Fission yeast Arp6 is required for telomere silencing, but functions independently of Swi6 - PubMed (original) (raw)

Fission yeast Arp6 is required for telomere silencing, but functions independently of Swi6

Masaru Ueno et al. Nucleic Acids Res. 2004.

Abstract

The actin-related proteins (Arps), which are subdivided into at least eight subfamilies, are conserved from yeast to humans. A member of the Arp6 subfamily in Drosophila, Arp4/Arp6, co-localizes with heterochromatin protein 1 (HP1) in pericentric heterochromatin. Fission yeast Schizosaccharomyces pombe possesses both an HP1 homolog and an Arp6 homolog. However, the function of S.pombe Arp6 has not been characterized yet. We found that deletion of arp6(+) impaired telomere silencing, but did not affect centromere silencing. Chromatin immunoprecipitation assays revealed that Arp6 bound to the telomere region. However, unlike Drosophila Arp4/Arp6, S.pombe Arp6 was distributed throughout nuclei. The binding of Arp6 to telomere DNA was not affected by deletion of swi6(+). Moreover, the binding of Swi6 to telomere ends was not affected by deletion of arp6(+). These results suggest that Arp6 and Swi6 function independently at telomere ends. We propose that the Arp6-mediated repression mechanism works side by side with Swi6-based telomere silencing in S.pombe.

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Figures

Figure 1

Figure 1

Deletion of arp6+ affects telomere silencing, but not centromere silencing. (A) Serial dilution assay on no histidine (SD-histidine) and non-selective (YPAD) plates. Telomere silencing in wild-type (FY1862), _arp6_Δ (TM001) and _taz1_Δ (KT21-UHA) cells was studied. (B) Quantitative RT–PCR was performed on RNA prepared from wild-type (FY1862) and _arp6_Δ cells (TM001) using primers to amplify His3 mRNA and to amplify Eno1 mRNA as a control. The RT–PCR products, His3 cDNA (his3) and Eno1 cDNA (eno1), separated by agarose gel are shown. (C) Serial dilution assay on low adenine and non-selective (YPAD) plates. Centromere silencing in wild-type cells (FY1862), _arp6_Δ (TM001) and _swi6_Δ (TK017) was studied.

Figure 2

Figure 2

Arp6-Myc is bound to telomere DNA in ChIP assay. Untagged wild-type control (JY746), Arp6-Myc (TM002), Arp6-Myc _swi6_Δ (TM004) and Arp6-Myc _taz1_Δ (TM005) cells were used. PCR was performed on whole-cell extracts (WCE) and on chromatin immunoprecipitates (IP: anti-Myc) using primers to amplify telomere DNA (telomere) and primers to amplify DNA from the eno1+ gene (eno1).

Figure 3

Figure 3

Arp6 is dispensable for localization of Swi6 to heterochromatin. (A) The binding of Swi6 to telomeres in _arp6_Δ cells was studied by the ChIP assay. Wild-type (JY746), _swi6_Δ (PY614) and _arp6_Δ (TM001) cells were used. PCR was performed on whole-cell extracts (WCE) and on chromatin immunoprecipitates (IP: anti-Swi6) using primers to amplify telomere DNA (telomere) and primers to amplify DNA from the eno1+ gene (eno1). (B) H3 Lys9 methylation at subtelomere region (∼2 kb away from telomere ends) was studied by the ChIP assay. Wild-type (JY746) and _arp6_Δ (TM001) cells were used. PCR was performed on whole-cell extracts (WCE) and on chromatin immunoprecipitates (with anti-dimethyl-histone H3-K9 antibody) (IP: anti-K9-Me) using primers to amplify subtelomere DNA (subtelomere) and primers to amplify DNA from the act1+ gene (act1). For control experiment (IP: –), extract of wild-type cells was immunoprecipitated without anti-dimethyl-histone H3-K9 antibody. (C) Localization of Swi6-GFP to silenced chromosomal locations was not affected by deletion of arp6+. Cells expressing swi6+-GFP in the wild-type strain (PY612) or in _arp6_Δ cells (TM006) were fixed with methanol and stained with DAPI. The positions of Swi6-GFP are indicated by arrows.

Figure 4

Figure 4

Schizosaccharomyces pombe Arp6 is distributed throughout nuclei. Indirect immunofluorescence microscopy of Arp6-Myc (wt, TM002) and Arp6-Myc _swi6_Δ cells (_swi6_Δ, TM004). Anti-Myc antibody was used to detect Myc-tagged Arp6.

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