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Farnham实验室的研究集中在C2H2锌指转录因子(TF)的ZFX家族上， 包括高度相关的ZFX，ZFY和ZNF711。此前，我们的实验室建立了CRISPR介导的敲打 ZFX和ZNF711在HEK293T细胞中的缺失和增殖缺陷及表达变化 成千上万的基因。来自Farnham实验室的其他初步数据已经确定ZFX和ZNF711 作为转录激活子，优先结合转录起始点下游+240个碱基对 (Tsss)在HEK293T细胞中的大多数CpG岛启动子。进一步的ZFX芯片-SEQ实验 几个人类细胞株显示，在所有测试中，大多数ZFX结合发生在相同的启动子上 细胞系。有趣的是，ZFX siRNA敲除后，这些细胞系中的rna-seq实验揭示了细胞类型。 一组特定的ZFX结合、下调的基因。然而，ZFX通过其转录机制 调节细胞类型特异性基因表达的机制尚不清楚。因此，在我的提案中，我将决定如何 当ZFX与几乎相同的一组靶基因结合时，它可以调节每种细胞类型中的一组不同的靶基因 不同细胞类型中的启动子。我假设ZFX与不同的蛋白质伙伴相互作用 每种细胞类型提供来自共同启动子结合位点的细胞类型特异性调节。在目标1中，我 建议使用IP-MS和灵敏邻近技术在多个细胞系中鉴定与ZFX相互作用的蛋白质 标记技术TurboID-MS候选ZFX绑定合作伙伴将通过co-IP和In 原位近距离连接实验(PLA)。确定候选人是否是ZFX监管的关键调解人 在相应的mRNAs的siRNA被敲除后，将进行转录、RNA-seq实验。我也是 目的构建ZFX突变体，首先定位N端高度保守的多肽区域 反式激活结构域，并执行co-IP实验以绘制蛋白质相互作用的位置，随后 转录激活分析以确定这些突变对转录的影响。在目标2中，我将表演 ZFX以细胞类型特异性方式结合和调节基因启动子中的基序分析 鉴定在不同细胞系中与ZFX协同结合的转录因子。对候选TF的约束将是 使用芯片顺序进行验证。CRISPR介导的候选转录因子结合突变细胞系 将建立位点，并进行qRT-PCR实验以确定候选转铁蛋白结合是否会影响 ZFx介导的细胞类型特异性基因的转录。我在多个方面的彻底机械化的描述 这种有趣的转铁蛋白细胞系具有独特的基因组结合模式，将对 转录领域。