Investigation of 3D Chromatin Architecture Rewiring During Muscle Satellite Cells activation and aging [CTCF ChIP-Seq]

3D genome architecture rewiring is known to influence spatiotemporal expression of lineage-specific genes and cell fate transition during multiple lineage progression and cell differentiation. However, it is yet unknown how nuclear architecture remodels and effects transcription changes during muscle stem cell (SC) development and ageing process. Here, we comprehensively map the 3D chromatin reorganization and integrate genome topology with transcriptional and epigenetic change during muscle SC lineage development and ageing process. Rewiring of compartmentalization is most pronounced when SC become activated, while striking loss in insulation of TAD borders and chromatin looping during early activation process. Meanwhile, super-enhancer containing TAD cluster reorganize in nuclear space and orchestrate stage-specific gene expression. In depth analysis identifies and verifies cis-regulatory elements at Pax7 locus controlling the local chromatin interactions and Pax7 expression. Geriatric cells display a more prominent gain in long-range contacts and loss insulation of TAD borders. Moreover, genome compartmentalization and chromatin loop has already altered for aged SC while geriatric SC display a more prominent loss in strength of TAD borders. Together, our results implicate 3D chromatin extensively reorganizes at multiple architectural levels and underpin the transcriptome remodeling and SC behaviors during SC lineage development and SC aging. Overall design: The in situ Hi-C libraries were prepared as previously reported. Briefly, isolated SCs were digested using 100U DpnII overnight at 37 °C and then filled in with biotin for 1.5 hours at 37 °C; ligated for 4.5 hours at room temperature. DNA was purified by ethanol precipitation, and then sheared into 300-400bp fragments using Covaris S220. DNA fragments containing biotin were enriched by Dynabeads MyOne Streptavidin C1 (Invitrogen 65001) for 15min at room temperature, followed by end repairing, adaptor ligation and PCR amplification as described. At least two biological replicates were performed for both control and sgMyc groups. The libraries were then sequenced via the Illumina HiSeq X Ten system at Genewiz company.

已知三维基因组（3D genome）结构重编程可影响谱系特异性基因的时空表达，以及多谱系进程与细胞分化过程中的细胞命运转变。然而，目前尚不清楚在肌肉干细胞（SC）发育与衰老过程中，细胞核结构如何重塑并影响转录组变化。本研究全面绘制了肌肉干细胞谱系发育与衰老过程中的三维染色质重编程图谱，并将基因组拓扑结构与转录组及表观遗传变化进行整合分析。当肌肉干细胞被激活时，区室化结构的重编程最为显著；而在早期激活过程中，拓扑关联结构域（Topologically Associating Domain, TAD）边界的绝缘性与染色质环的形成出现显著丢失。与此同时，包含超级增强子的TAD簇在细胞核空间内发生重排，并调控阶段特异性基因的表达。深入分析鉴定并验证了Pax7基因座处的顺式调控元件，其可调控局部染色质相互作用与Pax7基因的表达。老龄细胞展现出更为显著的长程相互作用增加，以及TAD边界绝缘性的丢失。此外，衰老肌肉干细胞的基因组区室化与染色质环已发生改变，而老龄肌肉干细胞的TAD边界强度则出现更为显著的下降。综上，本研究结果表明，三维染色质在多个结构层面发生广泛重排，这为肌肉干细胞谱系发育与衰老过程中的转录组重塑及干细胞行为奠定了基础。实验整体设计：原位Hi-C文库的构建方法参照已发表文献。简要步骤如下：分离得到的肌肉干细胞经100U DpnII于37℃消化过夜，随后在37℃下用生物素标记补平反应1.5小时，再于室温下连接4.5小时。通过乙醇沉淀法纯化DNA，随后使用Covaris S220超声破碎仪将DNA片段化至300-400bp。使用Dynabeads MyOne Streptavidin C1磁珠（Invitrogen 65001）在室温下富集含生物素的DNA片段，时长15分钟，随后按照标准流程进行末端修复、接头连接与PCR扩增。对照组与sgMyc组均至少设置2次生物学重复。最后，在Genewiz公司使用Illumina HiSeq X Ten测序平台对文库进行测序。