FBXL19 transcriptionally primes promoters of developmental genes via CDK8-Mediator recruitment [ChIP-seq 2]

Regulatory elements called CpG islands (CGIs) are associated with the majority of mammalian gene promoters and have been proposed to play an important role in gene expression. The regulatory capacity of CGIs is thought to rely upon a family of proteins that recognise CGIs through their ZF-CxxC DNA binding domains to recruit chromatin-modifying activities to gene promoters. Through studying FBXL19, a poorly characterized ZF-CxxC domain-containing protein, we have discovered that it specifically interacts with the CDK8-Mediator complex in mouse embryonic stem cells (ESCs). Intriguingly, FBXl19 recruits CDK8-Mediator to a subset of CGI-associated promoters of repressed developmental genes in ESCs. We show that FBXL19-dependent recruitment of CDK8 in ESCs is required for the proper induction of the associated genes during ESC differentiation. This is consistent with early embryonic lethality of in FBXl19 deficient mice. Together, our observations highlight a novel role for FBXL19 in priming developmental gene expression, via recruitment of CDK8 to their CGI promoters, in order to support normal gene induction during differentiation and development. Overall design: Fbxl19fl/fl embryonic stem cells were used to delete the CXXC domain of FBXL19. CDK8 occupancy was then profiled by ChIP-seq in biological duplicates to assess the contribution of FBXL19 to CDK8 binding to chromatin.被称为CpG岛（CpG islands，CGIs）的调控元件与大多数哺乳动物基因启动子相关，且被认为在基因表达中发挥重要作用。CGIs的调控能力被认为依赖于一类蛋白质家族：这类蛋白质通过其ZF-CxxC DNA结合结构域（ZF-CxxC DNA binding domains）识别CGIs，从而将染色质修饰活性招募至基因启动子。我们通过研究功能尚未得到充分表征的含ZF-CxxC结构域的蛋白质FBXL19，发现其在小鼠胚胎干细胞（embryonic stem cells，ESCs）中可与CDK8-中介体复合物（CDK8-Mediator complex）特异性相互作用。值得注意的是，FBXL19可将CDK8-中介体复合物招募至ESCs中一类受抑制的发育基因的CGI相关启动子子集。我们证实，ESCs中依赖FBXL19的CDK8招募，对于ESC分化过程中相关基因的正确诱导是必需的，这与FBXL19缺陷小鼠的早期胚胎致死表型一致。综上，我们的研究结果揭示了FBXL19的一种全新功能：通过将CDK8招募至发育基因的CGI启动子区域，实现发育基因表达的预激活，从而支持分化与发育过程中正常的基因诱导。本研究的总体实验设计如下：利用Fbxl19fl/fl胚胎干细胞删除FBXL19的CXXC结构域，随后通过生物学重复的染色质免疫共沉淀测序（ChIP-seq）分析CDK8的染色质结合情况，以评估FBXL19对CDK8染色质结合的贡献。