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细胞功能以及各种正常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位点转录活性的重要决定因素。DOT 1 L（端粒沉默1样破坏因子）组蛋白H3赖氨酸-79（H3 K79）甲基转移酶参与了几种不同的生物学过程，包括转录的正调控。 本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DOT 1 L有助于髓系/红系命运特化的机制。我们假设H3 K79甲基化通过阻止Gata 2位点抑制性转录复合物的形成而允许GATA 2表达。因此，在不存在H3 K79甲基化的情况下，Gata 2表达增加。使用体外功能获得和功能丧失实验，我们将测试GATA 2直接受DOT 1 L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Dot 1 L影响细胞命运的发育阶段。我们假设GATA 2在红系/髓系多能祖细胞中的表达受H3 K79甲基化调控。为了研究这种可能性，我们将使用来自Dot 1 L条件性KO小鼠的诱导多能干细胞（iPS），在体外开发基于胚胎干（ESC）细胞的红系分化模型。目的3-检测Dot 1 L缺陷细胞中促红细胞生成素的反应。我们将直接评估Dot 1 L缺陷细胞的反应，检查它们的生长和分化能力以及信号，以确定红细胞发育中的缺陷。目的4-确定DOT 1 L在成人红细胞生成中的作用。在这个目标中，我们将使用Dot 1 L的诱导型条件突变体来特异性地删除成年小鼠造血干细胞（HSC）中的基因。我们将使用胎肝和骨髓来源的HSC的集落测定来确定DOT 1 L缺失对HSC功能和分化的影响。

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.