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。
项目成果
期刊论文数量(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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