MeCP2 represses the rate of transcriptional initiation of highly methylated long genes (ChIP-Seq II)

Mutations in the methyl-DNA-binding repressor protein MeCP2 cause the devastating neurodevelopmental disorder Rett syndrome. It has been challenging to understand how MeCP2 regulates transcription because MeCP2 binds broadly across the genome, and MeCP2 mutations are associated with widespread small-magnitude changes in neuronal gene expression. We demonstrate here that MeCP2 represses nascent RNA transcription of highly methylated long genes in the brain through its interaction with the NCoR co-repressor complex. By measuring the rates of transcriptional initiation and elongation directly in the brain, we find that MeCP2 has no measurable effect on transcriptional elongation, but instead represses the rate at which Pol II initiates transcription of highly methylated long genes. These findings suggest a new model of MeCP2 function in which MeCP2 binds broadly across highly methylated regions of DNA, but acts at transcription start sites to attenuate transcriptional initiation. ChIP-seq was performed from WT and MeCP2 KO forebrain for H3K36me3 (n=10), H3K27ac (n=10), MeCP2 Ab1 (n=2), MeCP2 Ab2 (n=2), and pre-immune serum for MeCP2 Ab1 (n=2) with input controls. ChIP-seq was performed from WT and MeCP2 R306C forebrain for H3K9ac (n=5), H3K27ac (n=2), H4K12ac (n=3), and MeCP2 (n=4) with input controls. ChIP-seq was peformed from WT cortical neurons for YY1, and from WT forebrain for H3K79me2 (n=2) with input controls.

甲基化DNA结合阻遏蛋白MeCP2的突变会引发毁灭性神经发育障碍瑞特综合征（Rett syndrome）。长期以来，阐明MeCP2调控转录的具体机制颇具挑战：一方面MeCP2可在全基因组范围内广泛结合，另一方面其突变会导致神经元基因表达出现广泛的小幅变化。本研究证实，MeCP2可通过与NCoR辅阻遏复合物（NCoR co-repressor complex）相互作用，特异性抑制大脑内高度甲基化长基因的新生RNA转录。通过直接检测脑组织内转录起始与延伸的速率，我们发现MeCP2对转录延伸并无可检测到的调控效应，反而能够抑制RNA聚合酶II（Pol II）启动高度甲基化长基因转录的速率。上述研究结果提出了一种全新的MeCP2功能模型：MeCP2虽广泛结合于DNA高度甲基化区域，但仅在转录起始位点发挥作用，以衰减转录起始过程。针对野生型（WT）及MeCP2敲除（KO）小鼠前脑组织，本研究开展了染色质免疫共沉淀测序（ChIP-seq, chromatin immunoprecipitation sequencing）实验，检测靶标包括H3K36me3（n=10）、H3K27ac（n=10）、MeCP2抗体1（n=2）、MeCP2抗体2（n=2），以及MeCP2抗体1的预免疫血清（n=2），所有实验均设置了input对照。针对野生型及MeCP2 R306C突变型小鼠前脑组织，本研究开展了针对H3K9ac（n=5）、H3K27ac（n=2）、H4K12ac（n=3）及MeCP2（n=4）的ChIP-seq实验，并均设置了input对照。本研究还针对野生型皮层神经元开展了阴阳因子1（YY1）相关ChIP-seq实验，针对野生型小鼠前脑组织开展了H3K79me2（n=2）相关ChIP-seq实验，两类实验均设置了input对照。