MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription

Summary: Loss-of-function mutations in methyl-CpG binding protein 2 (MECP2) cause the neurodevelopmental disorder, Rett syndrome (RTT). The molecular mechanisms underlying MeCP2 function remain poorly understood. Here, using a MECP2 gain-of-function Drosophila model, we screened for genetic modifiers of MECP2-induced toxicity. Our approach identified several subunits of the Drosophila super elongation complex (SEC), a P-TEFb containing elongation factor that releases promoter-proximally paused RNA polymerase II (RNA pol II), as genetic interactors of MECP2. MeCP2 directly interacts with AFF4, the scaffold of the SEC, and facilitates its binding on a subset of genes that regulate neuronal development and synaptic function in the mouse cortex. Furthermore, genes with reduced AFF4 binding showed reduced RNA pol II binding in their genebody and decreased RNA expression in Mecp2 null mice. Taken together, we propose that MeCP2 interacts with the SEC to facilitate the release of RNA pol II and thereby support gene expression. Experimental protocols (extraction, library construction) and data processing steps are described in the GEO repository repository GSE275329. Experiment type Genome binding/occupancy profiling by high throughput sequencing. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) of pSer2 in wildtype and Mecp2 null cortex at 7-weeks of age Organism Mus musculus Platform Illumina NovaSeq 6000 Design Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) of AFF4 and total RNA Pol II in wildtype and Mecp2 null cortex at 7-weeks of age Tissue Cortex Genotypes Wildtype (WT), Mecp2 null (Null) Molecule Genomic DNA ChIP Ab pSer2 RNA pol II antibody (EMD Millipore; 04-1571) Sequencing strategy Single End, x100 bases Genome build mm10 Raw files type fastq Processed files type bigWig摘要：甲基CpG结合蛋白2（methyl-CpG binding protein 2, MECP2）的功能丧失型突变会引发神经发育疾病雷特综合征（Rett syndrome, RTT）。目前学界对MeCP2发挥功能的分子机制仍知之甚少。本研究借助MECP2功能获得性果蝇模型，筛选可调控MECP2诱导毒性的遗传修饰因子。研究鉴定出果蝇超级延伸复合物（super elongation complex, SEC）的多个亚基作为MECP2的遗传互作因子——该复合物是一类包含P-TEFb的延伸因子，可释放启动子近端暂停的RNA聚合酶II（RNA pol II）。MeCP2可直接与SEC的支架亚基AFF4结合，并促进其在小鼠皮层中调控神经元发育与突触功能的部分基因上的结合。此外，在Mecp2敲除小鼠中，AFF4结合水平降低的基因，其基因体上的RNA pol II结合水平与RNA表达量均出现下调。综上，我们提出MeCP2可与SEC互作以促进RNA pol II的释放，进而维持基因的正常表达。 实验方案（包括样本提取、文库构建）与数据处理步骤已在GEO数据库数据集GSE275329中详述。 实验类型 基于高通量测序的基因组结合/占据谱分析。对7周龄野生型与Mecp2敲除小鼠皮层开展pSer2位点RNA聚合酶II的染色质免疫沉淀测序（ChIP-seq） 实验生物 小家鼠（Mus musculus） 测序平台 Illumina NovaSeq 6000 实验设计 对7周龄野生型与Mecp2敲除小鼠皮层分别进行AFF4与总RNA聚合酶II的染色质免疫沉淀测序（ChIP-seq） 实验组织 大脑皮层 基因型 野生型（Wildtype, WT）、Mecp2敲除型（Mecp2 null, Null） 检测分子 基因组DNA ChIP抗体 pSer2 RNA pol II抗体（EMD Millipore；04-1571） 测序策略 单端测序，读长100 bp 基因组版本 mm10 原始文件格式 fastq 处理后文件格式 bigWig