Understanding heterochromatin boundary architecture by dissecting the spatial regulation of the putative histone demethylase Epe1

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This proposal aims to dissect the layers of regulation that control the local accumulation of a boundary factor on chromatin, ensuring its function as a barrier against the expansion of heterochromatin domains. Chromatin boundaries are crucial to separate ‘silent’ heterochromatin from ‘active’ euchromatin to ensure proper regulation of gene expression and cellular identity. While DNA elements have been identified that contribute to the recruitment of boundary factors, the spatial regulation of boundaries is highly complex. We previously demonstrated that the putative histone demethylase Epe1, which prevents ectopic spreading of heterochromatin in Schizosaccharomyces pombe (fission yeast), is spatiotemporally controlled on chromatin through ubiquitin-dependent degradation, confining Epe1 localization to the boundaries. This epigenetic concept of boundary formation differs from chromatin barriers established by DNA-binding sequences previously described. However, how Epe1 is protected from degradation at the boundaries and how it exerts its boundary function have remained elusive. Here, we seek to elucidate the mechanism of Epe1 regulation by dissecting the dynamic interactions with its multiple binding partners that control its recruitment, degradation, and protection. Our unpublished data indicate that removing the unstructured C-terminal region prevents Epe1 degradation, causing its accumulation on chromatin that triggers silencing defects at heterochromatin. We hypothesize that the C-terminal region, besides other functions, mediates Epe1 degradation but is masked at the boundaries. We seek to refine and separate these functions and identify the mechanisms protecting Epe1 from degradation. A candidate is the bromodomain protein Bdf2, which is recruited by Epe1 to the heterochromatin boundaries. We find that the loss of Bdf2 (bdf2∆) reduces the chromatin association of Epe1. Intriguingly, Epe1 levels can be restored in bdf2∆ cells when expressing truncated Epe1 that lacks the C-terminal part. Thus, we seek to test the hypothesis that Bdf2 protects Epe1 from ubiquitin-dependent degradation by masking its recognition or ubiquitylation sites. In addition, we will examine the dynamic interactions with Bdf2 and HP1 proteins, both contributing to Epe1 recruitment but at different chromatin regions. In particular, we seek to understand how differences in local Bdf2 and HP1 abundance drive Epe1’s ability to establish heterochromatin boundaries. The proposed work will shed light on the architecture of heterochromatin boundaries by providing mechanistic insights into how spatial regulation is coordinated through dynamic interactions across multiple protein partners.