Interaction of APC/C-E3 Ligase with Swi6/HP1 and Clr4/Suv39 in Heterochromatin Assembly in Fission Yeast*♦
- Rudra Narayan Dubey12,
- Nandni Nakwal13,
- Kamlesh Kumar Bisht3,
- Ashok Saini3,
- Swati Haldar3 and
- Jagmohan Singh4
- 4 To whom correspondence should be addressed. Tel.: 91-172-2636680, Ext. 3228; Fax: 91-172-2690585, 2690632; E-mail: jag{at}imtech.res.in.
Abstract
Heterochromatin assembly in fission yeast is initiated by binding of Swi6/HP1 to the Lys-9-dimethylated H3 followed by spreading via cooperative recruitment of Swi6/HP1. Recruitment of Cohesin by Swi6/HP1 further stabilizes the heterochromatin structure and integrity. Subsequently, polyubiquitylation of Cut2 by anaphase-promoting complex-cyclosome (APC/C)-ubiquitin-protein isopeptide ligase (E3 ligase) followed by degradation of Cut2 releases Cut1, which cleaves the Rad21 subunit of Cohesin, facilitating sister chromatid separation during mitosis. Here, we demonstrate a surprising role of APC/C in assembly of heterochromatin and silencing at mating type, centromere, and ribosomal DNA loci. Coincidentally with the loss of silencing, recruitment of Swi6, H3-Lys-9-Me2, and Clr4 at dg-dh repeats at cen1 and the K region of mat locus is abrogated in mutants cut4, cut9, and nuc2. Surprisingly, both Cut4 and Cut9 are also highly enriched at these regions in wild type and depleted in swi6Δ mutant. Cut4 and Cut9 interact directly with Swi6/HP1 and Clr4, whereas the mutant Cut4 does not, suggesting that a direct physical interaction of APC subunits Cut4 and Cut9 with Swi6 and Clr4 is instrumental in heterochromatin assembly. The silencing defect in APC mutants is causally related to ubiquitylation activity of APC-E3 ligase. Like swi6 mutant, APC mutants are also defective in Cohesin recruitment and exhibit defects like lagging chromosomes, chromosome loss, and aberrant recombination in the mat region. In addition, APC mutants exhibit a bidirectional expression of dh repeats, suggesting a role in the RNA interference pathway. Thus, APC and heterochromatin proteins Swi6 and Clr4 play a mutually cooperative role in heterochromatin assembly, thereby ensuring chromosomal integrity, inheritance, and segregation during mitosis and meiosis.
Footnotes
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↵5 The abbreviations used are: rDNA, ribosomal DNA; APC/C, anaphase-promoting complex-cyclosome; E3, ubiquitin-protein isopeptide ligase; Ub, ubiquitin; Swi, switching; Clr, cryptic loci regulator; HP1, heterochromatin protein 1; RE, repression element; DAPI, 4,6-diamidino-2-phenylindole; Sng, silencing not governed; Cut, chromosomes untimely torn; Cdc, cell division control; DSB, double strand break; pol, polymerase; RNAi, RNA interference; HA, hemagglutinin; ts, temperature-sensitive; FOA, fluoroorotic acid; PMA, phorbol 12-myristate 13-acetate; IP, immunoprecipitation; ChIP, chromatin IP; Ni-NTA, nickel-nitrilotriacetic acid; RT-PCR, reverse transcription-PCR; WT, wild type; YE, yeast extract.
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↵6 A. Saini and J. Singh, unpublished results.
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↵* This work was supported by a grant from the Department of Science and Technology, New Delhi. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The on-line version of this article (available at http://www.jbc.org) contains a supplemental table.
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↵♦ This article was selected as a Paper of the Week.
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↵1 These authors contributed equally to the work.
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↵2 Present Address: Dept. of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854.
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↵3 Supported by the Council for Scientific and Industrial Research senior research fellowships.
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- Received August 20, 2008.
- Revision received December 22, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
