Molecular Mechanisms of Heterochromatin Establishment and Maintenance in Fission Yeast

Abstract

In most eukaryotes, H3K9-mediated heterochromatin is found at repetitive DNA elements and serves to facilitate chromosome segregation, maintain genome stability, and silence transposons. While many of its molecular effectors have been identified, it is still unclear how heterochromatin located at pericentromeric repeats is established in mammalian cells. Fission yeast Schizosaccharomyces pombe heterochromatin shares molecular features with mammalian heterochromatin and provides a useful model system for studying mechanisms of heterochromatin establishment. In S. pombe, the RNAi machinery is first recruited to nascent transcripts emerging from pericentromeric DNA repeats, and in turn, recruits the histone methyltransferase Clr4, which catalyzes histone H3 lysine 9 di- and tri-methylation (H3K9me2 and H3K9me3). H3K9me recruits chromo domain proteins to stabilize the RNAi machinery and to promote transcriptional gene silencing (TGS). Clr4 recruitment requires pol II transcription as the RNAi machinery is recruited via siRNA base-pairing with nascent transcripts. Given that H3K9me also recruits factors that promote TGS, it has remained unclear how Clr4 recruitment can proceed concomitantly with transcriptional silencing. In Chapter 2, I demonstrate that this process is coordinated by H3K9me2 and H3K9me3. H3K9me2 robustly recruits the RNAi machinery via Chp1, a chromo domain protein required for RNAi, while H3K9me3 recruits HP1 homologs, Swi6 and Chp2, to promote TGS. During heterochromatin establishment, the appearance of H3K9me2 precedes H3K9me3, suggesting a temporal separation of active transcription and TGS. In RNAi mutants, the level of pericentromeric H3K9me is decreased to around 5 – 20% of wild-type level. In Chapter 3, I exclude the possibility that this residual H3K9me results from an alternative pathway for Clr4 recruitment, and demonstrate that 1) a functional Clr4 chromo domain and 2) the ability of Clr4 to catalyze H3K9me3 are both required for epigenetically maintaining residual H3K9me. Furthermore, these requirements were confirmed using an inducible system designed to examine epigenetic maintenance at an ectopic locus. Given that the Clr4 chromo domain has higher affinity for H3K9me3 over H3K9me2, these results suggest that in the absence of RNAi-mediated Clr4 recruitment, the interaction between the chromo domain and H3K9me3 becomes critical for Clr4 to recognize pre-existing H3K9me and subsequently catalyze H3K9me on newly deposited histones.