The Role of the Histone Methyltransferase DOT1L in Erythropoiesis

描述(由申请人提供)：精确调节的基因表达对于正常的细胞功能以及各种正常的哺乳动物发育过程至关重要。组蛋白甲基化状态已成为基因座转录活性的重要决定因素。DOT1L(端粒沉默1-样中断因子)组蛋白H3赖氨酸-79(H3K79)甲基转移酶参与多种不同的生物学过程，包括正向转录调控。这项研究的总体科学目标是了解这种甲基转移酶在红细胞生成中的作用。我们设计了四个具体的目标来解决这个问题：目标1-确定DOT1L对髓系/红系命运规范的贡献机制。我们假设H3K79甲基化通过防止在GATA2基因上形成抑制性转录复合体来允许GATA2的表达。因此，在没有H3K79甲基化的情况下，Gata2的表达增加。通过功能获得和功能丧失的体外实验，我们将验证GATA2直接受DOT1L调控的假设，并确定该基因沉默的机制。目的2-确定红细胞生成过程中DOT1L影响细胞命运的发育阶段。我们假设，在这个发育中的细胞群体中，GATA2在红系/髓系多潜能祖细胞中的表达受到H3K79甲基化的调节。为了验证这种可能性，我们将利用DOT1L条件性KO小鼠的诱导多能干细胞(IPS)，在体外建立基于胚胎干细胞(ESC)的红系分化模型。目的3-检测DOT1L缺陷细胞的促红细胞生成素反应。我们将直接评估DOT1L缺陷细胞的反应，检查它们的生长和分化能力，以及信号，以确定红系发育中的缺陷。目的4-确定DOT1L在成人红细胞生成中的作用。为此，我们将使用DOT1L的一个可诱导的条件突变体来特异性地删除成年小鼠造血干细胞(HSC)中的该基因。我们将使用胎肝和骨髓来源的HSC的集落分析来确定DOT1L缺失对HSC功能和分化的影响。 与公共卫生相关：了解控制红细胞产生的基本机制对于有效治疗各种病理疾病至关重要，包括那些导致贫血的疾病。我们发现了组蛋白甲基转移酶DOT1L在早期红细胞发育过程中的新作用。这些目标的完成将大大提高我们对这一过程的理解，并使我们能够设计干预贫血病例的治疗方法。

DESCRIPTION (provided by applicant): Precisely tuned gene expression is critical for normal cellular functions as well as a variety of normal mammalian developmental processes. Histone methylation status has emerged as an important determinant of gene locus transcriptional activity. The DOT1L (Disruptor of Telomere Silencing 1-Like) histone H3 lysine-79 (H3K79) methyltransferase has been implicated in several distinct biological processes, including positive regulation of transcription. The overall scientific goal of this study is to understand the role fr this methyltransferase in erythropoiesis. We have devised four specific aims to address this: Aim 1-To determine the mechanism by which DOT1L contributes to myeloid/erythroid fate specification. We hypothesize that H3K79 methylation is permissive for GATA2 expression, by preventing the formation of repressive transcriptional complexes at the Gata2 locus. In the absence of H3K79 methylation, thus, Gata2 expression is increased. Using gain-of- function and loss-of-function experiments in vitro, we will test the hypothesis that GATA2 is directly regulated by DOT1L, and determine the mechanism by which the gene is silenced. Aim 2-To determine the developmental stage during erythropoiesis at which Dot1L acts to influence cell fate. We hypothesize that GATA2 expression in erythroid/myeloid, multipotential progenitors in this developing cell population is regulated by H3K79 methylation. To examine this possibility, we will develop an embryonic stem (ESC) cell- based erythroid differentiation model in vitro, using induced Pluripotent Stem (iPS) cells derived from Dot1L conditional KO mice. Aim 3-To examine erythropoietin responses in Dot1L-deficient cells. We will assess the responses of Dot1L-deficient cells directly, examining their ability to grow and differentiate, as well as signa, in order to identify the defect in erythroid development. Aim 4-To determine a role for DOT1L in adult erythropoiesis. In this aim, we will use an inducible, conditional mutant of Dot1L to specifically delete the gene in hematopoietic stem cells (HSC) in adult mice. We will determine the effects of DOT1L loss on HSC function and differentiation using colony assays from fetal liver- and bone marrow-derived HSC. PUBLIC HEALTH RELEVANCE: Understanding the fundamental mechanisms controlling the production of red blood cells is essential to effectively treating a variety of pathological conditions, including those that result in anemia. We have a found a novel role for the histone methyltransferase, DOT1L in the process of early red blood cell development. The completion of these aims will significantly improve our understanding of this process and will enable us to design therapies to intervene in cases of anemia.