Muscle progenitor specification and myogenic differentiation are associated with changes in chromatin topology.. Muscle progenitor specification and myogenic differentiation are associated with changes in chromatin topology.

Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in inducible Pax7-expressing skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterized at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. We identified key promoter-enhancer interaction motifs, namely, cliques and networks, and interactions that were dependent on Pax7 binding. Remarkably, we found that the majority of super-enhancers were bound by Pax7 and a cadre of associated factors that maintained an epigenetic memory of active enhancers in the absence of Pax7. Lastly, we identified a previously uncharacterized Pax7-bound enhancer hub that simultaneously regulates the essential myosin heavy chain cluster during skeletal muscle cell differentiation. Our studies lay the groundwork for understanding the three-dimensional conformation of chromatin in muscle stem cells. Overall design: Examination of chromatin structure re-organizaion during myogenic differentiation in the inducible Pax7-expressing (iPax7) skeletal muscle progenitors. (1) in situ Hi-C in both undifferentiated progenitor (doxycyline treated, +Dox) and differentiated (-Dox) iPax7 cells; (2) promoter capture Hi-C (pCHi-C) in both undifferentiated progenitor (+Dox) and differentiated (-Dox) iPax7 cells; (3) ChIP-seq of CTCF and Smc3 in both undifferentiated progenitor (+Dox) and differentiated (-Dox) iPax7 cells; (4) ATAC-seq of iPax7 ESC (-Dox), wild-type (WT) and Myod1 knock-out primary myoblasts.

本研究在可诱导表达主转录因子配对盒蛋白7（Paired box 7, Pax7）的骨骼肌祖细胞中，借助Hi-C、启动子捕获Hi-C（promoter-capture Hi-C, pCHi-C）及其他全基因组学技术，以高分辨率系统性表征了成肌定向与分化过程中，染色质区室及启动子锚定相互作用的时空重塑特征。研究团队鉴定出关键的启动子-增强子相互作用模体——即相互作用簇与调控网络，以及依赖于Pax7结合的相互作用。值得注意的是，绝大多数超级增强子均被Pax7及一组关联调控因子结合，这些因子可在Pax7缺失的情况下维持活性增强子的表观遗传记忆。最后，本研究鉴定出一个此前未被表征的Pax7结合增强子枢纽，其可在骨骼肌细胞分化过程中同时调控关键的肌球蛋白重链基因簇。本研究为理解肌肉干细胞中染色质的三维构象奠定了重要研究基础。总体实验设计：在可诱导表达Pax7（iPax7）的骨骼肌祖细胞中，探究成肌分化过程中的染色质结构重塑。具体实验分为四部分：(1) 对未分化祖细胞（经多西环素处理，+Dox）与分化细胞（-Dox）的iPax7细胞开展原位Hi-C（in situ Hi-C）检测；(2) 对上述两组细胞进行启动子捕获Hi-C（pCHi-C）检测；(3) 对上述两组细胞开展CCCTC结合因子（CCCTC-binding factor, CTCF）与结构维持蛋白3（Structural Maintenance of Chromosomes 3, Smc3）的染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）；(4) 对iPax7胚胎干细胞（embryonic stem cell, ESC，-Dox）、野生型（WT）及肌分化因子1（Myogenic differentiation 1, Myod1）基因敲除原代成肌细胞进行转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）。