Heterochromatic 3D genome organization is directed by HP1a and H3K9-dependent and independent mechanisms [RNA-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 transposon and protein coding gene expression in total RNA-seq data collected from HP1a chromdomain mutant and H3.2-K9R mutant wing imaginal discs and their controls.

组蛋白翻译后修饰及其结合蛋白被视作三维基因组（3D genome）组织的驱动因子，然而其是否发挥该调控作用以及具体机制仍未阐明。本研究以果蝇组织为研究对象，评估了H3K9甲基化组成型异染色质（constitutive heterochromatin）对三维基因组组织的贡献。研究发现，野生型果蝇组织的核心基因组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分离。通过对异染色质蛋白1a（HP1a）染色质结构域（chromodomain）引入点突变，或是将复制型组蛋白H3替换为H3K9R突变型组蛋白，对HP1a与H3K9的结合进行双向扰动后，结果显示：组成型异染色质中，HP1a结合甲基化H3K9是限制着丝粒周边区域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，着丝粒周边异染色质的自身聚集现象仍基本得以保留。由此可见，HP1a与H3K9的相互作用对细胞核内常染色质与异染色质的分离具有促进作用，但并非该过程的唯一驱动因素。整体实验设计：对HP1a染色质结构域突变体、H3.2-K9R突变型果蝇翅成虫盘及其对照样本的总RNA测序（RNA-seq）数据，开展转座子与蛋白编码基因的表达水平比较分析。