Uncovering Transcriptional Regulation of a Master Hematopoietic Transcription Factor at Single Molecule Resolution
在单分子分辨率下揭示主要造血转录因子的转录调控
基本信息
- 批准号:9258128
- 负责人:
- 金额:$ 4.9万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2016
- 资助国家:美国
- 起止时间:2016-12-15 至 2019-12-14
- 项目状态:已结题
- 来源:
- 关键词:Acute Myelocytic LeukemiaAddressAge-MonthsBiological ProcessBloodBlood CellsCell CompartmentationCell physiologyCellsCellular biologyComplexDataDevelopmentDimensionsDoseEmbryoErythrocytesEventFishesGasesGene ActivationGene ExpressionGene Expression ProfilingGene Expression RegulationGenesGenetic TranscriptionGoalsHematologic NeoplasmsHematological DiseaseHematologyHematopoiesisHematopoieticHematopoietic stem cellsHemostatic functionHeterogeneityHomeostasisImageImaging DeviceIn SituKineticsLeadLinkLymphoidMaintenanceMalignant - descriptorMeasurementMeasuresMegakaryocytesMessenger RNAModelingMolecularMusNodalNoiseNormal CellNormal tissue morphologyOutputPathologicPlayPopulationPopulation HeterogeneityPrecipitating FactorsProcessProductionProteinsRNAReactionRegulationRegulatory ElementReporterResearchResolutionRoleSeriesShapesStem cellsSystemTechniquesTechnologyTimeTissuesTranscriptional RegulationUpstream Enhancerbasecohortdosagegranulocyteimmune functioninsightleukemialeukemic stem cellleukemogenesismolecular imagingmonocytenovelnovel therapeuticspressurepreventprogenitorprogramsprotein foldingsingle moleculestoichiometrytooltranscription factortumorigenesis
项目摘要
ABSTRACT
Gene expression, which encompasses a series of reactions from initial gene activation to final protein folding,
is an inherently noisy process for any cell population under study. As each step in the process is subject to
independent regulatory pressures, small between cell differences in the levels of these regulators can produce
substantial transcriptional heterogeneity, which may then propagate into substantial functional diversity. It is
therefore challenging to understand how complex multi-cellular tissues faithfully develop given the number of
genes that must be coordinately expressed to establish cellular identity. Master regulatory transcription factors
(TF) are the proposed solution to this teleological dilemma. These molecules have been shown to control
cohorts of genes required for normal cell function, and achieving the appropriate level and stoichiometry
between different TF appears to be critical for fate decisions during normal tissue development. Moreover, the
deregulation in either the expression or function of these factors appears to play a substantial role in malignant
transformation. TF have been extensively studied in hematopoiesis, the highly arborized differentiation network
that robustly and dynamically produces a spectrum of functionally distinct blood cell populations responsible for
hemostasis, gas exchange, and immune function. In order to achieve this complex cellular output,
hematopoietic differentiation is postulated to occur as a series of nodal fate decisions in increasingly
oligopotent stem and progenitor cell compartments (HSPC), each with distinct gene expression programs
governed by TF. Understanding how HSPC achieve the appropriate dose and activity of these TF is therefore
vital to our understanding of steady state blood differentiation and may expose novel therapeutic windows in
hematological disease. Complicating these efforts, however, is the finding that HSPC are functionally and
transcriptionally heterogeneous, which have limited the field's ability to uncover definitive regulation of TF
based on ensemble measurements. This project is intended to quantify the origins of that heterogeneity with
single molecule, quantitative techniques to uncover the regulation and expression of a master hematopoietic
TF, PU.1. Our proposal is to (1) determine how PU.1 mRNA and protein production is dynamically changed
during differentiation in single primary HSPC from mice by RNA FISH/IF and to (2) independently measure
how a highly conserved cis regulatory element (URE) controls the rate, magnitude, and dynamics of PU.1
transcription. Our preliminary findings have indicated that not only is our experimental approach feasible, it has
already revealed intriguing findings about PU.1 mRNA synthesis that were previously unknown. Using these
tools and sophisticated analytical techniques, this proposal will provide the highest resolution, quantitative
study to date of the regulation and activity of a master regulatory transcription factor in primary HSPC. We
anticipate that our approach will provide novel and fundamental insight into the molecular paradigms regulating
hematopoiesis and leukemogenesis.
摘要
基因表达,包括从初始基因激活到最终蛋白质折叠的一系列反应,
对于任何被研究的细胞群体来说都是一个固有的噪声过程。由于过程中的每一步都受到
独立的调节压力,这些调节剂水平的细胞间差异很小,
实质性的转录异质性,其然后可以传播成实质性的功能多样性。是
因此,理解复杂的多细胞组织如何忠实地发育具有挑战性,
必须协调表达以建立细胞身份的基因。主调节转录因子
(TF)是解决这个目的论困境的建议。这些分子已经被证明可以控制
正常细胞功能所需的基因群,并达到适当的水平和化学计量
在正常组织发育过程中,不同TF之间的差异似乎对命运决定至关重要。而且
这些因子的表达或功能的失调似乎在恶性肿瘤中起重要作用,
转型TF已被广泛研究的造血,高度树枝状分化网络
它能有力地、动态地产生一系列功能不同的血细胞群,
止血气体交换和免疫功能。为了实现这种复杂的细胞输出,
造血分化被假定为一系列淋巴结命运的决定,
寡能干细胞和祖细胞区室(HSPC),每个区室具有不同的基因表达程序
由TF管理。因此,了解HSPC如何达到这些TF的适当剂量和活性,
这对我们理解稳态血液分化至关重要,并可能揭示新的治疗窗口,
血液病然而,使这些努力复杂化的是发现HSPC在功能上和功能上是不稳定的。
转录异质性,这限制了该领域的能力,以揭示明确的调节TF
基于集合测量。该项目旨在量化这种异质性的起源,
单分子,定量技术来揭示调控和表达的主造血
TF,PU. 1.我们的建议是(1)确定PU.1 mRNA和蛋白质的产生是如何动态变化的
在通过RNA FISH/IF在来自小鼠的单个原代HSPC中分化期间,以及(2)独立地测量
高度保守的顺式调控元件(URE)如何控制PU的速率、大小和动力学。
转录。我们的初步研究结果表明,我们的实验方法不仅是可行的,
已经揭示了以前未知的关于PU.1 mRNA合成的有趣发现。使用这些
工具和先进的分析技术,这项建议将提供最高的分辨率,定量
到目前为止对原代HSPC中主调节转录因子的调节和活性的研究。我们
预计我们的方法将提供新的和基本的见解的分子范式调节
造血和白血病发生。
项目成果
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