Mechanisms of Heterochromatin Regulation by HP1 Proteins and the Chromatin Remodeler Mit1

Heterochromatin is a specialized form of chromatin defined by histone H3 lysine 9 methylation (H3K9me), which promotes transcriptional repression and drives spatial compartmentalization within the nucleus. A central effector of this silenced chromatin state is the conserved family of Heterochromatin Protein 1 (HP1) proteins, which bind H3K9me and contribute to both the establishment and maintenance of heterochromatin through a combination of effector recruitment and biophysical mechanisms such as phase separation. Despite decades of research, the distinct molecular functions of HP1 paralogs and their precise roles in heterochromatin regulation remain incompletely understood.This dissertation investigates the mechanisms by which HP1 proteins regulate chromatin in the fission yeast Schizosaccharomyces pombe, a genetically tractable model with two HP1 paralogs, Swi6 and Chp2. Using an inducible system to generate synthetic heterochromatin in vivo, I dissect the relative contributions of Swi6’s protein-protein interactions and phase separation capacity to its silencing function. I demonstrate that Swi6’s role in heterochromatin maintenance relies on its ability to form condensates, while effector recruitment is more critical during establishment. In parallel, I investigate the mechanism of the Chp2 interactor, Mit1, a CHD-family chromatin remodeler. Through genetic analysis, I uncover a previously undiscovered role for Mit1 in positioning nucleosomes to recruit and facilitate Clr4 spreading at both heterochromatic and euchromatic regions, revealing its broader impact on genome regulation.