Condensin IDC, H4K20me1, and perinuclear tethering maintain X chromosome repression in C. elegans

Dosage compensation in Caenorhabditis elegans equalizes X-linked gene expression between XX hermaphrodites and XO males. The process depends on a condensin-containing dosage compensation complex (DCC), which binds the X chromosomes in hermaphrodites to repress gene expression. Condensin IDC and an additional five DCC components must be present on the X during early embryogenesis in hermaphrodites to establish dosage compensation. However, whether the DCC's continued presence is required to maintain the repressed state once established is unknown. Beyond the role of condensin IDC in X chromosome compaction, additional mechanisms contribute to X-linked gene repression. DPY-21, a non-condensin IDC DCC component, is an H4K20me2/3 demethylase whose activity enriches the repressive histone mark, H4 lysine 20 monomethylation on the X chromosomes. In addition, CEC-4 tethers H3K9me3-rich chromosomal regions to the nuclear lamina, which also contributes to X-linked gene repression. To investigate the necessity of condensin IDC during the larval and adult stages of hermaphrodites, we used the auxin-inducible degradation system to deplete the condensin IDC subunit DPY-27. While DPY-27 depletion in the embryonic stages resulted in lethality, DPY-27 depleted larvae and adults survive. In these DPY-27 depleted strains, condensin IDC was no longer associated with the X chromosome, the X became decondensed, and the H4K20me1 mark was gradually lost, leading to X-linked gene derepression. These results suggest that the stable maintenance of dosage compensation requires the continued presence of condensin IDC. A loss-of-function mutation in cec-4, in addition to the depletion of DPY-27 or the genetic mutation of dpy-21, led to even more significant increases in X-linked gene expression, suggesting that tethering heterochromatic regions to the nuclear lamina helps stabilize repression mediated by condensin IDC and H4K20me1. Overall design: RNA-seq profiles of L3 larval stage C. elegans samples. Synced L1s were placed on HGM plates with or without auxin, and NA22. 24-hours later, L3 larvae were collected for RNA extraction. Genotype and treatment for each sample are listed separately.

秀丽隐杆线虫（Caenorhabditis elegans）的剂量补偿机制可使XX型雌雄同体与XO型雄性的X连锁基因表达水平趋于均等。该过程依赖于一种包含凝缩蛋白（condensin）的剂量补偿复合物（dosage compensation complex, DCC），该复合物结合雌雄同体的X染色体以抑制基因表达。在雌雄同体的早期胚胎发育阶段，必须在X染色体上存在凝缩蛋白IDC（Condensin IDC）以及另外5种DCC组分，方可建立剂量补偿。然而，剂量补偿建立后，是否仍需要DCC持续存在以维持基因抑制状态，目前尚不明确。除凝缩蛋白IDC在X染色体凝缩中的功能外，尚有其他机制参与X连锁基因的抑制过程。DPY-21作为一种非凝缩蛋白IDC的DCC组分，是一种H4K20me2/3去甲基化酶，其活性可使X染色体上的抑制性组蛋白标记——H4赖氨酸20单甲基化（H4K20me1）水平富集。此外，CEC-4可将富含H3K9me3的染色体区域锚定至核纤层，这同样有助于X连锁基因的抑制。为探究雌雄同体幼虫期与成虫期凝缩蛋白IDC的必要性，我们利用生长素诱导降解系统（auxin-inducible degradation system）降解凝缩蛋白IDC亚基DPY-27。尽管胚胎时期DPY-27的降解会导致个体致死，但经DPY-27降解的线虫品系仍可存活。在这些DPY-27降解的样本中，凝缩蛋白IDC不再与X染色体结合，X染色体发生解凝，H4K20me1标记逐渐丢失，最终引发X连锁基因的去抑制。上述结果表明，剂量补偿的稳定维持需要凝缩蛋白IDC的持续存在。与单独降解DPY-27或dpy-21基因突变相比，cec-4功能丧失突变会使X连锁基因的表达水平出现更显著的上调，这提示将异染色质区域锚定至核纤层，有助于稳定凝缩蛋白IDC与H4K20me1介导的基因抑制。整体实验设计：本研究对L3期幼虫阶段的秀丽隐杆线虫样本开展RNA测序（RNA-seq）分析。将同步化的L1期幼虫接种至添加或不添加生长素的HGM平板（含NA22菌株）上，24小时后收集L3期幼虫用于RNA提取。各样本的基因型及处理方式均单独列出。