H2A.Z has a function reminiscent of an activator required for preferential binding to intergenic DNA

Abstract

H2A.Z has been shown to regulate transcription in yeast, and that function resides in its C-terminal region as the reciprocal portion of H2A cannot substitute for the latter. We show that fusion of a transcriptional activating region to the C-terminal region of H2A, which is substituted for that of H2A.Z, can allow the chimera to fulfil the special role of H2A.Z in positive gene regulation, as well as complement growth deficiencies of htz1delta cells. We further show that the 'transcription' function of H2A.Z is linked to its ability to preferentially localize to certain intergenic DNA regions. Our results suggest that H2A.Z modulates functional interactions with transcription regulatory components, and thus increases its localization to promoters where it helps poise chromatin for gene activation.

Fig. 1. Structure and expression of variant histone protein chimeras used in this study. (A) Schematic representation of the chimeras. The location of the HA tag is shown as a gray bar. (B) Immunoblot analysis of H2A.Z chimeras. Histone protein levels were determined by immunoblotting with an anti-HA antibody.

Fig. 2. Fusion of a transcriptional activating region to ZA is sufficient to restore GAL1 and GAL7 transcription, as well as the HU-sensitivity phenotype of htz1Δ yeast cells. (A) Ability of H2A.Z derivatives to induce GAL1 and GAL7 expression in htz1Δ cells. Primer extension analyses of GAL1 and GAL7 expression upon galactose induction. The 25S rRNA is shown at the bottom of the figure as a loading control. (B) Ability of variant H2A.Z derivatives to complement htz1Δ- mediated HU growth deficiency. Ten-fold serial dilutions of yeast cell suspensions were dropped on YPDA plates supplemented or not with either 100 or 200 mM HU. Plates were incubated at 30°C for 2–5 days. (C) Overexpression of Gal80 in Δhtz1 cells expressing ZA-rII′. Δhtz1 cells containing the indicated chimeras were transformed with either an empty vector or a Gal80 expression vector under the control of the ACT1 promoter on a 2µ plasmid. Serial dilutions of cells were spotted on plates without or with HU and grown for 3–6 days at 30°C.

Fig. 3. Effect of H2A.Z derivatives on PUR5 and PHO5 transcription. (A) Growth of cells in the absence or presence of 6-AU. Cells were grown and then spotted onto plates containing no 6-AU, 10 µg/ml 6-AU or 25 µg/ml 6-AU. (B) Primer extension analysis of the PUR5 gene in the presence of 6-AU. Induction time in the presence of 6-AU is shown in minutes. The 25S rRNA is shown at the bottom of the figure as a loading control. (C) Transcription of the PUR5 gene in the presence of HU. Primer extension analysis of the PUR5 gene after yeast cells were grown and incubated or not for 3.5 h with 200 mM HU. The 25S rRNA is shown at the bottom of the figure as a loading control. (D) Transcription of the PHO5 gene. Primer extension analyses of the PHO5 locus after yeast cells were grown in the presence (+) or absence (–) of phosphate (Pi). The 25S rRNA is shown at the bottom of the figure as a loading control.

Fig. 4. Binding of H2A.Z derivatives at the PUR5 gene. ChIP experiments were performed using anti-HA antibodies to monitor the binding of H2A.Z, ZA and ZA-rII′ to PUR5 under repressed (–6-AU) or induced (+6-AU) conditions. All PCRs contain ARN1 primers used here as an internal control to normalize signals for each lane. (A) Top: representation of the PUR5 locus. Transcription start site (arrows with +1), open reading frame (open shaded rectangle) and regions amplified by PCR (black bars) are represented. Bottom: PCR titration of input (lanes 1–4 and 9–12) and immunoprecipitated material (lanes 5–8 and 13–16) for H2A.Z (lanes 1–8) and ZA (lanes 9–16) at the PUR5 promoter. (B) ChIP analysis of the binding of H2A.Z derivatives at the PUR5 promoter. Binding of the derivatives under repressed (–6-AU, lanes 1–6) and induced (+6-AU, lanes 7–14) conditions (left panel). The right part of the figure depicts a quantification of that experiment. Binding of each H2A.Z derivative under repressed conditions is normalized to 100%. (C) Same experiment as in (B) with the exception that the PUR5 region analyzed was in the open reading frame.

Fig. 5. Localization of H2A.Z, ZA and H2A at the PUR5 locus. (A) Top: representation of the PUR5 locus. Regions amplified by PCR in the promoter region (P-A and P-B) or in the open reading frame (O-A to O-D) are represented. Bottom: ChIP analysis of the binding of H2A.Z and ZA at the PUR5 locus. All PCRs contain ARN1 primers used here as an internal control to normalize signals for each lane. (B and C) Distribution of H2A.Z and ZA at the PUR5 locus. Quantification of (B) H2A.Z or (C) ZA under repressed (–6-AU, open bar) and induced (+6-AU, black bar) conditions. (D) ChIP analysis of the binding of Myc-H2A at the PUR5 locus. Cells were grown under repressed condition and chromatin was immunoprecipitated with (+) or without (–) an anti-Myc antibody. (E) Distribution of H2A over the PUR5 locus.

Fig. 6. Distribution of H2A.Z and ZA at the PHO5 genomic region. (A) Top: representation of a 4.3 kb genomic region spanning the PHO5 gene. The transcription start sites (arrows with +1), open reading frames (open shaded rectangle) and regions amplified by PCR (black bars) are represented. Bottom: ChIP analysis of H2A.Z and ZA binding over the PHO5 genomic region. Cells were grown under PHO5 repression conditions. All PCRs contain ARN1 primers used here as an internal control to normalize signals for each lane. (B and C) Distribution of (B) H2A.Z and (C) ZA over the PHO5 genomic region.

Fig. 7. Distribution of the ZA-rII′ fusion over the PUR5 and PHO5 genomic regions. (A) Binding of ZA-rII′ at PUR5 gene under repressed conditions. (B) Distribution of ZA-rII′ at the PUR5 gene. (C) Same experiment as in (A) but the PHO5 genomic region was analyzed. (D) Distribution of ZA-rII′ at the PHO5 genomic region. All PCRs contain ARN1 primers used as an internal control to normalize signals for each lane.