Hematopoietic Regulation via GATA Switches

通过 GATA 开关进行造血调节

• 批准号: 8610293
• 负责人: Emery H Bresnick
• 金额: $ 32.3万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8610293
• 关键词: Adult; Affect; Amino Acids; Binding; Biological; Biological Assay; Blood Cells; Blood Vessels; Cell Differentiation process; Cells; ChIP-seq; Chromatin; Chromatin Structure; Complex; DNA; Data Set; Development; Developmental Process; Elements; Embryo; Embryonic Development; Employee Strikes; Enhancers; Erythrocytes; Erythropoiesis; Failure; Family; Fetal Liver; Funding; Gene Targeting; Genetic; Genetic Transcription; Genome; Grant; Hematologic Neoplasms; Hematopoiesis; Hematopoietic; Human Development; Knock-in Mouse; Knowledge; Life; Mediating; Megakaryocytes; Mining; Modeling; Molecular; Mus; Mutant Strains Mice; Mutation; Output; Physiological; Protein Binding Domain; Recruitment Activity; Regulation; Repression; Retinoblastoma Protein; Site; Staging; Testing; Transgenic Mice; Translating; Up-Regulation; base; cell type; chromatin remodeling; embryonic stem cell; genome-wide; homologous recombination; human GATA1 protein; human disease; in vivo; insight; leukemia; leukemia/lymphoma; member; mutant; novel therapeutics; prospective; stem cell differentiation; transcription factor

DESCRIPTION (provided by applicant): Transcriptional networks orchestrate stem cell differentiation into blood cells. Master regulators of hematopoiesis, including GATA-2, establish these networks, which are disrupted in leukemias. GATA-2 is required for the genesis and/or survival of multipotent hematopoietic precursors, while erythropoiesis is associated with reduced GATA-2 and increased GATA-1. GATA-1 directly represses Gata2 transcription through GATA switches in which GATA-1 replaces GATA-2 from chromatin sites. We will test hypotheses regarding mechanisms and consequences of GATA switches. Specific Aim 1 - To test models for how physiological levels of GATA-2 that control hematopoiesis are established and regulated in vivo. We deleted a GATA switch site from the Gata2 locus (-1.8 kb), and analysis of the mutant mice revealed normal Gata2 activation early in embryogenesis, normal Gata2 repression as development proceeds, but reactivation thereafter. The -1.8 kb site requirement for maintaining repression represents the first example of a cis-element that maintains versus initiates repression in vivo. Gata2 reactivation in -1.8 kb mice is associated with impaired erythropoiesis. We will dissect how the -1.8 kb site maintains repression and test whether a distinct Gata2 enhancer (+9.5 kb) is required for induction of Gata2 transcription. Specific Aim 2 - To determine how GATA factors select chromatin target sites. We hypothesize that GATA-2 levels decline during erythropoiesis to permit unopposed GATA-1 occupancy of GATA switch sites. Computational approaches will be used to mine our genome-wide GATA factor ChIP-seq datasets to define molecular determinants of GATA factor chromatin occupancy. We shall test whether the -1.8 kb site deletion, which reactivates Gata2, creates a reverse GATA switch in vivo. Specific Aim 3 - To establish the molecular basis for defective Gata2 repression by a GATA-1 mutant associated with the development of human megakaryoblastic leukemia. We found that the leukemogenic mutant of GATA-1 ( 1-83) is a hyperactive activator, but severely impaired in its capacity to repress Gata2. We hypothesize that 1-83 is defective in recruiting critical co-repressors, and residues 81-85 constitute a Retinoblastoma Protein-binding motif. We shall test whether 1-83 is compromised in specific steps leading to Gata2 repression. These studies will elucidate mechanisms that control normal levels of a master regulator of hematopoiesis, how GATA factors select sites in a complex genome, and how a leukemogenic GATA-1 mutation dysregulates Gata2 expression and function. As GATA switches involving other GATA factors are likely to occur in a wide spectrum of cells, the results are expected to have broad biological and pathophysiological importance.

描述（由申请人提供）：转录网络协调干细胞分化为血细胞。包括GATA-2在内的造血主要调控因子建立了这些在白血病中被破坏的网络。GATA-2对于多能造血前体的发生和/或存活是必需的，而红细胞生成与GATA-2的减少和GATA-1的增加有关。GATA-1通过GATA开关直接抑制GATA的转录，GATA-1在染色质位点取代GATA-2。我们将测试关于GATA开关的机制和后果的假设。特异性目的1 -测试控制造血的GATA-2生理水平如何在体内建立和调节的模型。我们从Gata2基因座上删除了一个GATA开关位点（-1.8 kb），对突变小鼠的分析显示，在胚胎发生早期正常的Gata2激活，随着发育的进行，正常的Gata2抑制，但在此之后重新激活。维持抑制所需的-1.8 kb位点是在体内维持或启动抑制的顺式元件的第一个例子。-1.8 kb小鼠的Gata2再激活与红细胞生成功能受损有关。我们将分析-1.8 kb位点如何维持抑制，并测试是否需要一个独特的Gata2增强子（+9.5 kb）来诱导Gata2转录。特异性目标2 -确定GATA因子如何选择染色质靶点。我们假设GATA-2水平在红细胞生成过程中下降，以允许GATA-1无对抗地占据GATA开关位点。计算方法将用于挖掘我们的全基因组GATA因子ChIP-seq数据集，以定义GATA因子染色质占用的分子决定因素。我们将测试重新激活Gata2的-1.8 kb位点缺失是否会在体内产生反向的GATA开关。特异性目的3 -建立与人类巨核细胞白血病发展相关的GATA-1突变体对gata - 2抑制缺陷的分子基础。我们发现，致白血病突变体GATA-1（1-83）是一种过度活跃的激活因子，但其抑制gata - 2的能力严重受损。我们假设1-83在招募关键的共抑制因子方面存在缺陷，残基81-85构成了一个视网膜母细胞瘤蛋白结合基序。我们将测试1-83是否在导致Gata2抑制的具体步骤中受到损害。这些研究将阐明控制正常造血主调节因子水平的机制，GATA因子如何在复杂基因组中选择位点，以及导致白血病的GATA-1突变如何失调GATA的表达和功能。由于涉及其他GATA因子的GATA开关可能发生在广泛的细胞中，因此预计该结果具有广泛的生物学和病理生理学意义。