation codon of AGA2 denoted.

Figure 5. ChIP Analysis of H2A.Z Enrichment at Selected Euchromatic Promoters in Wild-Type, Histone Acetylation-Defective Mutants, and bdf1Δ Mutants

(A–C) Triplicate average HA₃-Htz1 enrichment ratios of HAT mutants (A), histone H4 mutants (B), and histone H3 mutants (C) compared to those of wild-type strains plotted on a log scale with SEM error bars. (D) Tetrad analysis of meiotic products of BDF1/bdf1Δ BDF2/bdf2-utrΔ heterozygous diploids. Genotypes of first column of spores are shown. Their phenotypes are representative. (E) Quadruplicate normalized average H2A.Z enrichment values for mutant compared to wild-type with SEM error bars.

Figure 6. High-Resolution Substitution Mutagenesis of the BPH1-SNT1 Intergenic Region Defines Sequences Necessary for H2A.Z Deposition In Vivo

(A) Summary of substitution mutants. Shown is the SNT1-BPH1 interval and microarray data from Figure 2 showing the position of the two H2A.Z nucleosomes that lie in the SNT1 promoter region. The regions defined as intervals 5 and 6 in Figure S7 were subjected to further mutagenesis. Shown are the sequences that were replaced with heterologous sequences (Table S7). Mutants are designated mu1–mu14. To the right are shown the normalized H2A.Z enrichments as determined by standard ChIP and QPCR. Experiments were performed in triplicate. Mean values and their SEM are displayed. (B) Detail of 3′ signal identified by substitution mutagenesis of interval 5. Shown is wild-type sequence corresponding to the right end of interval 5 (underlined in [A]). Consensus DNA binding site for Reb1 is shown; residues shown in large font are invariant (Liaw and Brandl, 1994). Adjacent dT:dA tract is indicated in blue. Shown below are right endpoints of the mu3 and mu4 mutants from (A) and their H2A.Z deposition levels. Substituted sequences are indicated by dashes. Mean values and their SEM from (A) are displayed.

Figure 7. A 22 bp Bipartite DNA Sequence from the SNT1 Promoter Sufficient to Direct the Deposition of Two H2A.Z Nucleosomes and the Formation of a Nucleosome-Free Region

(A) Experimental Design. Shown is the sequence from the SNT1 promoter and its arbitrarily chosen site of insertion in the PRM1 ORF. PCR probes used in (B) are indicated. (B) Demonstration that 22 bp element from SNT1 promoter is sufficient to promote H2A.Z deposition: standard ChIP analysis. Shown are the normalized H2A.Z enrichment values for the indicated probes for a wild-type strain and three isogenic strains containing either the 22 bp insertion shown in (A), a GGG→TAA mutant in the Reb1 site, or a mutant that precisely deletes the T tract. Experiments were performed in triplicate. Mean values and their SEM are displayed. (C) Nucleosome-scanning analysis of histone H3 positions in the PRM1 ORF in wild-type cells. Shown is the analysis of mononucleosomes immunoprecipitated using anti-H3 antibodies. The y axis corresponds to a mononucleosome:genomic ratio normalized to the median. Plotted is a four-window moving average for two replicate experiments (thin red and green lines) and their averages (thick black line). The moving average is plotted such that the first data point is relative to position 30, which is the center of the window. Indicated below are deduced positions of nucleosomes. Also marked is the site of the 22 bp insertion from the SNT1 promoter, which was placed into the middle of the +4 nucleosome. Peak-to-peak distance between the two nucleosomes flanking the site of insertion is indicated. (D) Nucleosome-scanning analysis of histone H3 positions in the PRM1 ORF in cells containing the 22 bp insertion. Strain containing the insertion was analyzed as in (C). Peak-to-peak distance between the two nucleosomes flanking the site of insertion is indicated. (E) Nucleosome-scanning analysis of histone H2A.Z positions in the PRM1 ORF in wild-type cells. Mononucleosomal material from the indicated strains was immunoprecipitated with anti-H2A.Z antibodies and analyzed as in (C). The y axis corresponds to a mononucleosome: genomic ratio. (F) Nucleosome-scanning analysis of histone H2A.Z positions in the PRM1 ORF in cells containing the 22 bp insertion. Mononucleosomal material from the indicated strains was immunoprecipitated with anti-H2A.Z antibodies and analyzed as in (E). The y axis is the same as in (C). Peak-to-peak distance between the two nucleosomes flanking the site of insertion is indicated.