High-throughput functional characterization of human enhancers
人类增强子的高通量功能表征
基本信息
- 批准号:10241101
- 负责人:
- 金额:$ 75.05万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-08-18 至 2023-01-31
- 项目状态:已结题
- 来源:
- 关键词:AddressAffectArchitectureBase PairingBindingBinding SitesBiological AssayCRISPR/Cas technologyCell LineCellsCharacteristicsChromatinChromatin Remodeling FactorClassificationClustered Regularly Interspaced Short Palindromic RepeatsComplementComplexComputing MethodologiesCuesDNADNA Polymerase IIDNA-Directed RNA PolymeraseDefectDeoxyribonuclease IDevelopmentDiseaseDistalDrosophila genusElementsEnhancersEnvironmentFailureGene ExpressionGene Expression ProfileGene Expression RegulationGenesGenetic TranscriptionGenomeGenomic DNAGoalsHCT116 CellsHSF1Heat-Shock ResponseHistonesHumanHypersensitivityK-562K562 CellsKnock-inLeadMaintenanceMammalian CellMass Spectrum AnalysisMeasuresMechanicsMessenger RNAMethodsModelingMolecularMutateMutationNutritionalOncogenesOrganismPatternPersonal SatisfactionPhenotypePositioning AttributeProcessProductionProteinsRNARNA SequencesRegulationRegulator GenesResearchResolutionRoleRun-On AssaysStructureSystemTestingTo specifyTransactivationTranscription Initiation SiteTranscriptional RegulationTransgenic OrganismsUndifferentiatedWorkaptamerbasebiological adaptation to stresscell typechromatin immunoprecipitationchromatin remodelingchromosome conformation capturecomparativedesignembryo cellfollow-upgenome-widehigh throughput screeninghistone modificationin vivoinsightmutantnovelnovel therapeuticsnutritionpromoterrecruitresponsespatiotemporaltranscription factor
项目摘要
PROJECT SUMMARY/ABSTRACT:
The genomes of higher organisms and their expression are highly regulated in response to a variety of
developmental, environmental, and nutritional cues. The failure to execute proper gene regulation can lead to
developmental defects and disease states. The overarching goal of this research is to understand basic
regulatory mechanisms of genes at the level of transcription, the stage where RNA polymerase II (Pol II)
transcribes the genes into mRNA. This regulation is conducted by the concerted effort of DNA elements
(promoters and enhancers) and many protein machineries such as Pol II, TFs, and chromatin/histone modifying
complexes. Recent studies in Drosophila and mammalian cells have revealed that transcription is primarily
regulated at two rate-limiting steps; 1) Recruitment of Pol II to promoter and Pol II pausing at 20-50bp
downstream of transcription initiation site, and 2) Release of paused Pol II to productive elongation. A battery of
complementary approaches (some novel) are designed to identify human TFs and chromatin remodelers that act
at these steps and then rigorously address the mechanism by which they exert transcriptional regulation. by TFs
and chromatin modifiers with the highest possible spatiotemporal resolution genome-wide in distinct cell lines,
including K562, HCT116, HEK293, and undifferentiated and differentiated WTC-11. The wealth of existing
information in these cell lines will be complemented with Run-On assays (RO-seq), and others when necessary,
to identify TFs that act primarily on one or both of the rate-limiting regulated steps in transcription. These
observational assays include multiple highly-sensitive RO-seq assays for mapping the position and amount of
transcription across genes and enhancers genome-wide, ChIP-exo for detecting TF binding and occupancy,
STARR-seq assays to measure transcription regulatory activity of enhancers and promoters, and a degron
system to rapidly degrade TFs to assess their primary effects by these ‘-seq’ assays. Together, these systems
will test our regulatory models and assess their generality. In Aim 1, using a novel aptamer-based pulldown and
mass spectrometry approach, we will identify pioneering TFs that work through remodeling complexes such as
PBAP (SWI/SNF) to open chromatin for subsequent TF and Pol II recruitment. In Aim 2, we seek to identify cell-
type-specific TFs that act at major regulated steps of transcription, Steps 1, 2, or both. This will be achieved by a
comparative analysis of cell line specific nascent transcription profiles. Aim 3 will test the role of TF candidates,
which are associated with one or both of the regulatory steps in the transcription cycle, in living cells using a
Degron approach and multiple ‘-seq’ assays. In Aim 4, we will assess the roles of TFs in the interplay of
enhancers and promoters interactions using high-throughput episomal and chromosomal enhancer assays. The
proposal will identify TFs that act at specific steps in transcription regulation and provide insights to the
mechanics of their action.
