Regulation of boundaries through chromatin sculpting
We previously discovered an unprecedented mechanism, by which chromatin domains are shaped through the removal of a heterochromatin-associated factor by ubiquitin-dependent degradation, a process we coined ‘chromatin sculpting’ (Braun et al. Cell 2011). In S. pombe, the boundary factor Epe1 belonging to the conserved family of Jumonji demethylases prevents the spreading of H3K9 methylation beyond the natural borders of heterochromatin. Paradoxically, while antagonizing silent chromatin, Epe1 itself is recruited to heterochromatin through binding to HP1 proteins, raising the question of how Epe1 is prevented from interfering with silent chromatin. Through a genetic screen for mutants with silencing defects, we identified the conserved ubiquitin ligase complex Cul4-Ddb1Cdt2 and demonstrated that Epe1 acquires its specific chromatin distribution through ubiquitin-dependent degradation. Remarkably, Epe1 degradation takes place exclusively within the body of heterochromatin — but not at its boundaries. The selective degradation restricts Epe1’s accumulation to the boundaries between active and silent chromatin, thereby specifying its function as a boundary factor, and prevents its invasion into heterochromatin. How degradation of Epe1 is counteracted at the boundaries remains unknown, though. We are currently testing several hypotheses on how ubiquitylation can be spatially controlled.
Maintaining the integrity of chromatin domains through anchoring
Another factor opposing silent chromatin is the histone acetyl transferase (HAT) Mst2 that resides in a multi-subunit complex. Surprisingly, one of its components, the PWWP domain protein Pdp3, was isolated by our genetics screens as a factor that actually contributes to silencing. Using genetic interaction studies, we found that the silencing defect of the pdp3 mutant can be suppressed by deleting other components of the Mst2 complex including the HAT. This implies that Pdp3 negatively regulates the anti-silencing activity of Mst2. In a collaborative effort with the lab of Mark Bühler (FMI), we demonstrated that Pdp3 anchors the Mst2 complex to euchromatin via its PWWP domain, which recognizes methylated H3K36, a histone mark of actively transcribed euchromatin. Loss of the Pdp3 anchor results in the mistargeting of Mst2 to heterochromatin, causing a silencing defect. Intriguingly, while Pdp3 protects heterochromatin from Mst2 encroachment, the HAT vice versa prevents the ectopic heterochromatin assembly by acetylating the non-histone target Brl1, a subunit of the histone H2B ubiquitin ligase complex. Brl1 acetylation increases histone H2B ubiquitination, which positively feeds back on transcription. These findings illustrate how opposing feedback loops maintain the partitioning of chromatin into transcriptionally active and inactive states (Flury et al. Mol Cell 2017).