Interaction of APC/C-E3 Ligase with Swi6/HP1 and Clr4/Suv39 in Heterochromatin Assembly in Fission Yeast

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 complexcyclosome (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 swi6⌬ 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.

Role of Swi6/HP1 Self-association-mediated Recruitment of Clr4/Suv39 in Establishment and Maintenance of Heterochromatin in Fission Yeast

“…Further, the swi6 -115 mutant does express a drastically reduced albeit detectable level of Swi6 (23), whereas the swi6⌬ mutant does not. Thus, earlier results may have had a contribution from the residual Swi6 in initiating the heterochromatin mark by recruiting Clr4, whereas the lack of Swi6 in the present study and our previous study (26) implies a complete lack of Clr4 recruitment in the swi6⌬ mutant, leading to a defect in both establishment and maintenance of heterochromatin.…”

Involvement of Telomerase Reverse Transcriptase in Heterochromatin Maintenance

“…Prior work has demonstrated that the maintenance of centromeric heterochromatin is necessary for proper chromosomal segregation during mitotic progression in both S. pombe (28) and human cells (29), and regulation of expression of alphoid transcripts is required for mitosis (49). Since both centromeric alphoid regions and transposable elements are maintained in a heterochromatic state during mitosis (Fig.…”

“…In S. pombe (28) and C. elegans (5,6), the recruitment of RITS to heterochromatic regions requires the synthesis of dsRNAs and processing to siRNAs. To assess hTERT-associated RdRP activity, we established an IP-RdRP assay by modifying a method used to analyze the C. elegans RdRP (21).…”

“…regions is essential for proper chromosomal segregation during mitosis in S. pombe (28), C. elegans (5,6), and human cells (29). Using nocodazole treatment to arrest cells in mitosis as assessed by staining for pHH3 Ser10 (Fig.…”

The Anaphase Promoting Complex Regulates Yeast Lifespan and rDNA Stability by Targeting Fob1 for Degradation

“…This would provide a molecular mechanism to explain how the APC controls lifespan and how the cell overcomes Fob1's potential negative effects on rDNA recombination and longevity. Recent work has demonstrated that the APC is required for rDNA silencing, assembly, and segregation in budding and fission yeast (Sullivan et al 2008;Dubey et al 2009;Rodríguez-Sánchez et al 2011). Thus, the identification of Fob1 as a novel APC target would provide insight into how the APC may influence rDNA biology.…”