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主要集中在C2H2锌指转录因子（TFs）的ZFX家族，