Expression data from mouse myogenic differentiation and ectopic MeCP2. Mus musculus

The methyl-cytosine binding protein 2 (MeCP2) is a reader of epigenetic DNA methylation marks and necessary and sufficient to reorganize 3D heterochromatin structure during cellular differentiation, e.g., myogenesis. In addition to global expression profile changes, myogenic differentiation is accompanied by 3D-heterochromatin reorganization that is dependent on MeCP2. MeCP2 is enriched at pericentric heterochromatin foci (chromocenters). During myogenesis, the total heterochromatin foci number per nucleus decreases while foci volumes and MeCP2 protein levels increase. Ectopic MeCP2 is able to mimic similar heterochromatin restructuring in the absence of differentiation. We compared expression profile changes during myogenic differentiation to changes related to MeCP2-induced heterochromatin reorganization in the absence of differentiation. We used the Affymetrix 430.2 microarray system to study the expression profile changes during myogenic differentiation (myotubes vs myoblast) and MeCP2 related expression changes in transiently transfected myoblasts (high vs. low MeCP2 protein levels) in five independent experiments for each condition. Overall design: To obtain expression profile changes during myogenic differentiation, five biological replicas of undifferentiated Pmi 28 myoblasts (MB) and in vitro differentiated Pmi 28 myotubes (MT) were cultured as described elsewhere. To obtain MeCP2 dependent and differentiation independent expression profile changes, five biological replica of undifferentiated Pmi 28 myoblasts were transiently transfected with MeCP2-YFP. Subsequently, transfected cells were FACSorted to generate MeCP2-high (R4) and -low (R5) level fractions. Total RNA was prepared for each sample out of cell pellets containing 6.5x10exp5 to 1.7x10exp6 cells and further processed according to the GeneChip Expression Analysis Technical Manual by Affymetrix and hybridized to Affymetrix 430.2 microarrays.甲基胞嘧啶结合蛋白2（methyl-cytosine binding protein 2，MeCP2）是表观遗传DNA甲基化标记的识别蛋白，在细胞分化（如肌发生过程）中，其对于重塑3D异染色质结构既是必需的，也是充分的。除全局基因表达谱发生改变外，肌发生过程中的3D异染色质重塑同样依赖于MeCP2。MeCP2富集于着丝粒周边异染色质焦点（染色质中心）。在肌发生过程中，每个细胞核内的异染色质焦点总数会减少，而焦点体积与MeCP2蛋白水平则会升高。异位表达的MeCP2可在未发生分化的细胞中模拟类似的异染色质重构。 我们将肌发生分化过程中的表达谱变化，与未发生分化时MeCP2诱导的异染色质重构相关的表达变化进行了对比。本研究使用Affymetrix 430.2微阵列系统，针对两种实验条件开展各5次独立重复实验，以分别探究肌发生分化过程中的表达谱变化（肌管 vs 成肌细胞），以及瞬时转染成肌细胞中与MeCP2相关的表达变化（高MeCP2蛋白水平 vs 低MeCP2蛋白水平）。 整体实验设计：为获取肌发生分化过程中的表达谱变化，我们按照已发表方法培养了5批未分化的Pmi 28成肌细胞（MB）与体外分化获得的Pmi 28肌管（MT）生物学重复样本。为获取依赖于MeCP2且不依赖于分化的表达谱变化，我们对5批未分化的Pmi 28成肌细胞生物学重复样本进行MeCP2-YFP瞬时转染。随后通过荧光激活细胞分选（FACS）对转染细胞进行分选，得到MeCP2高表达（R4）与低表达（R5）的细胞组分。从每批样本的细胞沉淀（细胞量为6.5×10^5至1.7×10^6个细胞）中提取总RNA，随后参照Affymetrix公司的《GeneChip表达分析技术手册》进行后续处理，并与Affymetrix 430.2微阵列进行杂交。