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.