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 swi6Delta 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.
Highlights
Heterochromatin plays a central role in the structural integrity of chromosomes and their faithful segregation during mitosis
A direct physical interaction of Cut4 and Cut9 with Swi6 and Clr4 plays an important role in heterochromatin assembly, and the silencing and other defects of APC mutants may be ascribed to the inability of the mutant proteins to bind and recruit Swi6 and Clr4
In light of the results shown above, we infer that the defects observed in cut4 and cut9 mutants at permissive temperature can be ascribed to the loss of heterochromatin and, thereby, of Cohesin to the centromeric and mating type heterochromatin
Summary
Strains and Plasmids—The following parental strains were used in most experiments. SP1152 (genotype: Msmto REII⌬mat2::ura ura4D18 leu, ade6-210) and PG1649 (genotype: mat1P⌬17::LEU2 REIII⌬mat3MEcoRV::ade, leu132, ura4D18, ade6-210) were used to monitor mating type silencing. Chromosomal loss rates were determined by using a strain with ade210 mutation and carrying an artificial chromosome Ch-16 that contains ade216 allele [16]. Expression of the ade reporter was monitored by streaking the cultures on YE plates and incubating at 25 °C for 4 –5 days, and cut665 mutant was grown at 30 °C. Beads were incubated overnight with 2–5 mg of the protein extract prepared from strains carrying HA-tagged cut or cut genes. Samples were boiled for 5–10 min and subjected to SDS-PAGE followed by Western blotting with anti-HA antibody (1:1000) or anti-Swi antibody (1:10,000). For pull-down assays, extracts were prepared from Escherichia coli strains expressing His6-tagged Clr and allowed to bind to Ni-NTA resin (Qiagen (binding capacity 5–10 mg of protein/ml resin)), which was equilibrated with binding buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole). Incubations with antibodies and substrates were performed as per the manufacturer’s instructions (Santa Cruz Biotechnology, Sigma, Millipore)
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