H2A.Z is required for global chromatin integrity and for recruitment of RNA polymerase II under specific conditions

Abstract

Evolutionarily conserved variant histone H2A.Z has been recently shown to regulate gene transcription in Saccharomyces cerevisiae. Here we show that loss of H2A.Z in this organism negatively affects the induction of GAL genes. Importantly, fusion of the H2A.Z C-terminal region to S phase H2A without its corresponding C-terminal region can mediate the variant histone's specialized function in GAL1-10 gene induction, and it restores the slow-growth phenotype of cells with a deletion of HTZ1. Furthermore, we show that the C-terminal region of H2A.Z can interact with some components of the transcriptional apparatus. In cells lacking H2A.Z, recruitment of RNA polymerase II and TATA-binding protein to the GAL1-10 promoters is significantly diminished under inducing conditions. Unexpectedly, we also find that H2A.Z is required to globally maintain chromatin integrity under GAL gene-inducing conditions. We hypothesize that H2A.Z can positively regulate gene transcription, at least in part, by modulating interactions with RNA polymerase II-associated factors at certain genes under specific cell growth conditions.

FIG. 1

Deletion of HTZ1 confers slow growth, Gal⁻ phenotypes, and reduced GAL1-10 induction in yeast. (A) htz1Δ cells have a Gal⁻ phenotype. W303α, htz1Δ cells (MAY424), or htz1Δ cells containing a plasmid expressing a wild-type allele of HTZ1 were serially diluted by a factor of 10 on SD media containing either glucose (Glu) or galactose (Gal) as a sole carbon source. Cells were incubated for approximately 2 to 3 days on glucose plates and approximately 4 to 6 days on galactose plates. (B) H2A.Z is required for proper induction of a GAL1::lacZ reporter gene. The strains used in this experiment (W303 and MAY424) contain an integrated reporter template bearing the GAL1 UAS_G upstream of the GAL1 promoter fused to lacZ. Cells were grown in minimal media with either glucose (Glu) or galactose/raffinose (Gal), and β-galactosidase assays (9) were carried out to measure the extent of gene induction. (C) Ability of htz1Δ cells to induce the GAL1-10 genes. Primer extension analyses were carried out with purified RNA from wild-type (WT) and htz1Δ cells. Yeast cells were grown in raffinose (R), and then galactose (G) to a final concentration of 5% was added to one-half of the culture volume for 6 h in order to induce GAL gene expression.

FIG. 2

ChIP analysis of the binding of Gal4 to the GC-rich region of the GAL1-10 locus. The binding of Gal4 to the GC-rich region and to the GAL1 open reading frame over time after addition of galactose is shown for both the wild-type (WT) and the htz1Δ strains.

FIG. 3

The H2A.Z C-terminal region is sufficient to mediate the special function of the variant histone. (A) Drawing of H2A-H2A.Z chimeras used in these experiments. (B) The AZ fusion (H2A [aa 1 to 90]-H2A.Z [aa 98 to 134]) is sufficient to complement the Gal⁻ phenotype of htz1Δ cells. All histone derivatives (H2A.Z, H2A, AZ, and ZA; see text for description) are expressed from the ACT1 (β-actin) promoter on ARS-CEN plasmids and introduced into htz1Δ cells (MAY424). The growth assay was performed as described in the legend to Fig. 1A. (C) The AZ fusion (H2A [aa 1 to 90]-H2A.Z [aa 98 to 134]) is sufficient to fully activate the GAL1-10 genes. The H2A-H2A.Z fusions were assayed by primer extension analyses as for Fig. 2. (D) Histone protein levels were determined by immunoblotting with an anti-HA antibody.

