Hematopoietic Regulation via GATA Switches

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。

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. PUBLIC HEALTH RELEVANCE: This competitive renewal application seeks to renew our grant "Hematopoietic Regulation via GATA Switches". Members of the GATA factor family of transcription factors (GATA-1-6) regulate critical aspects of mammalian development and are dysregulated in human diseases including leukemias. These factors bind to a simple DNA motif distributed abundantly within genomes, but our studies revealed that GATA-1 and GATA-2 occupy only a small fraction of the total GATA motifs in cells. GATA-1 and GATA-2 can occupy the same sites of target genes at distinct stages of development, inducing distinct functional outputs. Mechanisms controlling chromatin site selection and differential activities of GATA factors are poorly understood. We aim to elucidate these mechanisms, which will yield fundamental insights into the control of hematopoiesis, hematologic malignancies involving GATA factor dysregulation, and diverse developmental processes. In the long-term, we aim to develop creative strategies to translate knowledge on GATA factor mechanisms into novel therapeutics.