Stc1: a critical link between RNAi and chromatin modification required for heterochromatin integrity.
Bottom Line: Our analyses show that Stc1 is specifically required to establish H3K9 methylation via RNAi, and interacts both with the RNAi effector Ago1, and with the chromatin-modifying CLRC complex.Moreover, tethering Stc1 to a euchromatic locus is sufficient to induce silencing and heterochromatin formation independently of RNAi.We conclude that Stc1 associates with RITS on centromeric transcripts and recruits CLRC, thereby coupling RNAi to chromatin modification.
Affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.Show MeSH
Mentions: It is known that RNAi is required to establish, but not to maintain, silent heterochromatin over the mat2-mat3 and subtelomeric regions. Consequently, deletion of Dcr1 has little or no impact on silencing of embedded genes (Hall et al., 2002; Kanoh et al., 2005). We crossed stc1Δ, dcr1Δ, clr4Δ, rik1Δ, dos1Δ, or dos2Δ cells with cells harboring a silent ura4+ gene close to mat3 (mat3-M:ura4+). The CLRC components Clr4, Rik1, Dos1, and Dos2 are required to maintain silencing of mat2-mat3; consequently, cells lacking these proteins lose mat3-M:ura4+ silencing, resulting in growth on plates lacking uracil (−URA) and loss of resistance to counterselective FOA (+FOA). qRT-PCR confirms that ura4+ transcript levels increase by around 20-fold in CLRC mutants (Figures 2A–2C). In contrast, mat3-M:ura4+ remained silent in both dcr1Δ and stc1Δ cells, as indicated by continued growth on FOA (equivalent to the wild-type) and low transcript levels. Maintenance of silencing at telomeres was similarly unaffected by deletion of Dcr1 or Stc1 but was disrupted by deletion of CLRC components (Figure S2). So that the role of Stc1 in establishing silencing of mat3-M:ura4+ could be assessed, the clr4+ gene was removed (causing loss of silencing) and then reintroduced in wild-type, dcr1Δ or stc1Δ backgrounds by crossing (Figure 2A). Silencing of mat3:ura4+ could not be re-established in the absence of Dcr1 or Stc1 (Figures 2B and 2C). This indicates that, like Dcr1, Stc1 is required for the establishment, but not the maintenance, of silencing over mat2-mat3, strongly suggesting that Stc1 is specifically required for RNAi-dependent heterochromatin establishment.
Affiliation: Wellcome Trust Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, The University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.