Abstract
Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct 'territories,' in which 'intra-arm' interactions are the most prevalent, followed by 3D contacts between specific PCH regions on different chromosomes. Strikingly, H3K9me2/3-enriched regions embedded in euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2/3 enrichment, are likely mediated by liquid-liquid phase separation, and influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.
