Developmental dynamics of C. elegans dosage compensation

In Caenorhabditis elegans, the dosage compensation complex (DCC) specifically binds to and represses transcription from both X chromosomes in hermaphrodites. The DCC is composed of an X-specific condensin complex that interacts with several proteins. During embryogenesis, DCC starts localizing to the X chromosomes around the 40-cell stage, and is followed by X-enrichment of H4K20me1 between 100-cell to comma stage. Here, we analyzed dosage compensation of the X chromosome between sexes, and the roles of dpy-27 (condensin subunit), dpy-21 (non-condensin DCC member), set-1 (H4K20 monomethylase) and set-4 (H4K20 di-/tri-methylase) in X chromosome repression using mRNA-seq and ChIP-seq analyses across several developmental time points. We found that the DCC starts repressing the X chromosomes by the 40-cell stage, but X-linked transcript levels remain significantly higher in hermaphrodites compared to males through the comma stage of embryogenesis. Dpy-27 and dpy-21 are required for X chromosome repression throughout development, but particularly in early embryos dpy-27 and dpy-21 mutations produced distinct expression changes, suggesting a DCC independent role for dpy-21. We previously hypothesized that the DCC increases H4K20me1 by reducing set-4 activity on the X chromosomes. Accordingly, in the set-4 mutant, H4K20me1 increased more from the autosomes compared to the X, equalizing H4K20me1 level between X and autosomes. H4K20me1 increase on the autosomes led to a slight repression, resulting in a relative effect of X derepression. H4K20me1 depletion in the set-1 mutant showed greater X derepression compared to equalization of H4K20me1 levels between X and autosomes in the set-4 mutant, indicating that H4K20me1 level is important, but X to autosomal balance of H4K20me1 contributes only slightly to X-repression. Thus H4K20me1 by itself is not a downstream effector of the DCC. In summary, X chromosome dosage compensation starts in early embryos as the DCC localizes to the X, and is strengthened in later embryogenesis by H4K20me1. RNA-Seq profiles of C. elegans wild type hermaphrodite, mixed sex, at 5 time points and dpy-27, set-4, dpy-21, set-1 and RNAi at 2-3 time points with 3-4 replicates each. RNA-Seq profiles of C. elegans. Strains are N2 (wild type), BS553 fog-2(oz40) V, CB428 dpy-21(e428) V, MK4 dpy-27(y56) III, MT14911 set-4 (n4600) II, SS1075 set-1(tm1821)/hT2g[bli-4(e937) let-?(q782) qIs48] (I;III). Set-1 collections made as heterozygotes and dpy-27(y56) homozygotes. Spike in RNA-seq libraries have AF16 wild type C. briggsae L1s added at a 1:10 ratio. Timepoints are early embryos (synchronized collection, <40 cells), comma embryos (synchronized early embryos aged for 4 hours), mixed embryos (mixed stage embryos from unsynchronized population), L1 (synchronized L1 larvae), L3 (synchronized L3 larvae), and YA (synchronized young adults, collected before embryos present in hermaphrodite gonad). Biological replicates for each strain/stage listed separately. ChIP-seq profiles of C. elegans DCC subunit dpy-27, H4K20me1 histone modification and RNA pol II large subunit ama-1 in 3-6 replicates from mixed stage (unsynchronized) embryos and synchronized L3 larvae. Corresponding inputs are labelled with "Input_" plus ChIP name.

在秀丽隐杆线虫（Caenorhabditis elegans）中，剂量补偿复合体（dosage compensation complex, DCC）可特异性结合并抑制雌雄同体个体的两条X染色体的转录活性。该复合体由一种X染色体特异性的黏连蛋白（condensin）复合物与多种蛋白质相互作用组装而成。在胚胎发育过程中，DCC约于40细胞阶段开始定位至X染色体，随后在100细胞至逗号期阶段，X染色体上的H4K20单甲基化（H4K20me1）出现富集。
本研究通过多发育时间点的mRNA测序（mRNA-seq）和染色质免疫共沉淀测序（ChIP-seq）分析，解析了雌雄个体间X染色体的剂量补偿效应，并探究了dpy-27（黏连蛋白亚基）、dpy-21（非黏连蛋白型DCC成员）、set-1（H4K20单甲基化酶）与set-4（H4K20二/三甲基化酶）在X染色体转录抑制中的功能。研究发现，DCC于40细胞阶段即开始抑制X染色体转录，但直至胚胎发育至逗号期，雌雄同体的X连锁转录本水平仍显著高于雄性个体。dpy-27与dpy-21是整个发育过程中X染色体转录抑制所必需的，且二者的突变会引发显著的转录组变化，这提示dpy-21存在不依赖于DCC的功能。
我们此前曾提出假说，认为DCC可通过降低X染色体上set-4的活性来提升H4K20me1水平。据此，在set-4突变体中，常染色体上的H4K20me1水平提升幅度高于X染色体，最终使X染色体与常染色体间的H4K20me1水平趋于均衡。常染色体上H4K20me1的升高会引发轻度的转录抑制，进而相对表现为X染色体的去抑制效应。与set-4突变体中X染色体与常染色体H4K20me1水平均衡的表型相比，set-1突变体中H4K20me1缺失会引发更显著的X染色体去抑制，这表明H4K20me1水平本身对X染色体抑制至关重要，但X与常染色体间的H4K20me1平衡仅对X染色体转录抑制起到微弱贡献。由此可见，H4K20me1本身并非DCC的下游效应分子。综上，X染色体剂量补偿起始于早期胚胎阶段，随DCC定位至X染色体而启动，并在后续胚胎发育过程中通过H4K20me1得以强化。
本数据集包含秀丽隐杆线虫野生型雌雄同体、混合性别样本的5个发育时间点转录组测序（RNA-seq）数据，以及dpy-27、set-4、dpy-21、set-1突变体与RNA干扰（RNAi）样本的2~3个发育时间点转录组测序数据，每个样本设置3~4次生物学重复。
本次测序使用的秀丽隐杆线虫品系包括：N2（野生型）、BS553 fog-2(oz40) V、CB428 dpy-21(e428) V、MK4 dpy-27(y56) III、MT14911 set-4(n4600) II、SS1075 set-1(tm1821)/hT2g[bli-4(e937) let-?(q782) qIs48] (I;III)。其中set-1样本以杂合子形式收集，dpy-27(y56)样本以纯合子形式收集。
转录组测序文库中均按1:10的比例加入了AF16野生型布氏隐杆线虫（Caenorhabditis briggsae）L1期幼虫作为外源内参。
发育时间点包括：早期胚胎（同步化收集，细胞数<40）、逗号期胚胎（同步化早期胚胎体外培养4小时后收集）、混合阶段胚胎（未同步化种群的多阶段胚胎）、L1期幼虫（同步化L1幼虫）、L3期幼虫（同步化L3幼虫）以及青年成虫（同步化收集，在雌雄同体性腺中出现胚胎前采集）。每个品系/发育时间点的生物学重复单独标注。
本数据集还包含秀丽隐杆线虫DCC亚基dpy-27、组蛋白修饰H4K20me1以及RNA聚合酶II大亚基ama-1的染色质免疫共沉淀测序（ChIP-seq）数据，样本来自未同步化混合阶段胚胎与同步化L3期幼虫，每个样本设置3~6次生物学重复，对应的输入对照样本以"Input_+ChIP名称"的格式命名。