Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel.
RNA. 2018 Oct;24(10):1351-1362. doi: 10.1261/rna.064865.117. Epub 2018 Jul 12.
Alternative splicing (AS) contributes to proteome diversity. As splicing occurs cotranscriptionally, epigenetic determinants such as DNA methylation likely play a part in regulation of AS. Previously, we have shown that DNA methylation marks exons and that a loss of DNA methylation alters splicing patterns in a genome-wide manner. To investigate the influence of DNA methylation on splicing of individual genes, we developed a method to manipulate DNA methylation in vivo in a site-specific manner using the deactivated endonuclease Cas9 fused to enzymes that methylate or demethylate DNA. We used this system to directly change the DNA methylation pattern of selected exons and introns. We demonstrated that changes in the methylation pattern of alternatively spliced exons, but not constitutively spliced exons or introns, altered inclusion levels. This is the first direct demonstration that DNA methylation of exon-encoding regions is directly involved in regulation of AS.
可变剪接 (AS) 有助于蛋白质组的多样性。由于剪接是在共转录过程中发生的，因此表观遗传决定因素（如 DNA 甲基化）可能在 AS 的调控中发挥作用。此前，我们已经表明 DNA 甲基化标记外显子，并且 DNA 甲基化的丧失会以全基因组的方式改变剪接模式。为了研究 DNA 甲基化对个体基因剪接的影响，我们开发了一种使用与甲基化或去甲基化 DNA 的酶融合的失活内切酶 Cas9 在体内以特定方式操纵 DNA 甲基化的方法。我们使用该系统直接改变选定外显子和内含子的 DNA 甲基化模式。我们证明，可变剪接外显子的甲基化模式的变化，但不是组成性剪接外显子或内含子的变化，改变了包含水平。这是第一个直接证明编码外显子的区域的 DNA 甲基化直接参与 AS 的调控的证明。
