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.
Highlights
Centromeres are specialized regions of eukaryotic chromosomes that direct the assembly of kinetochores and are essential for the equal segregation of chromosomes during mitosis and meiosis [1,2,3]
We characterized the function of the JmjC domain protein Msc1 in fission yeast, which is required for proper chromosome segregation [14]
We found that Msc1 is an integral component of the Swr1 complex and that both Msc1 and Swr1 are required for the preferential incorporation of H2A.ZPht1 at gene promoters
Summary
Fission Yeast Strains—Msc1-FLAG, Swr1-FLAG, Swr1-Myc, Pht1-Myc, Cnp1-FLAG, Cnp1-GFP, Cnp3-Myc, swr1⌬, pht1⌬, and cnp3⌬ strains were constructed using a PCR-based module method [35]. Chromosome Segregation Assay—Assaying the rate of chromosome loss was performed as described previously [36]. If chromosome loss occurs in the first division, half of the resultant colony carrying Ch16 will be white, whereas the half without Ch16 will be red. The number of half-sectored red/white colonies was determined, and the rate of chromosome loss per cell division was calculated by dividing the number of half-sectored colonies by the total number of white colonies plus half-sectored colonies. Protein Purification and Mass Spectrometry Analysis—Affinity purification of Msc1-FLAG and Swr1-FLAG complexes and MudPIT mass spectrometry analysis were performed as described previously [37]. Western Blots and Antibodies—Protein extracts were prepared by lysis of cells with glass beads, followed by sonication to dissolve chromatin [37]. Chromatin Immunoprecipitation—ChIP analysis was performed as described previously [36].
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