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。造血的主要调节因子，包括加塔-2，建立了这些网络，这些网络在白血病中被破坏。加塔-2是多能造血前体的发生和/或存活所必需的，而红细胞生成与加塔-2减少和加塔-1增加有关。加塔-1通过加塔开关直接抑制Gata 2转录，其中加塔-1从染色质位点取代加塔-2。我们将测试有关加塔开关的机制和后果的假设。具体目的1 -测试控制造血的加塔-2的生理水平如何在体内建立和调节的模型。我们从Gata 2基因座（~ 1.8 kb）中删除了一个加塔开关位点，对突变小鼠的分析揭示了在胚胎发生早期正常的Gata 2激活，随着发育的进行正常的Gata 2抑制，但此后重新激活。维持阻遏所需的~ 1.8kb位点代表了在体内维持阻遏与启动阻遏的顺式元件的第一个实例。在~ 1.8kb小鼠中Gata 2再活化与受损的红细胞生成相关。我们将剖析-1.8 kb位点如何维持抑制，并测试是否需要一个独特的Gata 2增强子（+9.5 kb）来诱导Gata 2转录。具体目标2 -确定加塔因子如何选择染色质靶位点。我们假设，在红细胞生成过程中，加塔-2水平下降，从而允许加塔-1不受阻碍地占据加塔转换位点。计算方法将用于挖掘我们的全基因组加塔因子ChIP-seq数据集，以定义加塔因子染色质占有率的分子决定因素。我们将测试重新激活Gata 2的-1.8 kb位点缺失是否在体内产生反向加塔开关。具体目标3 -建立与人巨核细胞白血病发展相关的加塔-1突变体对缺陷性Gata 2抑制的分子基础。我们发现加塔-1（1-83）的致白血病突变体是一种过度活跃的激活剂，但其抑制GATA 2的能力严重受损。我们假设1-83在募集关键的共阻遏物方面是有缺陷的，并且残基81-85构成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Gata 2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节因子正常水平的机制，加塔因子如何在复杂基因组中选择位点，以及致白血病加塔-1突变如何失调Gata 2表达和功能。由于涉及其他加塔因子的加塔开关可能在广泛的细胞中发生，因此预期结果具有广泛的生物学和病理生理学重要性。 公共卫生相关性：此竞争性更新申请旨在更新我们的赠款“通过加塔开关造血调节”。转录因子的加塔因子家族的成员（加塔-1-6）调节哺乳动物发育的关键方面，并且在包括白血病的人类疾病中失调。这些因子与基因组中大量分布的简单DNA基序结合，但我们的研究表明，加塔-1和加塔-2仅占细胞中总加塔基序的一小部分。加塔-1和加塔-2可以在发育的不同阶段占据靶基因的相同位点，诱导不同的功能输出。控制染色质位点选择和加塔因子差异活性的机制知之甚少。我们的目标是阐明这些机制，这将产生基本的见解控制造血，血液恶性肿瘤涉及加塔因子失调，和不同的发展过程。从长远来看，我们的目标是开发创造性的策略，将加塔因子机制的知识转化为新的治疗方法。

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.