MeCP2 represses the rate of transcriptional initiation of highly methylated long genes (RNA-Seq I)

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. Using multiple approaches, we demonstrate 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 in the brain directly, 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 it binds broadly across highly methylated regions of DNA, but acts from a distance to attenuate transcriptional initiation. Total RNA-seq was performed on whole-cell, nuclear, and chromatin-associated RNA from forebrain tissue from 10 MeCP2 KO and 10 WT and 10 MeCP2 R306C and 10 WT mice. Total RNA-seq was performed on visual cortex tissue from 6 Dnmt3a cKO and 6 WT mice.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调控转录的分子机制始终颇具挑战：一方面MeCP2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。本研究通过多种实验策略证实，MeCP2可通过与NCoR辅阻遏复合物（NCoR co-repressor complex）相互作用，抑制脑内高度甲基化长基因的新生RNA转录过程。通过直接检测脑内转录起始与延伸速率，我们发现MeCP2对转录延伸过程无显著可检测影响，反而会抑制RNA聚合酶II（Pol II）起始高度甲基化长基因转录的速率。上述研究结果提出了MeCP2功能的全新模型：该蛋白可广泛结合DNA的高度甲基化区域，却能通过远距离作用减弱转录起始过程。我们对10只MeCP2敲除（KO, knockout）小鼠、10只野生型（WT, wild type）小鼠，以及10只MeCP2 R306C突变型小鼠和10只野生型小鼠的前脑组织的全细胞RNA、细胞核RNA及染色质结合RNA进行了总RNA测序（Total RNA-seq）。此外，我们还对6只Dnmt3a条件性敲除（cKO, conditional knockout）小鼠与6只野生型小鼠的视觉皮层组织开展了总RNA测序。