Exploring high-resolution chromatin interaction changes and functional enhancers of myogenic marker genes during myogenic differentiation

Skeletal muscle differentiation (myogenesis) is a complex and highly coordinated biological process regulated by a series of myogenic marker genes. Chromatin interactions between gene's promoters and their enhancers have an important role in transcriptional control. However, the high-resolution chromatin interactions of myogenic genes and their functional enhancers during myogenesis remain largely unclear. Here, we used circularized chromosome conformation capture coupled with next-generation sequencing (4C-seq) to investigate eight myogenic marker genes in C2C12 myoblasts (C2C12-MBs) and C2C12 myotubes (C2C12-MTs). We revealed dynamic chromatin interactions of these marker genes during differentiation, and identified 163 and 314 significant interaction sites (SISs) in C2C12-MBs and C2C12-MTs, respectively. The interacting genes of SISs in C2C12-MTs were mainly involved in muscle development, and histone modifications of the SISs changed during differentiation. Through functional genomic screening, we also identified 25 and 41 putative active enhancers in C2C12-MBs and C2C12-MTs, respectively. Using luciferase reporter assays for putative enhancers of Myog and Myh3, we identified eight activating enhancers. Furthermore, dCas9-KRAB epigenome editing and RNA-seq revealed a role for Myog enhancers in the regulation of Myog expression and myogenic differentiation in the native genomic context. Taken together, this study lays the groundwork for understanding 3D chromatin interaction changes of myogenic genes during myogenesis and provides insights that contribute to our understanding of the role of enhancers in regulating myogenesis. Overall design: We performed gene expression profiling analysis using data obtained from RNA-seq of dCas9-KRAB cells and Myog-En5 cells on days 5 of differentiation

骨骼肌分化（肌发生，myogenesis）是一类由一系列肌源性标记基因调控的复杂且高度协调的生物学过程。基因启动子与其增强子之间的染色质相互作用，在转录调控中发挥关键作用。然而，肌发生过程中肌源性基因与其功能性增强子的高分辨率染色质相互作用，目前仍未得到充分阐明。本研究采用环形染色质构象捕获结合高通量测序（4C-seq）技术，对C2C12成肌细胞（C2C12-MBs）与C2C12肌管（C2C12-MTs）中的8个肌源性标记基因开展研究。研究揭示了这些标记基因在分化过程中的动态染色质相互作用，并分别在C2C12-MBs和C2C12-MTs中鉴定出163个和314个显著相互作用位点（SISs）。C2C12-MTs中显著相互作用位点的相互作用基因主要参与肌肉发育过程，且显著相互作用位点的组蛋白修饰在分化进程中发生动态改变。通过功能基因组筛选，本研究还分别在C2C12-MBs和C2C12-MTs中鉴定出25个和41个推定活性增强子。针对Myog与Myh3的推定增强子开展荧光素酶报告基因实验，本研究鉴定出8个具有激活功能的增强子。此外，借助dCas9-KRAB表观基因组编辑与RNA测序（RNA-seq）分析，证实了Myog增强子在天然基因组背景下，可调控Myog的表达并参与肌发生过程。综上，本研究为阐明肌发生过程中肌源性基因的三维染色质相互作用变化奠定了基础，并为理解增强子在肌发生调控中的作用提供了新的研究视角。整体实验设计：本研究对分化第5天的dCas9-KRAB细胞与Myog-En5细胞的RNA测序数据进行了分析。