Histone variant H2A.Z regulates centromere silencing and chromosome segregation in fission yeast

Abstract

The incorporation of histone variant H2A.Z into nucleosomes plays essential roles in regulating chromatin structure and gene expression. A multisubunit complex containing chromatin remodeling protein Swr1 is responsible for the deposition of H2A.Z in budding yeast and mammals. Here, we show that the JmjC domain protein Msc1 is a novel component of the fission yeast Swr1 complex and is required for Swr1-mediated incorporation of H2A.Z into nucleosomes at gene promoters. Loss of Msc1, Swr1, or H2A.Z results in loss of silencing at centromeres and defective chromosome segregation, although centromeric levels of CENP-A, a centromere-specific histone H3 variant that is required for setting up the chromatin structure at centromeres, remain unchanged. Intriguingly, H2A.Z is required for the expression of another centromere protein, CENP-C, and overexpression of CENP-C rescues centromere silencing defects associated with H2A.Z loss. These results demonstrate the importance of H2A.Z and CENP-C in maintaining a silenced chromatin state at centromeres.

FIGURE 1.

Msc1 is an integral component of the fission yeast Swr1 complex. A, Msc1-FLAG is functional. The loss rates of a nonessential chromosome (Ch16) in the indicated strains were measured. wt, wild type. B, cell extract prepared from an Msc1-FLAG strain was immunoprecipitated with anti-FLAG resin and subjected to Western blot analysis with anti-FLAG antibody. C, cell extract prepared from an Msc1-FLAG strain was used to perform affinity purification with anti-FLAG resin, resolved by SDS-PAGE, and silver-stained. D, shown are the results from MudPIT mass spectrometry analysis of proteins associated with Msc1-FLAG. The numbers of peptides, as well as the percentages of each protein these peptides cover, are indicated. The corresponding proteins of the S. cerevisiae Swr1 complex were also shown for comparison. E, protein extracts prepared from the indicated strains were immunoprecipitated (IP) with anti-FLAG resin and analyzed by Western blot analysis with anti-FLAG and anti-Myc antibodies. F, an affinity-purified complex containing Msc1-FLAG and Swr1-Myc was subjected gel filtration analysis with a Superose 6 column. Fractions were trichloroacetic acid-precipitated and subjected to Western blot analysis with anti-FLAG and anti-Myc antibodies. The elution volumes of protein standards (in kilodaltons) are also indicated.

FIGURE 2.

Msc1 is not required for the integrity of the Swr1 complex. A, cell extract prepared from an Swr1-FLAG strain was immunoprecipitated with anti-FLAG resin and subjected to Western blot analysis with anti-FLAG antibody. B, extracts prepared from Swr1-FLAG strains in the presence or absence of Msc1 were used to perform affinity purification with anti-FLAG resin, resolved by SDS-PAGE, and silver-stained. C, shown are the results from mass spectrometry analysis of proteins associated with Swr1-FLAG. The numbers of peptides, as well as the percentages of each protein these peptides cover, are indicated.

FIGURE 3.

Msc1 and Swr1 are required for H2A.Z^Pht1 incorporation into chromatin. A, scatter plots of Pht1-Myc ChIP-chip analysis. Log values of Pht1 ChIP and WCE signals for each probe were plotted against each other. wt, wild type. B, H2A.Z^Pht1 is enriched at gene promoters. The 42,708 probes are divided into three categories: open reading frame (ORF; coding region), promoter (within 500 bp upstream of the coding region), and intergenic (noncoding regions that do not belong to the promoter category). Pht1-enriched represents the top 5% (2,134) of probes that are enriched for Pht1, all of which have enrichment values of >3-fold. C, H2A.Z^Pht1 is enriched at gene promoters. Left panel, example of H2A.Z^Pht1 ChIP-chip analysis across a 10-kb region that includes vid21⁺. Gene coding regions are indicated on top, and the positions of PCR fragments used in the calculation are shown as short bars. Middle panel, quantification of H2A.Z^Pht1 enrichment at vid21⁺ by real-time PCR. Right panels, quantification of H2A.Z^Pht1 enrichment at the vid21⁺ promoter by competitive PCR. -Fold enrichment is shown below each lane. D, Msc1 is required for targeting of Swr1 to the vid21⁺ promoter. Quantification by both real-time PCR and competitive PCR is shown. E, Msc1 does not affect Swr1 protein levels. Cell extracts prepared from the indicated strains were subjected to Western blot analysis with anti-Myc antibody.

FIGURE 4.

Msc1, Swr1, and H2A.Z^Pht1 are required for proper chromosome segregation. A, Msc1 and Swr1 are required for the maintenance of a nonessential minichromosome. wt, wild type. B, shown is a diagram of the fission yeast centromere 1. The insertion site of the cnt1::ura4⁺ reporter gene is indicated. C, serial dilution plating analysis of the indicated strains were performed to measure the expression of ura4⁺. FOA, 5-fluoroorotic acid; N/S, non-selective medium. D, RT-PCR analyses were performed to measure the RNA levels of ura4⁺ and act1⁺. −RT indicates the reverse transcription step was omitted. E, ChIP analysis was performed to measure the levels of Cnp1-FLAG at centromere regions. -Fold enrichment is shown below each lane. F, shown are the results from live cell imaging analysis of the indicated yeast strains expressing Cnp1-GFP.

FIGURE 5.

H2A.Z^Pht1 regulates CENP-C^Cnp3 expression. A, ChIP-chip analysis of H2A.Z^Pht1 enrichment at the centromere of chromosome 2 (cnt2) and a 10-kb region that includes cnp3⁺. wt, wild type. B, ChIP analysis of H2A.Z^Pht1 at centromeres (cnt) and the cnp3 promoter. C, RT-PCR analysis of Cnp3 mRNA levels. D, CENP-C^Cnp3 is required for silencing at centromeres. RT-PCR analysis were performed to measure the expression of cnt1::ura4⁺.

FIGURE 6.

PHDs are required for Msc1 function. A, shown is the domain architecture of Msc1. Zf, zinc finger. B, shown is sequence alignment of the JmjC domains of the JARID1 family proteins. Arrows indicate amino acid residues mutated in Msc1. Asterisks represent residues that are essential for histone demethylase activity (17, 60). sp, S. pombe; hs, Homo sapiens; dm, Drosophila melanogaster. C, cell extracts prepared from the indicated strains were subjected to Western blot analysis with anti-Myc antibody. Ponceau S staining of part of the membrane is shown below as a control for loading. wt, wild type. D, shown is the maintenance of a nonessential minichromosome in Msc1 mutants. E, serial dilution plating analysis of the indicated strains was performed to measure the expression of cnt1::ura4⁺. FOA, 5-fluoroorotic acid; N/S, non-selective medium.