Assessing the basis of chromatin opening by hematopoietic pioneer factors
通过造血先锋因子评估染色质开放的基础
基本信息
- 批准号:10368949
- 负责人:
- 金额:$ 3.42万
- 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:2020-03-01 至 2023-02-28
- 项目状态:已结题
- 来源:
- 关键词:AddressAffinityBindingBiochemicalBiologyCCAAT-Enhancer-Binding Protein-alphaCell Fate ControlCellsChromatinChromatin StructureChromosomesDNADNA Binding DomainDNA SequenceDataDeoxyribonuclease IDependenceDevelopmentEctopic ExpressionEukaryotic CellFibroblastsGene ExpressionGene Expression RegulationGenerationsGenomic approachGoalsHematopoieticHematopoietic stem cellsHistonesHypersensitivityIn VitroLabelLengthLiverMapsMass Spectrum AnalysisMolecularMyelogenousNucleosomesPatternPositioning AttributeProcessProteinsRegulationResearchRoleTertiary Protein StructureTestingTherapeuticactivating transcription factorcell typecellular developmentclinically relevantcrosslinkdeletion analysisgenome-widein vivoinsightmacrophagemutantnanomolarnanoporeprogramsprotein structurereconstitutionscreeningtranscription factor
项目摘要
Project Summary/Abstract
The goal of this proposal is to investigate the intrinsic ability of hematopoietic transcription factors (TFs) to
engage closed chromatin and initiate chromatin opening during cell fate changes. Cell fate control is a
fundamental process in biology and de novo generation of clinically relevant cell types has enormous
implications for research and therapeutics; however, the general principles by which closed and unmarked
chromatin is initially accessed by these factors are not fully understood. In eukaryotic cells, chromatin
compaction serves as a mechanism for gene regulation by modulating the accessibility of TFs to target DNA
sequences. Development of macrophages from hematopoietic stem cells requires coordinated expression of
PU.1 with myeloid TFs C/EBPα and C/EBPβ, and ectopic expression of PU.1 and C/EBPα/β converts
fibroblasts to the macrophage lineage. I hypothesized that PU.1, C/EBPα, and C/EBPβ act as “pioneer” factors
and that initiating development requires these factors to directly bind compacted chromatin, initiate chromatin
opening, and allow subsequent binding of additional TFs that activate gene expression. Our lab identified
nucleosomes targeted by PU.1, C/EBPα, and C/EBPβ in vivo, and reconstituted these nucleosomes in vitro. To
assess the interactions of these factors with higher-order chromatin substrates, I reconstituted fluorescently
end-labeled synthetic nucleosome arrays and inserted a central nucleosome capable of binding by PU.1,
C/EBPα, and C/EBPβ. I have found that PU.1, C/EBPα, and C/EBPβ bind and initiate accessibility of H1-
compacted nucleosome arrays with different efficiencies. Furthermore, I have found that the DNA binding
domains of PU.1 and C/EBPα open chromatin with less efficiency compared to the full-length proteins. I am
now investigating the ability of these pioneer factors to open chromatin and how their mechanisms of action
compare to another established pioneer factor, FoxA. I hypothesize that PU.1, C/EBPα, and C/EBPβ possess
protein domains that enable chromatin interactions, and that initiating chromatin accessibility is fundamental to
the ability of these factors to direct cell fate in vivo. To address this hypothesis I will 1) use nanopore
sequencing to define TF-chromatin interactions and determine if PU.1, C/EBPα, and C/EBPβ interact with
histones using crosslinking mass spectrometry 2) perform a deletion analysis of PU.1, C/EBPα, and C/EBPβ
to map domains required for chromatin opening in vitro and determine if chromatin opening by pioneer factors
is required for TF directed macrophage transdifferentiaiton. My project will provide insights into the
mechanisms of chromatin structure regulation by hematopoietic TFs during cellular development and
reprogramming. Furthermore, biochemical analysis will provide details of the molecular features that endow
TFs with pioneering activity.
项目摘要/摘要
这项提议的目的是研究造血转录因子(TF)的内在能力
在细胞命运改变的过程中,启动关闭的染色质并启动染色质的打开。细胞命运控制是一种
生物学的基本过程和临床相关细胞类型的从头产生有巨大的
对研究和治疗的影响;然而,封闭和未标记的一般原则
染色质最初是由这些因素获得的,目前还没有完全了解。在真核细胞中,染色质
紧凑作用是一种通过调节转录因子与靶DNA的可及性来调节基因调控的机制
序列。从造血干细胞发育成巨噬细胞需要协调表达
髓系转移因子C/eBPα和C/eBPβ,异位表达pU.1和C/eBPα/β
成纤维细胞向巨噬细胞转化。我假设PU.1、C/EBPα和C/EBPβ是“先锋”因子
而启动发育需要这些因素直接结合紧密的染色质,启动染色质
开放,并允许随后与激活基因表达的额外TF结合。我们的实验室鉴定出
在体内以PU1、C/eBPα和C/eBPβ为靶向的核小体,并在体外重组这些核小体。至
评估这些因子与高阶染色质底物的相互作用,I荧光重组
末端标记的合成核小体阵列并插入能够与PU结合的中心核小体,
C/eBPα和C/eBPβ。我发现PU.1、C/EBPα和C/EBPβ结合并启动H1的可及性-
不同效率的致密核小体阵列。此外,我发现DNA结合
与全长蛋白相比,PU.1和C/EBPα结构域开放染色质的效率较低。我是
现在正在研究这些先驱因子打开染色质的能力以及它们的作用机制
与另一个公认的先锋因素FoxA相比。我假设PU.1、C/EBPα和C/EBPβ具有
使染色质相互作用的蛋白质结构域,以及启动染色质可及性是
这些因素在体内决定细胞命运的能力。为了解决这个假设,我将1)使用纳米孔
测序以确定Tf-染色质相互作用并确定PU.1、C/EBPα和C/EBPβ是否相互作用
使用交联质谱的组蛋白2)执行PU.1、C/EBPα和C/EBPβ的缺失分析
绘制体外开放染色质所需的结构域,并确定先驱因子是否开放染色质
是转铁蛋白诱导的巨噬细胞转分化所必需的。我的项目将提供对
造血因子在细胞发育和发育过程中对染色质结构的调节机制
重新编程。此外,生化分析将提供赋予
具有开拓性活动的TFS。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Megan A. Frederick其他文献
Megan A. Frederick的其他文献
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{{ truncateString('Megan A. Frederick', 18)}}的其他基金
Assessing the basis of chromatin opening by hematopoietic pioneer factors
通过造血先锋因子评估染色质开放的基础
- 批准号:
10112745 - 财政年份:2020
- 资助金额:
$ 3.42万 - 项目类别:
Assessing the basis of chromatin opening by hematopoietic pioneer factors
通过造血先锋因子评估染色质开放的基础
- 批准号:
9907544 - 财政年份:2020
- 资助金额:
$ 3.42万 - 项目类别:
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