Incomplete MyoD-induced transdifferentiation is mediated by chromatin remodeling deficiencies [ChIP-Seq]. Homo sapiens

MyoD is known to transdifferentiate fibroblasts into muscle-like cells. Despite phenotypic resemblance and expression of myogenic marker genes in transdifferentiated cells, our global gene expression data suggests that ~100 genes, many involved in muscle development and function, remain non-reprogrammed. To understand this incomplete reprogramming, we characterized genome-wide chromatin accessibility and MyoD binding in human primary myoblasts and in MyoD-induced skin fibroblast cells. Our analyses revealed thousands of sites with incomplete chromatin reprogramming.Combined analyses of gene expression and epigenetic profiles revealed that many myogenic genes not upregulated during the transdifferentiation process have undergone MyoD-dependent chromatin remodeling, but to a significantly lower extent than reprogrammed genes. Our findings suggest that incomplete MyoD-induced transdifferentiation is due to chromatin-remodeling deficiencies, and that additional factors are required to transdifferentiate cells into a state more similar to myoblasts. Overall design: Fibroblast cells were transduced with a vector carrying human MyoD gene and induced by tetracycline for expression. Dnase-seq, ChIP-seq and RNA-seq were used to identify transcription factors binding, differential chromtin structural change, and differential expression at on-target and off-target sites.

肌分化因子（MyoD）可将成纤维细胞（fibroblasts）转分化为肌样细胞。尽管转分化获得的细胞在表型上与肌细胞高度相似，且表达肌源性标记基因，但我们的全局基因表达谱数据显示，约100个基因（其中多数参与肌肉发育与功能调控）仍未完成重编程。为解析这一不完全重编程现象，我们对人原代成肌细胞（human primary myoblasts）以及经MyoD诱导的皮肤成纤维细胞开展了全基因组染色质开放程度与MyoD结合位点的表征分析。分析结果显示，存在数千个染色质重编程不完全的位点。对基因表达与表观遗传谱的联合分析表明，诸多在转分化过程中未被上调的肌源性基因虽发生了MyoD依赖的染色质重塑，但其重塑程度显著低于已完成重编程的基因。本研究结果提示，MyoD诱导的转分化不完全源于染色质重塑缺陷，且需要额外的调控因子，方可将细胞转分化为更接近成肌细胞的状态。实验设计概述：将携带人MyoD基因的载体转导至成纤维细胞，通过四环素诱导其表达。采用脱氧核糖核酸酶I测序（Dnase-seq）、染色质免疫共沉淀测序（ChIP-seq）及RNA测序（RNA-seq）技术，分别鉴定转录因子结合位点、染色质结构差异变化以及靶位点与脱靶位点的差异表达。