How does the ubiquitously expressed ZFX mediate cell type-specific transcriptional regulation

普遍表达的 ZFX 如何介导细胞类型特异性转录调控

• 批准号: 10677572
• 负责人: Emily Hsu
• 金额: $ 6.91万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677572
• 关键词: Address; Affect; Binding; Binding Proteins; Binding Sites; Bioinformatics; Biological Assay; Biotinylation; C2H2 Zinc Finger; Canada; Cell Line; Cells; ChIP-seq; Clustered Regularly Interspaced Short Palindromic Repeats; CpG Islands; DNA-Binding Proteins; Data; Disease; Down-Regulation; Family; Family member; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genomics; Health; Human; Human Cell Line; In Situ; Individual; Knock-out; Label; Learning; Ligation; MCF7 cell; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Mediator; Mentors; Messenger RNA; Methods; Molecular Biology; Molecular Biology Techniques; Mutate; Mutation; N-terminal; Nuclear Proteins; Oncogenic; Pattern; Peptides; Physicians; Proliferating; Promoter Regions; Proteins; Proteomics; Proto-Oncogene Proteins c-myc; Publishing; Quantitative Reverse Transcriptase PCR; Regulation; Research; Site; Small Interfering RNA; Techniques; Testing; Training; Transactivation; Transcription Coactivator; Transcription Initiation Site; Transcriptional Regulation; Universities; ZFY protein; candidate identification; candidate validation; cdc Genes; cell type; experimental study; human disease; insight; knock-down; mutant; promoter; protein protein interaction; transcription factor; transcriptome; transcriptome sequencing; tumorigenesis

My research in the Farnham lab is centered on the ZFX family of C2H2 zinc finger transcription factors (TFs), including the highly related ZFX, ZFY, and ZNF711. Previously, our lab established CRISPR-mediated knock outs of ZFX and ZNF711 in HEK293T cells and demonstrated defective proliferation and altered expression of thousands of genes. Additional preliminary data from the Farnham lab has determined that ZFX and ZNF711 function as transcriptional activators which preferentially bind +240 bp downstream of transcription start sites (TSSs) at the majority of CpG island promoters in HEK293T cells. Further ZFX ChIP-seq experiments in several human cell lines revealed that the majority of ZFX binding occurs at the same promoters in all tested cell lines. Interestingly, RNA-seq experiments in these cell lines after ZFX siRNA knockdown revealed cell-type specific sets of ZFX-bound, downregulated genes. However, the transcriptional mechanisms by which ZFX regulates cell type-specific gene expression remain unclear. Therefore, in my proposal, I will determine how ZFX can regulate a distinct set of target genes in each cell type when it binds to mostly the same set of promoters in the different cell types. I hypothesize that ZFX interacts with different protein partners in each cell type to provide cell type-specific regulation from common promoter binding sites. In Aim#1, I propose to identify proteins that interact with ZFX in multiple cell lines using IP-MS and a sensitive proximity labeling technique TurboID-MS. Candidate ZFX binding partners will be individually validated with co-IP and in situ proximity ligation assay (PLA) experiments. To determine if candidates are key mediators of ZFX-regulated transcription, RNA-seq experiments after siRNA knockdown of corresponding mRNAs will be performed. I also aim to construct ZFX mutants, focusing first on the highly conserved peptide regions in the N-terminal transactivation domain, and perform co-IP experiments to map sites of protein interaction followed by transactivation assays to determine the effects of these mutations on transcription. In Aim#2, I will perform motif analysis in promoters of genes which are bound and regulated by ZFX in a cell type-specific manner to identify TFs that bind cooperatively with ZFX in the different cell lines. Binding of candidate TFs will be validated using ChIP-seq. Cell lines with CRISPR-mediated mutations of candidate transcription factor binding sites will be made, and qRT-PCR experiments will be performed to determine if candidate TF binding affects ZFX-mediated transcription of cell type-specific genes. My thorough mechanistic characterization in multiple cell lines of this interesting TF with a unique genomic binding pattern will make significant contributions to the field of transcription.

我在法纳姆实验室的研究集中在C2H2锌指转录因子（TFS）的ZFX家族中， 包括高度相关的ZFX，ZFY和ZNF711。以前，我们的实验室建立了CRISPR介导的敲门 HEK293T细胞中ZFX和ZNF711的出现，表现出缺陷的增殖和改变的表达 成千上万的基因。 Farnham实验室的其他初步数据确定了ZFX和ZNF711 功能充当转录激活剂，优先结合转录起始位点下游+240 bp （TSSS）在HEK293T细胞中大多数CpG岛启动子。进一步的ZFX ChIP-Seq实验 几种人类细胞系显示，大多数ZFX结合发生在所有测试中的同一启动子处 细胞系。有趣的是，ZFX siRNA敲低后这些细胞系中的RNA-seq实验揭示了细胞类型 特定的ZFX结合，下调的基因。但是，ZFX的转录机制 调节细胞类型特异性基因表达仍然不清楚。因此，在我的建议中，我将确定如何 当ZFX与每个单元格结合时，可以调节每个细胞类型中的一组不同的靶基因 不同细胞类型的启动子。我假设ZFX与不同的蛋白质伴侣相互作用 每种细胞类型可从共同启动子结合位点提供细胞类型特异性调节。在AIM＃1中，i 建议使用IP-MS识别多个细胞系中与ZFX相互作用的蛋白质和敏感的接近度 标签技术涡轮增压。候选ZFX绑定伙伴将通过Co-IP和IN单独验证 原位接近连接测定（PLA）实验。确定候选人是否是ZFX调节的关键调解人 将进行转录，siRNA敲低相应mRNA后的RNA-seq实验。我也是 旨在构建ZFX突变体，首先集中于N末端中高度保守的肽区域 反式激活结构域，并执行co-IP实验以映射蛋白质相互作用的位点，然后进行 进行反式激活测定，以确定这些突变对转录的影响。在AIM＃2中，我会表演 基因启动子的基序分析，这些启动子以细胞类型特异性方式与ZFX结合和调节 识别在不同细胞系中与ZFX合作结合的TF。候选TF的约束将是 使用chip-seq验证。具有CRISPR介导的候选转录因子结合突变的细胞系 将制作站点，并将执行QRT-PCR实验，以确定候选TF结合是否影响 ZFX介导的细胞类型特异性基因转录。我在多个的彻底机械表征 具有独特基因组结合模式的这种有趣的TF的细胞系将对 转录场。