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

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 FAIRE-seq data collected from HP1a chromdomain 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）的驱动因素，但其具体作用机制与实际贡献仍未阐明。本研究以果蝇组织为实验材料，评估了H3K9甲基化组成型异染色质（H3K9-methylated constitutive heterochromatin）在三维基因组组织中的贡献。研究发现，野生型果蝇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区的空间分离。通过两种方式对HP1a与H3K9的结合进行双向扰动：一是在HP1a染色质结构域（HP1a chromodomain）引入点突变，二是将依赖复制的H3组蛋白替换为H3K9R突变型组蛋白。结果显示，组成型异染色质中HP1a结合甲基化H3K9，是限制着丝粒周区与染色体臂之间长程相互作用的必要条件。令人意外的是，尽管着丝粒周区的H3K9甲基化与HP1a结合均已丧失，但HP1a与H3K9的结合被破坏后，着丝粒周区异染色质的自身缔合仍基本得以保留。由此可见，HP1a与H3K9的相互作用仅参与而非单独驱动细胞核内常染色质与异染色质的空间分离。
整体实验设计：对HP1a染色质结构域突变型翅成虫盘及其对照样本的FAIRE-seq（甲醛辅助调控元件分离测序，formaldehyde-assisted isolation of regulatory elements sequencing）数据开展比较分析。