Investigating the role of chromatin regulators in genome function

Abstract

Genome function relies on the dynamic regulation of chromatin, encompassing both its structural organization and epigenetic landscape. This thesis explores the roles of two crucial chromatin regulators, the chromatin remodeler HELLS and the structural protein complex cohesin, in safeguarding chromatin integrity and modulating genome function. In the first part of this work, we explore the role of HELLS in regulating DNA methylation in human pluripotent stem cells. Through genome-wide profiling of DNA methylation and chromatin accessibility in HELLS knockout cells, we reveal that HELLS is indispensable for maintaining DNA methylation, particularly at pericentromeric satellite repeats across all chromosomes, though not all classes are equally affected. Beyond these repeat regions, HELLS plays a broader role in sustaining global DNA methylation. It appears to act in synergy with DNMT3A and DNMT3B to maintain methylation levels, especially in compact chromatin regions where DNMT1-mediated maintenance is less efficient. Despite substantial disruptions to the chromatin landscape, human pluripotent stem cells exhibit a remarkable resilience to HELLS depletion, maintaining their ability to differentiate into all three germ layers. In contrast, HELLS knockout in mice results in more severe DNA methylation defects, emphasizing the critical developmental role of HELLS with perinatal lethality as a consequence of its absence. In the second part of this thesis, we investigate the role of cohesin in enhancer-promoter interactions by analyzing how the absence of cohesin impacts 3D chromatin contacts and enhancer function. Our findings reveal that enhancer-promoter contact frequency directly modulates gene expression, with cohesin loss significantly reducing enhancer-driven transcriptional activity. Notably, distal enhancers are particularly dependent on cohesin, exhibiting substantial decreases in activity upon cohesin depletion, whereas proximal enhancers remain largely unaffected. These results highlight the crucial role of cohesin in facilitating distal enhancer-promoter interactions and highlight the consequences of disrupting these interactions on gene expression. Interestingly, we find that most genes are regulated by proximal enhancers, which explains why the global transcriptional impact of cohesin loss is relatively modest. Only genes reliant on cohesin-dependent distal enhancers experience transcriptional changes, broadening our understanding of the relationship between chromatin architecture and enhancer function.