siRNAs in C2C12

The two catalytic subunits of the SWI/SNF chromatin remodeling complex - Brg1 and Brm - have been often implicated as essential components of common biological processes, suggesting a functional redundancy between these two proteins. For instance, earlier works indicated that both proteins are required for the activation of the myogenic program. However, their mutually exclusive pattern of incorporation in SWI/SNF complexes with variable composition and functional heterogeneity predicts that Brg1- and Brm-based SWI/SNF complexes execute specific functions to coordinate gene expression in multistep programs, such as skeletal myogenesis. We detected a distinct expression profile of Brg1 and Brm during the myogenic program, with Brm being upregulated in differentiating myocytes. Genetic knockdown of either Brg1 or Brm in skeletal myoblasts, followed by gene expression analysis, revealed discrete functions during myogenic differentiation. While Brm is required for the exit from the cell cycle at the early stages of differentiation, by repressing CyclinD1 transcription, Brg1 is required for the activation of muscle gene transcription. Interestingly, at later stages of differentiation Brg1 and Brm cooperate to the activation of a cluster of common target genes. Thus, Brg1 and Brm appear to coordinate activation and repression of distinct subsets of genes during initial phase myogenesis, and converge to cooperatively activate the expression of muscle genes at later stages. C2C12 myoblasts treated with siBRM or siBRG1 or siSCR in growth medium (GM) and differentiated by medium replacemnte (DM ). Celles were collected at 18h and 48h from the onset of DM for RNA extraction and microarray analysis. Arrays were used to generate 6 data sets in duplicate.To address the question of which genes were regulated by BRM and/or BRG1 the fold changes in gene expression were calculated between Scrambled siRNA and the BRM or BRG1 siRNA. The experiments were repeated at 2 different time points (18 hours and 48 hours) .

SWI/SNF染色质重塑复合物（SWI/SNF chromatin remodeling complex）的两个催化亚基——Brg1与Brm——常被认定为常见生物学过程的必需组分，提示二者间存在功能冗余。例如早期研究表明，两种蛋白均为肌源性程序的激活所必需。然而，它们在组成可变、功能异质性的SWI/SNF复合物中呈互斥整合模式，这预示基于Brg1和Brm的SWI/SNF复合物各自执行特定功能，以协调多步骤生物学程序（如骨骼肌发生）中的基因表达。我们在肌源性程序中检测到Brg1与Brm的独特表达谱，其中Brm在分化中的肌细胞内被上调。在骨骼肌成肌细胞中分别敲低Brg1或Brm，随后开展基因表达分析，结果显示二者在肌源性分化过程中发挥离散功能：Brm通过抑制CyclinD1的转录，在分化早期介导细胞周期退出；而Brg1则是肌肉基因转录激活所必需的。值得注意的是，在分化后期，Brg1与Brm协同激活一组共同的靶基因。综上，在肌发生初始阶段，Brg1与Brm分别协调不同基因子集的激活与抑制，而在后期则协同激活肌肉基因的表达。本研究将C2C12成肌细胞在生长培养基（GM）中分别用siBRM、siBRG1或阴性对照siSCR处理，随后更换为分化培养基（DM）以诱导分化。分别在分化启动后的18小时与48小时收集细胞，用于RNA提取与微阵列分析。微阵列实验共生成6组重复数据集。为探究哪些基因受BRM和/或BRG1调控，我们计算了阴性对照siSCR处理组与BRM或BRG1 siRNA处理组之间的基因表达倍数变化。实验在两个不同时间点（18小时与48小时）重复开展。