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C2 H2锌指转录因子（TF）的ZFX家族， 包括高度相关的ZFX、ZFY和ZNF 711。此前，我们的实验室建立了CRISPR介导的敲除 HEK 293 T细胞中ZFX和ZNF 711的表达出现缺陷，并表现出增殖缺陷和ZNF 711的表达改变 成千上万的基因。法纳姆实验室的其他初步数据已经确定，ZFX和ZNF 711 作为转录激活因子，优先结合转录起始位点下游+240 bp 在HEK 293 T细胞中，在大多数CpG岛启动子处存在TSSs。进一步的ZFX ChIP-seq实验 几种人类细胞系显示，在所有测试的细胞系中，大多数ZFX结合发生在相同的启动子处。 细胞系有趣的是，在ZFX siRNA敲低后，这些细胞系中的RNA-seq实验揭示了细胞类型 ZFX结合的下调基因的特定集合。然而，ZFX的转录机制 调节细胞类型特异性基因表达的机制尚不清楚。因此，在我的建议中，我将决定如何 当ZFX与大多数相同的靶基因结合时，它可以调节每种细胞类型中不同的靶基因组。 不同类型细胞中的启动子。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 我建议使用IP-MS和灵敏的邻近检测技术在多个细胞系中鉴定与ZFX相互作用的蛋白质。 候选ZFX结合配偶体将用co-IP单独验证，并在 原位邻位连接测定（PLA）实验。确定候选人是否是ZFX调节的 在转录后，将进行相应mRNA的siRNA敲低后的RNA-seq实验。我也 目的是构建ZFX突变体，首先关注N-末端高度保守的肽区， 反式激活结构域，并进行co-IP实验，以定位蛋白质相互作用的位点， 转录激活测定以确定这些突变对转录的影响。第2章我会执行 在基因的启动子中的基序分析，所述基因以细胞类型特异性的方式被ZFX结合和调节， 鉴定不同细胞系中与ZFX协同结合的TF。候选TF的约束力将 使用ChIP-seq进行验证。具有CRISPR介导的候选转录因子结合突变的细胞系 将建立位点，并进行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的转录。我对多重人格的彻底机械描述 具有独特基因组结合模式的这种有趣TF的细胞系将对基因组的表达做出重大贡献。 转录领域。