项目总结/摘要:
高等生物的基因组及其表达受到多种生物因子的高度调节,
发育、环境和营养线索。如果不能进行适当的基因调控,
发育缺陷和疾病状态。这项研究的首要目标是了解基本的
基因在转录水平上的调控机制,RNA聚合酶II(Pol II)
将基因转录成mRNA。这种调节是由DNA元件的协同努力进行的
(启动子和增强子)和许多蛋白质机制,如Pol II,TF和染色质/组蛋白修饰,
配合物最近在果蝇和哺乳动物细胞中的研究表明,转录主要是
Pol Ⅱ在启动子上的募集和Pol Ⅱ在20- 50 bp的停顿是两个限速步骤
转录起始位点下游,和2)暂停的Pol II释放到生产性延伸。的电池
互补的方法(有些是新的)被设计来鉴定人类TF和染色质重塑,
在这些步骤中,然后严格解决它们发挥转录调节的机制。由TFs
和在不同细胞系中具有全基因组最高可能时空分辨率的染色质修饰剂,
包括K562、HCT 116、HEK 293和未分化和分化的WTC-11。现有的财富
这些细胞系中的信息将用Run-On测定法(RO-seq)补充,必要时用其他测定法补充,
以鉴定主要作用于转录中的一个或两个限速调节步骤的TF。这些
观察性测定包括多种高灵敏度的RO-seq测定,用于定位蛋白质的位置和量。
跨基因和增强子的全基因组转录,用于检测TF结合和占用的ChIP-exo,
用于测量增强子和启动子的转录调节活性的STARR-seq测定,以及降解决定子
系统来快速降解TF,以通过这些“-seq”测定来评估它们的主要作用。总之,这些系统
将测试我们的监管模式并评估其通用性。在目标1中,使用一种新的基于适体的下拉,
质谱方法,我们将确定开拓性的TF,通过重塑复合物,如
PBAP(SWI/SNF)打开染色质,用于随后的TF和Pol II募集。在目标2中,我们试图识别细胞-
在转录的主要调节步骤,步骤1,2或两者中起作用的类型特异性TF。这将通过一个
细胞系特异性新生转录谱的比较分析。目标3将测试TF候选人的作用,
其与转录周期中的一个或两个调节步骤相关,
降解决定子方法和多重“-seq”测定。在目标4中,我们将评估TF在以下因素相互作用中的作用:
使用高通量附加体和染色体增强子测定进行增强子和启动子相互作用。的
该提案将确定在转录调控的特定步骤中起作用的转录因子,并为转录调控提供见解。
他们的行动机制。
项目成果
期刊论文数量(7)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Enhancer transcription: what, where, when, and why?
- DOI:10.1101/gad.311605.118
- 发表时间:2018-01-01
- 期刊:
- 影响因子:10.5
- 作者:Tippens ND;Vihervaara A;Lis JT
- 通讯作者:Lis JT
RNA polymerase mapping in plants identifies intergenic regulatory elements enriched in causal variants.
- DOI:10.1093/g3journal/jkab273
- 发表时间:2021-10-19
- 期刊:
- 影响因子:0
- 作者:Lozano R;Booth GT;Omar BY;Li B;Buckler ES;Lis JT;Del Carpio DP;Jannink JL
- 通讯作者:Jannink JL
Stress-induced transcriptional memory accelerates promoter-proximal pause release and decelerates termination over mitotic divisions.
- DOI:10.1016/j.molcel.2021.03.007
- 发表时间:2021-04-15
- 期刊:
- 影响因子:16
- 作者:Vihervaara A;Mahat DB;Himanen SV;Blom MAH;Lis JT;Sistonen L
- 通讯作者:Sistonen L
Pioneer-like factor GAF cooperates with PBAP (SWI/SNF) and NURF (ISWI) to regulate transcription.
- DOI:10.1101/gad.341768.120
- 发表时间:2021-01-01
- 期刊:
- 影响因子:10.5
- 作者:Judd J;Duarte FM;Lis JT
- 通讯作者:Lis JT
Chromatin-mediated epigenetic regulation of HSV-1 transcription as a potential target in antiviral therapy.
- DOI:10.1016/j.antiviral.2021.105103
- 发表时间:2021-08
- 期刊:
- 影响因子:7.6
- 作者:Schang LM;Hu M;Cortes EF;Sun K
- 通讯作者:Sun K
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{{ truncateString('JOHN T LIS', 18)}}的其他基金
Functional Architecture and Interplay of Transcription Regulatory Elements of the Human Genome
人类基因组转录调控元件的功能结构和相互作用
- 批准号:
10639574 - 财政年份:2023
- 资助金额:
$ 75.05万 - 项目类别:
High-throughput functional characterization of human enhancers
人类增强子的高通量功能表征
- 批准号:
10166068 - 财政年份:2020
- 资助金额:
$ 75.05万 - 项目类别:
Tissue biology studies of histone modification, nascent transcription, and post-transcription regulation
组蛋白修饰、新生转录和转录后调控的组织生物学研究
- 批准号:
10746577 - 财政年份:2018
- 资助金额:
$ 75.05万 - 项目类别:
High-throughput functional characterization of human enhancers
人类增强子的高通量功能表征
- 批准号:
9904754 - 财政年份:2017
- 资助金额:
$ 75.05万 - 项目类别:
Distance-Hi-C: Creating Photo Activated X-linkers To Define Nuclear Architecture
Distance-Hi-C:创建光激活 X 链接器来定义核架构
- 批准号:
9769846 - 财政年份:2015
- 资助金额:
$ 75.05万 - 项目类别:
Distance-Hi-C: Creating Photo Activated X-linkers To Define Nuclear Architecture
Distance-Hi-C:创建光激活 X 链接器来定义核架构
- 批准号:
9000948 - 财政年份:2015
- 资助金额:
$ 75.05万 - 项目类别:
Distance-Hi-C: Creating Photo Activated X-linkers To Define Nuclear Architecture
Distance-Hi-C:创建光激活 X 链接器来定义核架构
- 批准号:
9144434 - 财政年份:2015
- 资助金额:
$ 75.05万 - 项目类别:
Factor-general characterization of dynamic transcriptional stress responses
动态转录应激反应的因子一般特征
- 批准号:
8846643 - 财政年份:2013
- 资助金额:
$ 75.05万 - 项目类别:
Factor-general characterization of dynamic transcriptional stress responses
动态转录应激反应的因子一般特征
- 批准号:
8578768 - 财政年份:2013
- 资助金额:
$ 75.05万 - 项目类别:
Factor-general characterization of dynamic transcriptional stress responses
动态转录应激反应的因子一般特征
- 批准号:
8729397 - 财政年份:2013
- 资助金额:
$ 75.05万 - 项目类别:
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