Analysis of genome architecture during SCNT reveals a role of cohesin in impeding minor ZGA

Somatic cell nuclear transfer (SCNT) can reprogram a somatic nucleus to a totipotent state. However, the re-organization of three-dimensional chromatin structure in this process remains poorly understood. Using low-input Hi-C, we revealed that during SCNT, the transferred nucleus first enters a mitotic-like state (premature chromatin condensation). Unlike fertilized embryos, SCNT embryos show stronger TADs at the 1-cell stage. TADs become weaker at the 2-cell stage, followed by gradual consolidation. Compartments A/B are markedly weak in 1-cell SCNT embryos and become increasingly strengthened afterward. By the 8-cell stage, somatic chromatin architecture is largely reset to embryonic patterns. Unexpectedly, we found cohesin represses minor zygotic genome activation (ZGA) genes (2-cell specific genes) in pluripotent and differentiated cells, and pre-depleting cohesin in donor cells facilitates minor ZGA and SCNT. These data reveal multi-step reprogramming of 3D chromatin architecture during SCNT and support dual roles of cohesin in TAD formation and minor ZGA repression. Donor MEF/cumulus cells (MEF/CC), the SCNT embryos at the PCC stage (premature chromosome condensation) (1 hour after nuclear transfer) and SCNT embryos after PCC exit through “SCNT activation” using strontium chloride (SrCl2) at the 1-cell(4hours after activation, 6hours after activation and 10hours after activation), late 2-cell (28 hours after activation), and 8-cell (56 hours after activation) stages were obtained. Both MEF and CC are used for donor cells for the collection of SCNT 8-cell samples. HiC and RNA-seq were performed in these cells at various stages during SCNT embryogenesis. For the predepletion of CTCF/cohesin experiment, the short term differentiated cells with/without auxin treatment are collected for HiC experiments, RNA-seq and ChIP-seq experiments.

体细胞核移植（Somatic cell nuclear transfer, SCNT）可将体细胞核重编程至全能性状态，但该过程中三维染色质结构的重塑机制仍有待深入解析。本研究借助低起始量Hi-C（Hi-C）技术开展研究，结果显示：体细胞核移植过程中，移入的供体细胞核首先进入类有丝分裂状态，即早熟染色体凝集（premature chromosome condensation, PCC）。与受精胚胎不同，体细胞核移植胚胎在1细胞阶段即表现出更强的拓扑关联结构域（Topologically Associating Domains, TAD）；至2细胞阶段，TAD强度减弱，随后逐渐趋于稳定整合。A/B染色质区室（Compartment A/B）在1细胞期体细胞核移植胚胎中显著减弱，后续则逐渐增强；至8细胞阶段，体细胞的染色质架构已基本重编程为胚胎型模式。令人意外的是，本研究发现黏连蛋白（cohesin）在多能细胞与分化细胞中可抑制微量合子基因组激活（minor zygotic genome activation, ZGA）相关基因（即2细胞特异性基因）的表达，而在供体细胞中预先敲除黏连蛋白可促进微量ZGA与体细胞核移植进程。上述数据揭示了体细胞核移植过程中三维染色质架构的多阶段重编程过程，并证实黏连蛋白在TAD形成与微量ZGA抑制中发挥双重功能。本研究收集的样本包括：供体小鼠胚胎成纤维细胞/卵丘细胞（MEF/CC）、核移植后1小时的早熟染色体凝集阶段体细胞核移植胚胎，以及通过氯化锶（SrCl2）进行"SCNT激活"以脱离PCC阶段后的各阶段体细胞核移植胚胎：1细胞期（激活后4、6、10小时）、晚2细胞期（激活后28小时）与8细胞期（激活后56小时）。本研究同时使用MEF与CC作为供体细胞，以收集8细胞期体细胞核移植胚胎样本。针对体细胞核移植胚胎发生过程各阶段的上述样本，本研究均开展了Hi-C与RNA测序（RNA-seq）实验。针对CTCF/黏连蛋白预敲除实验，本研究收集了经/未经植物生长素（auxin）处理的短期分化细胞，用于Hi-C、RNA-seq与染色质免疫共沉淀测序（ChIP-seq）实验。