H3K4 demethylase KDM5B regulates global dynamics of transcription elongation and alternative splicing in embryonic stem cells [RNA-seq]

Epigenetic regulation of chromatin plays a critical role in controlling embryonic stem (ES) cell self-renewal and pluripotency. However, the roles of histone demethylases and activating histone modifications such as trimethylated histone 3 lysine 4 (H3K4me3) in transcriptional events such as RNA polymerase II (RNAPII) elongation and alternative splicing are largely unknown. In this study, we show that KDM5B, which demethylates H3K4me3, plays an integral role in regulating RNAPII occupancy, transcriptional initiation and elongation, and alternative splicing events in ES cells. Depletion of KDM5B leads to altered RNAPII promoter occupancy, and decreased RNAPII initiation and elongation rates at active genes and at genes marked with broad H3K4me3 domains. Moreover, our results demonstrate that spreading of H3K4me3 from promoter to gene body regions, which is mediated by depletion of KDM5B, modulates RNAPII elongation rates and RNA splicing in ES cells. We further show that KDM5B is enriched nearby alternatively spliced exons, and depletion of KDM5B leads to altered levels of H3K4 methylation in alternatively spliced exon regions, which is accompanied by differential expression of these ASEs. Altogether, our data indicate an epigenetic role for KDM5B in regulating RNAPII elongation and alternative splicing, which may support the diverse mRNA repertoire in ES cells. RNA-Seq of murine shLuc and shKdm5b ES cells.

染色质的表观遗传调控在调控胚胎干细胞（embryonic stem, ES）的自我更新与多能性方面发挥关键作用。然而，组蛋白去甲基化酶以及诸如三甲基化组蛋白H3赖氨酸4（trimethylated histone 3 lysine 4, H3K4me3）这类激活型组蛋白修饰，在RNA聚合酶II（RNA polymerase II, RNAPII）延伸及可变剪接等转录事件中的作用在很大程度上尚不明确。本研究表明，可去除H3K4me3的组蛋白去甲基化酶KDM5B，在胚胎干细胞中对RNAPII结合、转录起始与延伸以及可变剪接事件的调控中发挥不可或缺的核心作用。敲低KDM5B会使RNAPII在启动子区域的结合发生改变，并在活跃基因以及带有宽幅H3K4me3结构域标记的基因中，降低RNAPII的起始与延伸速率。此外，本研究结果证实，KDM5B敲低所介导的H3K4me3从启动子区向基因体区域的扩散，可调控胚胎干细胞中的RNAPII延伸速率与RNA剪接过程。本研究进一步发现，KDM5B在可变剪接外显子（alternative spliced exons, ASEs）附近富集；敲低KDM5B会使可变剪接外显子区域的H3K4甲基化水平发生改变，并伴随这些可变剪接事件的差异表达。综上，本研究数据表明KDM5B在调控RNAPII延伸与可变剪接中发挥表观遗传调控作用，这或有助于维持胚胎干细胞中多样的mRNA转录组谱。本研究对转染短发夹RNA靶向Luc基因（shLuc）与靶向Kdm5b基因（shKdm5b）的小鼠胚胎干细胞进行了RNA测序（RNA-Seq）。