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。DOT1L（端粒沉默1样干扰物）组蛋白H3赖氨酸-79 （H3K79）甲基转移酶与几个不同的生物学过程有关，包括转录的正调控。本研究的总体科学目标是了解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：目的1：确定DOT1L对髓系/红细胞命运规范的作用机制。我们假设H3K79甲基化是允许GATA2表达的，通过阻止在GATA2位点形成抑制性转录复合物。因此，在没有H3K79甲基化的情况下，Gata2的表达增加。通过体外功能获得和功能丧失实验，我们将验证GATA2受DOT1L直接调控的假设，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，在发育中的细胞群中，红细胞/髓系多潜能祖细胞中的GATA2表达受H3K79甲基化调节。为了验证这种可能性，我们将利用Dot1L条件KO小鼠的诱导多能干细胞（iPS），在体外建立基于胚胎干（ESC）细胞的红细胞分化模型。目的：观察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，以确定红细胞发育中的缺陷。目的4：探讨DOT1L在成人红细胞生成中的作用。为此，我们将在成年小鼠造血干细胞（HSC）中使用一种可诱导的条件Dot1L突变体特异性地删除该基因。我们将利用胎儿肝和骨髓源性HSC的集落测定来确定DOT1L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.