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， 包括高度相关的ZFX、ZFY和ZNF711。此前，我们实验室建立了CRISPR介导的基因敲除 HEK293T 细胞中 ZFX 和 ZNF711 的缺失，并表现出增殖缺陷和表达改变 数千个基因。 Farnham 实验室的其他初步数据已确定 ZFX 和 ZNF711 作为转录激活剂，优先结合转录起始位点下游 +240 bp (TSS) 在 HEK293T 细胞中的大多数 CpG 岛启动子上。进一步的 ZFX ChIP-seq 实验 几种人类细胞系显示，在所有测试中，大多数 ZFX 结合发生在相同的启动子处 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 特定组的 ZFX 结合、下调基因。然而，ZFX 的转录机制 调节细胞类型特异性基因表达仍不清楚。因此，在我的建议中，我将确定如何 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 每种细胞类型从共同的启动子结合位点提供细胞类型特异性调节。在目标#1中，我 提议使用 IP-MS 和敏感接近度来识别多个细胞系中与 ZFX 相互作用的蛋白质 标记技术 TurboID-MS。候选 ZFX 结合伙伴将通过 co-IP 进行单独验证，并在 原位邻近连接测定（PLA）实验。确定候选人是否是 ZFX 监管的关键调解人 相应 mRNA 的 siRNA 敲低后将进行转录、RNA-seq 实验。我也 旨在构建 ZFX 突变体，首先关注 N 末端高度保守的肽区域 反式激活结构域，并进行 co-IP 实验来绘制蛋白质相互作用位点，然后 反式激活测定以确定这些突变对转录的影响。在目标#2中，我将执行 对 ZFX 以细胞类型特异性方式结合和调节的基因启动子进行基序分析 识别不同细胞系中与 ZFX 协同结合的 TF。候选 TF 的绑定将是 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 将制作位点，并进行 qRT-PCR 实验以确定候选 TF 结合是否会影响 ZFX 介导的细胞类型特异性基因的转录。我对多个方面的彻底机械表征 这种有趣的 TF 细胞系具有独特的基因组结合模式，将为 转录领域。