FIG. 4

The C-terminal region of H2A.Z interacts with components of the transcriptional machinery. (A) Aligned amino acid sequences of yeast H2A.Z and H2A using BLAST (National Center for Biotechnology Information). Boxed areas represent the C-terminal regions that were fused to GST for the experiments illustrated in panels B and C (red and white) and the M6 region in Drosophila H2A.Z required for viability (yellow) (4). (B) GST, GST-H2A (aa 96 to 132), and GST-Z (GST-H2A.Z [aa 103 to 134]) proteins bound to glutathione-Sepharose beads were incubated with a chromatin-enriched yeast extract. L, 2% input of the mixture; S, 2% of the supernatant after pelleting the Sepharose beads; P, washed Sepharose pellet. Samples were analyzed by SDS-PAGE followed by immunoblotting with either an anti-RNA polII antibody or an anti-TBP antibody. (C) The H2A.Z-RNA polII interaction is not mediated by the indirect bridging effect of nucleic acids. The chromatin-enriched extract was treated with or without DNase and RNase and then loaded in a 500-μl glutathione-Sepharose column coupled to GST-H2A.Z (aa 103 to 134). The column was washed and eluted with potassium acetate. L, input of the total reaction; E1 and E2, elutions. Sup-40K is an extract not enriched in chromatin; Pel-40K −DNase is a chromatin-enriched extract not treated with nucleases; Pel-40K +DNase represents the chromatin-enriched extract treated with nucleases. Samples were analyzed as for panel B with an anti-RNA polII antibody.

FIG. 5

Effect of a htz1Δ mutation on recruitment of the transcriptional machinery to the GAL1-10 locus after galactose induction. (A) Representation of the GAL1-10 locus. GAL1 and GAL10 TATA boxes (TATA), transcriptional initiation sites (arrows with +1), and partial open reading frames are represented. The four Gal4 UASs (UAS_G) are shown by black crossbars. Circles, positioned nucleosomes covering both GAL1 and GAL10 promoters; stippling, remodeled nucleosomes during galactose induction (24); black bars, regions amplified by PCR in the ChIP experiments shown in panels B, C, and E. (B) Linear PCR amplification of DNA. (C) ChIP analysis of the binding of Rpb1 to the GAL1 and GAL10 promoters. The relative binding of Rpb1 over time after addition of galactose is shown for both wild-type (WT) and htz1Δ strains. ARN1 is used here as an internal control to normalize signals from each lane. (D) Binding of Rpb1 to the GAL1 and GAL10 promoters. Quantification of the experiment illustrated in panel C is shown. (E) ChIP analysis of the binding of TBP to the GAL1 and GAL10 promoters. The procedure was the same as for panel C except that the immunoprecipitation was carried out with an anti-TBP antibody. (F) Binding of TBP to the GAL1 and GAL10 promoters. Quantification of the experiment illustrated in panel E is shown.

FIG. 6

DNA binding of H2A.Z in vivo. (A) ChIP analysis of various loci with an anti-Myc antibody on strains expressing either Myc-H2A or Myc-H2A.Z fusion proteins as well as a nontagged strain. Shown are the PHO84, YJL100W, and SSB2 open reading frames, as well as the ACT1 and YHB1 promoters. (B) ChIP analysis of the binding of a Myc-tagged version of H2A.Z to the GAL1-10 promoters after induction by galactose. Lanes 1 to 4, linear PCR amplification of DNA (input DNA); lanes 5 to 10, binding of Myc-H2A.Z to the GAL1-10 promoters and the ARN1 promoter over time after addition of galactose.

FIG. 7

Global chromatin analyses of htz1Δ yeast cells. (A) htz1Δ cells have an increased sensitivity to MNase in the presence of galactose and raffinose but not raffinose alone. Yeast nuclei were digested with 25 U of MNase per ml for increasing amounts of time (up to 20 min as indicated). Chromatin DNA was then analyzed by agarose gel electrophoresis. (B) Plot of band intensities (from top to bottom) showing the relative differences in nuclease sensitivity of wild-type (WT) and htz1Δ cells. Bands were scanned from 5- and 10-min digests of WT and htz1Δ cells grown in either raffinose (Raf) or raffinose and galactose (Raf/Gal). (C) Adding glucose to nuclei prepared from galactose- and raffinose-grown htz1Δ cells restores the altered chromatin state. Lane 1, molecular weight marker; lanes 2 to 7, MNase digests (0, 1, 3, 5, 8, and 12 U/ml digested for 20 min) of nuclei prepared from cells grown in raffinose and galactose. In this part of the experiment, raffinose and galactose were added to the nucleus preparation buffers. Lanes 8 to 13, the same MNase digestions from cells also grown in raffinose and galactose but with the addition of glucose to the nuclei at the time of their preparation. Lanes 14 to 19, the same MNase digestions from cells grown in raffinose-galactose but with the addition of glucose 90 min prior to nucleus preparation and throughout their preparation. Samples were analyzed by agarose gel electrophoresis.