Chromatin landscape of budding yeast acquiring H3K9 methylation and its reader molecule HP1 [ATAC-Seq]

Histone H3 lysine 9 (H3K9) methylation and heterochromatin protein 1 (HP1) are well conserved epigenetic silencing mark and its reader molecule, and crucial for heterochromatin formation. However, the details of the importance of H3K9 methylation and HP1 in heterochromatin formation still remain unclear. One of the reason is the redundancy problem, as there are multiple reader molecules for H3K9 methylation, including HP1, and HP1 itself functions as a hub that recruits various effector molecules. To overcome the redundancy issue, we took synthetic biology approach and introduced H3K9 methylation and HP1 into budding yeast Saccharomyces cerevisiae, which does not have this system, and examined its impact on transcription and chromatin compaction. We observed that mammalian H3K9 methyltransferase can induce genome wide H3K9 di- and tri-methylation (H3K9me2,3) in budding yeast, and that HP1 accumulates over the H3K9 methylated regions. However, H3K9 methylation occurred mainly in the gene body region of the genes and excluded around TSS where H3K9ac pre-exists. Correspondingly, expression of H3K9 methyltransferase and HP1 did not affect transcription in budding yeast, including repression. ATAC-seq analysis also showed no impact on chromatin accessibility, and Hi-C-seq analysis of chromatin 3D structure revealed no significant differences. These results suggest that even though H3K9 methylation and recruitment of HP1 play essential roles in epigenetic regulation of heterochromatin, they are not sufficient to build up heterochromatin, at least at gene body regions, and further participation of effector molecules, including downstream factors of HP1, is required.组蛋白H3赖氨酸9（Histone H3 lysine 9，H3K9）甲基化与异染色质蛋白1（heterochromatin protein 1，HP1）是高度保守的表观遗传沉默标记及其识别分子，对异染色质形成至关重要。然而，H3K9甲基化与HP1在异染色质形成中的具体作用机制仍未明确。其中一个核心原因是冗余问题：H3K9甲基化存在多种识别分子，HP1即为其中之一，且HP1本身可作为枢纽招募各类效应分子。为攻克这一冗余难题，本研究采用合成生物学策略，将H3K9甲基化系统与HP1引入原本不具备该系统的酿酒酵母（Saccharomyces cerevisiae），并探究其对转录与染色质压缩的影响。研究结果显示，哺乳动物来源的H3K9甲基转移酶可在酿酒酵母中诱导全基因组范围的H3K9二甲基化与三甲基化（H3K9me2、H3K9me3），且HP1会在H3K9甲基化区域发生富集。但H3K9甲基化主要发生在基因体区域（gene body），且被排除在预先存在H3K9乙酰化（H3K9 acetylation，H3K9ac）的转录起始位点（transcription start site，TSS）周边区域。与之对应，表达H3K9甲基转移酶与HP1并不会对酿酒酵母的转录水平产生影响，包括基因沉默过程。ATAC测序（ATAC-seq）分析同样未检测到染色质开放度的显著变化，而染色质三维结构的Hi-C测序（Hi-C-seq）分析也未发现明显差异。上述结果表明，尽管H3K9甲基化与HP1的招募在异染色质的表观遗传调控中发挥关键作用，但二者单独并不足以构建异染色质——至少在基因体区域如此，还需要包括HP1下游因子在内的更多效应分子的参与。