Quiescence specific β-catenin transcriptional networks in myogenic cells

Wnt/β-catenin signaling is active during cellular quiescence in muscle myoblasts. Exposure of quiescent myoblasts to Wnt3a led to deregulation of genes associated with both myogenic differentiation and proliferation. Genome-wide analysis Ctnnb1 by ChIP-chip revealed quiescence-specific enrichment on genes of multiple functional classes. The major class of genes bound by Ctnnb1were Wnt pathway genes and all occupied promoters were highly enriched for TCF DNA binding motifs. Cross-comparison of ChIP-Chip with transcriptome data revealed both transcriptional activation as well as repression in Ctnnb1 occupied genes. Inhibition of Ctnnb1-mediated transactivation using shRNA and pharmacological agents led to de-regulation of the quiescence-associated transcriptional profile. Ctnnb1 binding is associated with repression of myogenic genes and cell cycle progression factors while maintaining differential response by these genes to Wnt signaling during quiescence. Microarray analysis of Wnt3A treated G0 myoblasts compared to untreated G0 cells Myogenic Cell were synchronized in G0 by resuspending them in semi-solid media and treated with purified Wnt3A for 48 hours and harvested for RNA isolation

Wnt/β-连环蛋白（Wnt/β-catenin）信号通路在肌肉成肌细胞的细胞静息状态下具有活性。将处于静息状态的成肌细胞暴露于Wnt3a后，会导致与肌源性分化及增殖相关的基因表达失调。通过染色质免疫沉淀-芯片（ChIP-chip）对Ctnnb1进行全基因组分析，发现其在多个功能类别的基因上存在静息状态特异性富集。Ctnnb1结合的基因中最主要的类别为Wnt通路相关基因，且所有被结合的启动子区域均高度富集TCF DNA结合基序。将ChIP-chip数据与转录组数据进行交叉比对后发现，Ctnnb1结合的基因同时存在转录激活与转录抑制两种调控模式。通过短发夹RNA（shRNA）及药理学试剂抑制Ctnnb1介导的反式激活作用，会导致静息状态相关的转录谱发生失调。Ctnnb1的结合与肌源性基因及细胞周期进程相关因子的抑制有关，同时可使这些基因在静息状态下对Wnt信号通路维持差异化应答。本研究针对经Wnt3A处理的G0期成肌细胞与未处理的G0期成肌细胞开展微阵列分析：实验中将成肌细胞重悬于半固体培养基中同步至G0期，经纯化的Wnt3A处理48小时后收集样本并提取RNA。