Reduction of ZFX levels decreases histone H4 acetylation and increases Pol2 pausing at target promoters [RNA-Seq]

The ZFX transcriptional activator binds to CpG island promoters, with a major peak at ~200-250bp downstream from transcription start sites. Because ZFX binds within the transcribed region, we investigated whether it regulates transcriptional elongation. We used GRO-seq to show that loss or reduction of ZFX increased the pause ratio at ZFX-regulated promoters. To further investigate the mechanisms by which ZFX regulates transcription, we determined regions of the protein needed for transactivation and for recruitment to the chromatin. Interestingly, although ZFX has 13 grouped zinc fingers, deletion of the first 11 fingers produces a protein that can still bind to chromatin and activate transcription. We next used TurboID-MS to detect ZFX-interacting proteins, identifying ZNF593, proteins that interact with the N-terminal transactivation domain (e.g. histone modifying proteins), and proteins that interact with ZFX when it is bound to the chromatin (e.g. TAFs and other histone modifying proteins). Our studies support a model in which ZFX enhances elongation at target promoters by recruiting H4 acetylation complexes and reducing pausing. Overall design: To profile the effects of ZFX on gene expression in human cell lines. siRNA knockdowns with siZFX and/or siZNF593 and a control siRNA oligo were performed in LN-229 cells with 3 biological replicates. 293T cells with ZFX and ZNF711 knockouts (DKO) were transfected with mutant ZFX constructs or the empty vector control pCMV6-Entry with 3 biological replicates.

ZFX转录激活因子结合CpG岛启动子，在转录起始位点下游约200-250bp处存在主要结合峰。由于ZFX结合于转录区域内，本研究旨在探究其是否调控转录延伸过程。本研究借助GRO-seq技术证实，ZFX的缺失或表达下调会提升ZFX调控启动子处的转录暂停比率。

为进一步解析ZFX调控转录的分子机制，本研究明确了该蛋白中参与反式激活以及招募至染色质的关键区域。值得注意的是，尽管ZFX含有13个成簇锌指结构域，删除前11个锌指的突变体蛋白仍可结合染色质并激活转录。

随后本研究借助TurboID-MS技术检测ZFX的互作蛋白，鉴定出ZNF593、与N端反式激活结构域互作的蛋白（如组蛋白修饰蛋白），以及ZFX结合染色质时所互作的蛋白（如TAFs及其他组蛋白修饰蛋白）。本研究支持如下模型：ZFX通过招募H4乙酰化复合物并降低转录暂停比率，增强靶启动子处的转录延伸。

实验总体设计：为分析ZFX对人细胞系基因表达的调控效应，本研究在LN-229细胞中分别开展siZFX、siZNF593、二者联合敲低以及对照siRNA寡核苷酸处理的敲低实验，每组设置3次生物学重复。在携带ZFX与ZNF711双敲除（DKO）的293T细胞中，将突变型ZFX表达构建体或空载体对照pCMV6-Entry转染至细胞中，同样设置3次生物学重复。