Maternal SMARCA5 is required for major ZGA in mouse [RNA-seq]

Zygotic genome activation in mice happens in two waves, a minor wave in the 1 cell embryo, and a major wave at the two-cell stage, both accompanied by global transcriptional and epigenetic reprogramming. However, the orchestration of these reprogramming events by maternal factors deposited in the oocyte is not yet entirely understood. We and others have recently shown that epigenetic modifiers such as SMARCA5 (the main ATPase in ISWI complexes), can initiate the ZGA transcriptional programme in vitro. So far, the role of SMARCA5 in ZGA in vivo has not been addressed, as constitutive knock-out mice lacking SMARCA5 are not viable. We have overcome this issue by using the targeted protein-depletion system Trim-Away. Degradation of SMARCA5 in early zygotes in combination with single cell multi-omics methods allow us to investigate its role in transcription, DNA methylation and chromatin accessibility during ZGA, as well as the wider impact of the lack of maternal SMARCA5 on embryonic development. We show that in absence of SMARCA5, major ZGA transcription is downregulated, whereas maternal and other genes are upregulated. Our results show that the global accessibility at the two-cell stage is higher than in a wild-type context, and that major ZGA gene promoters are not following that global trend but remain less accessible in SMARCA5 depleted embryos. Moreover, while the accessibility of repeat elements like MERVL increases, so does the methylation level in these regions, suggesting that SMARCA5 might indirectly influence more than just the chromatin accessibility during these stages. RNA-seq libraries were generated from Smarca5 Heterozygous 2 cell stage embryos (Smarca5 mat KO x WT male) (2 technical replicates, each replicate containing embryos from multiple matings, pooled). Same type of libraries generated from WT 2 cell stage embryos in which SMARCA5 was degraded at the zygotic stage, using the TrimAway system (libraries generated at least 24 hours post-injection). The Trim Away embryos libraries were also in 3 technical replicates (each replicate representing a different pool of injected embryos). The same samples were used to generate NOME-seq libraries (as part of the scNMT-seq protocol);小鼠的合子基因组激活（Zygotic genome activation, ZGA）分为两个波次：单细胞胚胎时期的次要波次，以及二细胞阶段的主要波次，二者均伴随全局性转录与表观遗传重编程。然而，由卵母细胞中沉积的母源因子调控这些重编程事件的完整机制尚未完全阐明。我们及其他团队近期研究表明，表观遗传修饰因子如SMARCA5（ISWI复合物的核心ATP酶）可在体外启动ZGA转录程序。但截至目前，SMARCA5在体内ZGA过程中的功能尚未被探究——因为SMARCA5组成型敲除小鼠无法存活。我们通过靶向蛋白降解系统Trim-Away解决了这一难题：对早期合子中的SMARCA5进行降解，并结合单细胞多组学技术，使我们得以探究其在ZGA过程中对转录、DNA甲基化及染色质可及性的调控作用，以及母源SMARCA5缺失对胚胎发育的更广泛影响。研究显示，SMARCA5缺失会导致主要ZGA转录过程下调，而母源基因及其他基因则出现上调。本研究的结果表明，二细胞阶段的全局染色质可及性高于野生型对照，而主要ZGA基因的启动子并未遵循这一全局趋势，在SMARCA5降解的胚胎中仍保持较低的可及性。此外，尽管MERVL等重复序列元件的可及性升高，这些区域的甲基化水平也同步上升，这提示SMARCA5可能在上述阶段间接参与调控不止染色质可及性这一过程。本研究的RNA测序（RNA-seq）文库构建自Smarca5杂合子二细胞阶段胚胎（Smarca5母源敲除 × 野生型雄性），设置2个技术重复，每个重复包含多次交配获得的混合胚胎。采用相同建库策略的文库还构建自合子阶段经Trim-Away系统降解SMARCA5的野生型二细胞胚胎（于注射后至少24小时制备文库）。Trim-Away处理组的胚胎文库同样设置3个技术重复，每个重复代表一批不同的注射胚胎混合样本。上述样本同时用于构建NOME-seq文库，属于scNMT-seq实验流程的一部分。