Hematopoietic Regulation via GATA Switches

描述(申请人提供)：转录网络协调干细胞分化为血细胞。造血的主要调节者，包括GATA-2，建立了这些网络，这些网络在白血病中被破坏。GATA-2是多能造血祖细胞发生和/或存活所必需的，而红细胞生成与GATA-2减少和GATA-1增加有关。GATA-1通过GATA开关直接抑制GATA2转录，其中GATA-1取代染色质上的GATA-2。我们将测试关于GATA开关的机制和后果的假设。具体目标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(-1.8kb)中删除了一个GATA开关位点，对突变小鼠的分析显示，在胚胎发育早期，Gata2正常激活，随着发育的进行，正常的Gata2抑制，但之后重新激活。维持抑制的-1.8kb位点要求代表了体内维持抑制与启动抑制的顺式元件的第一个例子。GATA2在-1.8kb小鼠中的重新激活与红细胞生成受损有关。我们将剖析-1.8kb位点如何维持抑制，并测试是否需要一个独特的Gata2增强子(+9.5kb)来诱导Gata2转录。特定目标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无竞争地占据GATA开关位点。计算方法将被用来挖掘我们的全基因组GATA因子芯片序列数据集，以定义GATA因子染色质占有率的分子决定因素。我们将测试重新激活GATA2的-1.8kb位点删除是否在体内创建了反向GATA开关。具体目的3-建立GATA-1突变体抑制GATA2缺陷的分子基础，该突变体与人类巨核细胞白血病的发生有关。我们发现GATA-1(1-83)的白血病突变体是一种高度活跃的激活剂，但其抑制GATA2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是有缺陷的，残基81-85构成了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抑制的特定步骤中受到损害。这些研究将阐明控制造血主调制子正常水平的机制，GATA因子如何在复杂基因组中选择位置，以及导致白血病的GATA-1突变如何失调GATA2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，因此该结果有望具有广泛的生物学和病理生理学意义。 公共卫生相关性：这项竞争性续签申请旨在续签我们的赠款“通过GATA开关进行造血调控”。GATA转录因子家族成员(GATA-1-6)调节哺乳动物发育的关键方面，在包括白血病在内的人类疾病中调节失调。这些因子与基因组中广泛分布的一个简单的DNA基序相结合，但我们的研究表明，GATA-1和GATA-2只占细胞中GATA基序的一小部分。GATA-1和GATA-2可以在不同的发育阶段占据目标基因的相同位置，诱导不同的功能输出。控制染色质位置选择和GATA因子差异活性的机制尚不清楚。我们的目标是阐明这些机制，这将为控制造血、涉及GATA因子失调的血液系统恶性肿瘤以及不同的发育过程提供基本的见解。从长远来看，我们的目标是开发创造性的战略，将GATA因子机制方面的知识转化为新的疗法。

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.