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
基本信息
- 批准号:10677572
- 负责人:
- 金额:$ 6.91万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AddressAffectBindingBinding ProteinsBinding SitesBioinformaticsBiological AssayBiotinylationC2H2 Zinc FingerCanadaCell LineCellsChIP-seqClustered Regularly Interspaced Short Palindromic RepeatsCpG IslandsDNA-Binding ProteinsDataDiseaseDown-RegulationFamilyFamily memberGene ExpressionGene Expression RegulationGenesGenetic TranscriptionGenomicsHealthHumanHuman Cell LineIn SituIndividualKnock-outLabelLearningLigationMCF7 cellMalignant NeoplasmsMapsMass Spectrum AnalysisMediatingMediatorMentorsMessenger RNAMethodsMolecular BiologyMolecular Biology TechniquesMutateMutationN-terminalNuclear ProteinsOncogenicPatternPeptidesPhysiciansProliferatingPromoter RegionsProteinsProteomicsProto-Oncogene Proteins c-mycPublishingQuantitative Reverse Transcriptase PCRRegulationResearchSiteSmall Interfering RNATechniquesTestingTrainingTransactivationTranscription CoactivatorTranscription Initiation SiteTranscriptional RegulationUniversitiesZFY proteincandidate identificationcandidate validationcdc Genescell typeexperimental studyhuman diseaseinsightknock-downmutantpromoterprotein protein interactiontranscription factortranscriptometranscriptome sequencingtumorigenesis
项目摘要
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,
项目成果
期刊论文数量(0)
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科研奖励数量(0)
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Emily Hsu其他文献
Emily Hsu的其他文献
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{{ truncateString('Emily Hsu', 18)}}的其他基金
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:
10458396 - 财政年份:2022
- 资助金额:
$ 6.91万 - 项目类别:
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