Semple, J. | bioRxiv Science

bioRxiv2025

HP1 dimerization but not LLPS drives the condensation and segregation of H3K9me-marked chromatin

Heterochromatin protein 1 (HP1) is a conserved chromatin-associated factor implicated in the establishment and maintenance of H3K9me-marked heterochromatin, potentially through phase separation-mediated condensation. Whether HP1 promotes heterochromatin condensation primarily through its dimerization or liquid-liquid phase separation (LLPS) remains unresolved. Using the C. elegans HP1 orthologue HPL-2 and a combined in vitro-in vivo approach, we systematically dissected the molecular determinants of HPL-2 function in heterochromatin condensation. Through specific mutants, we demonstrate that HPL-2 dimerization is essential for condensing H3K9me chromatin arrays in vitro and for maintaining HPL-2 and H3K9me heterochromatin foci in C. elegans embryos, while LLPS enhances or stabilizes these foci. We further show that HPL-2 dimerization is sufficient to mediate segregation of H3K9me from unmodified chromatin arrays in vitro, forming spatially distinct H3K9me-enriched condensates. Surprisingly, HPL-2 mutants defective in heterochromatin foci formation cause only minor transcriptional changes among genes associated with H3K9me-marked heterochromatin, implying that HP1-dependent heterochromatin foci and gene silencing are not tightly coupled in vivo. Nonetheless, these mutant C. elegans exhibit profound physiological and developmental defects. Our findings establish dimerization as the principal molecular mechanism of HP1-driven H3K9me-chromatin condensation, elucidate an auxiliary role of LLPS, and reveal the uncoupling between HP1-dependent heterochromatin and transcriptional regulation.

biochemistry↗

bioRxiv2023

Cohesin forms fountains at active enhancers in C. elegans

Transcriptional enhancers must locate their target genes with both precision and efficiency. In mammals, this specificity is facilitated by topologically associated domains (TADs), which restrict the enhancer search space through three-dimensional genome organization. In contrast, the nematode genome lacks such TAD-based segmentation despite harboring over 30000 sequences with chromatin signature characteristic of enhancers, thereby raising the question of how enhancer-promoter specificity is achieved. Using high-resolution Hi-C in C. elegans, we identify distinct 3D chromatin structures surrounding active enhancers, which we term fountains. These structures span 38 kb in average, are unique to active enhancers, and are enriched for the major somatic cohesin complex. Fountains collapse upon in vivo cohesin cleavage, indicating their cohesin dependency. Notably, fountains accumulate topological stress, as evidenced by the enrichment of topoisomerases and the psoralen-binding signature of negatively-supercoiled DNA. Functionally, fountain disassembly correlates with transcriptional upregulation of active enhancer-proximal genes, suggesting that fountains act as spatial repressors of enhancer activity. This repression is particularly pronounced for neuronal genes, including the skn-1/Nrf gene, which becomes upregulated, switches isoform and transcription start site upon cohesin loss in a pair of head neurons. Behaviorally, cohesin cleavage alters nematode movement and foraging behavior, linking enhancer-driven transcriptional changes to neural circuit function and organismal phenotypes, reminiscent of pathologies caused by cohesin mutations in humans. Together, our findings uncover fountains as a novel 3D chromatin feature that modulates enhancer activity in a TAD-less genome, establishing a mechanistic link between genome architecture, gene regulation and behavior.

genomics↗