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. 2011 Jan 15;10(2):293-300.
doi: 10.4161/cc.10.2.14536. Epub 2011 Jan 15.

γH2A is a component of yeast heterochromatin required for telomere elongation

Tasuku Kitada  1 , Thomas Schleker, Adam S Sperling, Wei Xie, Susan M Gasser, Michael Grunstein
Affiliations

γH2A is a component of yeast heterochromatin required for telomere elongation

Tasuku Kitada et al. Cell Cycle. .

Abstract

Histones of heterochromatin are deacetylated in yeast and methylated in more complex eukaryotes to regulate heterochromatin structure and gene silencing. Here, we report that histone H2A phosphorylated at serine 129 (γH2A) in Saccharomyces cerevisiae is a conceptually new type of heterochromatin modification that functions downstream of silent chromatin assembly. We show that γH2A is enriched throughout yeast telomeric and silent mating locus (HM) heterochromatin where γH2A results from the action of kinases Tel1 and Mec1. Interestingly, mutation of γH2A has no apparent effect on the binding of Sir (silent information regulator) complex or on gene silencing. In contrast, deletion of SIR3 abolishes the formation of γH2A at heterochromatin. To address the function of γH2A, we used a Δrif1 mutant strain in which telomeres are excessively elongated to show that γH2A is required for the optimal recruitment of Cdc13, a regulator of telomere elongation, and for telomere elongation itself. Thus, a histone modification that parallels Sir3 protein binding is shown here to be dispensable for the formation of a silent structure but is important for a crucial heterochromatin-specific downstream function in telomere homeostasis.

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Figures

Figure 1
Figure 1
γH2A overlaps with Sir3 throughout heterochromatin. (A) γH2A distribution of a representative chromosome (Chromosome III). γH2A level was obtained by ChIP-chip of asynchronously growing wild-type H2A cells (YDS2). γH2A level was smoothed (500 bp intervals) and shown in red bars for signals above a certain threshold (one standard deviation above the genome-wide average level of γH2A) and in light grey bars for signals below it. The positions of telomeres (grey text), ARSs (orange text/rectangles), centromere (green text/oval) and pseudogenes and ORFs (blue rectangles) are also depicted. (B and C) γH2A level and Sir3 binding at four representative telomeres (TEL03L, TEL06R, TEL11R and TEL15L; 12 kb region from the telomeric ends) and the silent mating type loci (HML, HMR; 12 kb regions). γH2A level [obtained as explained in (A)] and Sir3 level from ChIP-chip of asynchronously growing wild-type H2A cells (RMY200) are compared. The smoothed (200 bp interval) γH2A level is indicated in red and smoothed Sir3 binding in blue.
Figure 2
Figure 2
Telomeric γH2A is dependent on the Sir complex and the PI-3-kinase-related kinases Tel1 and Mec1. (A) Schematic representation of the genes at the subtelomere of TEL06R and the positions of the probes used for PCR. (B and C) ChIP of Sir3 (B) and Sir4 (C) at TEL06R in a wild-type H2A strain (TKY307) and an hta1S129A mutant strain (TKY308). Binding levels were first normalised to input DNA and further to an internal control region (ACT1). (D) ChIP of γH2A normalized to H2A level at TEL06R in a wild-type Sir3 strain (YDS2) and a sir3Δ strain (ASY110). The level of γH2A and H2A were first normalized to input DNA and further to an internal control region (ACT1). Then γH2A level was normalized by H2A level. (E) ChIP of γH2A at TEL6R in wild-type (Y300), tel1Δ (TKY301), mec1-21 (Y604) and tel1Δ mec1-21 (TKY605) strains. The level of γH2A was normalized to input DNA.
Figure 3
Figure 3
(A) γH2A level at TEL06R was assessed by ChIP throughout the cell cycle. bar1Δ (MMY001) cells were synchronized in G1 phase by α-factor arrest and released into YPD media containing protease by filtration. Samples were taken every 20 min. PCR probes are as described in Figure 2A and γH2A levels were normalized to input DNA. A representative result of three independent experiments is shown. Cell cycle phases were assessed by determining the binding of RNAP II on cell cycle regulated genes by ChIP. (B) γH2A level at TEL06R was assessed by ChIP in G1, S and M phases by cell synchronization. bar1Δ (MMY001) cells were synchronized in G1, S and M phases by treatment with α-factor, hydroxyurea (HU) and nocodazole, respectively. Quantitation was as described in (A).
Figure 4
Figure 4
γH2A is important for telomere elongation. (A) Schematic representation of the subelement structure of a telomere. All telomeres contain X elements and they may be followed by up to four repeats of Y′ elements. The core X element is followed by subtelomeric repeats (STRs) and TG1–3 repeats exist in between X elements and Y′ elements and also at the very end of the telomere. The XhoI restriction enzyme site is located approximately 850 bp away from the terminal TG1–3 repeats within Y′ elements. (B) Southern blot analysis showing the length of TG1–3 repeats. Genomic DNA isolated from strains HTA1 (TKY307), hta1S129A (TKY308), hta1S129E (TKY309), HTA1 yku70Δ (TKY507), hta1S129A yku70Δ (TKY508), hta1S129E yku70Δ (TKY509), HTA1 rif1Δ (TKY517), hta1S129A rif1Δ (TKY518), hta1S129E rif1Δ (TKY519), HTA1 rif2Δ (TKY527), hta1S129A rif2Δ (TKY528), hta1S129E rif2Δ (TKY529) were digested with XhoI, run on a agarose gel, transferred to a nylon membrane and probed with a radioactive TG repeat probe (A). A representative result of five experiments is shown. (C) The average telomere repeat lengths of Y′ elements in hta1S129 mutant strains were quantified from (B) and are shown relative to wild-type H2A strains. (D and E) ChIP of Cdc13-myc (D) and yKu80-myc (E) at TEL06R in an HTA1 rif1Δ strain (TKY547B for Cdc13-myc, TKY577B for yKu80-myc) and an hta1S129A rif1Δ mutant strain (TKY548B for Cdc13-myc, TKY578B for yKu80-myc).
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References

    1. Gottschling DE, Aparicio OM, Billington BL, Zakian VA. Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell. 1990;63:751–762. - PubMed
    1. Chang CR, Wu CS, Hom Y, Gartenberg MR. Targeting of cohesin by transcriptionally silent chromatin. Genes Dev. 2005;19:3031–3042. - PMC - PubMed
    1. Taddei A, Hediger F, Neumann FR, Gasser SM. The function of nuclear architecture: a genetic approach. Annu Rev Genet. 2004;38:305–345. - PubMed
    1. Taddei A, Gasser SM. Repairing subtelomeric DSBs at the nuclear periphery. Trends Cell Biol. 2006;16:225–228. - PubMed
    1. Rusche LN, Kirchmaier AL, Rine J. The establishment, inheritance and function of silenced chromatin in Saccharomyces cerevisiae. Annu Rev Biochem. 2003;72:481–516. - PubMed

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