Heterochromatic 3D genome organization is directed by HP1a and H3K9-dependent and independent mechanisms [Hi-C]

Histone post-translational modifications and the proteins that bind them are proposed to be drivers of 3D genome organization, but whether and how they do so remain unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wildtype tissues is segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1a•H3K9 binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to restrict long-range interactions between pericentromeres and chromosome arms. Surprisingly, self-association of pericentromeric heterochromatin is largely preserved upon disruption of HP1a•H3K9 binding despite loss of pericentromeric H3K9 methylation and HP1a occupancy. Thus, the HP1a•H3K9 interaction contributes to, but does not solely drive, segregation of euchromatin and heterochromatin inside the nucleus. Overall design: Comparative analysis of Hi-C data collected from HP1a chromdomain mutant and H3.2-K9R mutant wing imaginal discs and their controls.

组蛋白翻译后修饰（Histone post-translational modifications）及其结合蛋白被认为是三维基因组组织（3D genome organization）的驱动因子，但其具体作用机制与是否存在该调控功能仍未阐明。本研究针对果蝇组织中H3K9甲基化组成型异染色质（constitutive heterochromatin）对三维基因组组织的贡献展开评估。研究发现，野生型组织的核心组织特征为常染色质染色体臂与异染色质着丝粒周边区域的相互分离。通过HP1a染色质结构域（chromodomain）点突变，或用H3K9R突变型组蛋白替换依赖复制的组蛋白H3，对HP1a与H3K9的结合进行双向扰动，结果表明：组成型异染色质中HP1a结合甲基化H3K9，是限制着丝粒周边区域与染色体臂间长程相互作用的必要条件。令人意外的是，尽管着丝粒周边区域的H3K9甲基化与HP1a结合均已丧失，但HP1a-H3K9结合被破坏后，着丝粒异染色质的自身聚集仍基本得以保留。综上，HP1a与H3K9的相互作用仅参与细胞核内常染色质与异染色质的分离过程，而非其唯一驱动因素。整体实验设计：对HP1a染色质结构域突变型、H3.2-K9R突变型果蝇翅成虫盘（wing imaginal discs）及其对照样本的Hi-C测序数据（Hi-C data）开展比较分析。