Effects of dynamic cytosine methylation on alternative pre-mRNA splicing in T lymphocytes

Intragenic 5-methylcytosine and CTCF mediate opposing affects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF binding sites were unclear. In this study, we identify the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. Overall design: Transcriptional profiling of human T-lymphocytes in the naïve and activated states was performed by RNA-Seq. Methylation status (5mC and 5hmC) was assayed by medIP-Seq. CTCF binding sites were identified by ChIP-Seq.

基因内5-甲基胞嘧啶（5-methylcytosine）与CTCF（CCCTC结合因子）对前体mRNA（pre-mRNA）剪接具有相反调控效应：CTCF通过RNA聚合酶II（RNA polymerase II）暂停，促进弱上游外显子的保留；而5-甲基胞嘧啶则会驱逐CTCF，进而引发外显子排除。然而，调控CTCF结合位点处DNA动态甲基化的机制此前尚不明确。本研究鉴定发现，甲基胞嘧啶双加氧酶TET1与TET2可通过将5-甲基胞嘧啶转化为其氧化衍生物，作为CTCF介导的可变剪接的活性调控因子。
实验设计概述：
采用RNA测序（RNA-Seq）对幼稚态与活化态人T淋巴细胞开展转录组分析；通过甲基化DNA免疫沉淀测序（medIP-Seq）检测基因组甲基化状态（涵盖5-甲基胞嘧啶（5mC）与5-羟甲基胞嘧啶（5hmC））；借助染色质免疫沉淀测序（ChIP-Seq）鉴定CTCF结合位点。