Establishing the role and mechanisms of LSD1 during megakaryocytic and erythroid fate commitment

PROJECT SUMMARY/ABSTRACT Hematopoiesis is responsible for producing the varied cell types found in blood. In adults, this process primarily takes place in the bone marrow and is characterized by successive rounds of differentiation beginning with the hematopoietic stem cell through to lineage commitment. This process involves progressive narrowing of lineage potency as progenitors eventually commit to the production of a single cell lineage. Disruption of hematopoiesis can lead to benign and malignant pathologies. We focus on the bipotent megakaryocytic-erythroid progenitor (MEP), which has the potential to differentiate into a lineage committed erythroid progenitor (ErP) or a lineage committed megakaryocytic progenitor (MkP). MEP fate specification has been studied to a limited extent, uncovering only a small number of influences that contribute to this process. One understudied aspect of human MEP fate specification is the role of epigenetics. KDM1A (LSD1) is known to be important for erythroid maturation, but its role in MEP fate specification is unknown. Preliminary data using primary human cells show that inhibition of LSD1 in primary human ErP results in the ability of erythroid committed progenitors to undergo granulocytic-monocytic and megakaryocytic commitment. In contrast, LSD1 inhibition does not affect MkP commitment to the megakaryocytic fate. This suggests that LSD1 only antagonizes alternative lineage potential during erythropoiesis, and not megakaryopoiesis, even though LSD1 mRNA is expressed at similar levels in MEP, ErP, and MkP. The goal of this proposal is to establish the mechanism by which LSD1 promotes erythroid commitment while silencing alternative myeloid and megakaryocytic lineage potential. I will achieve these goals utilizing a mix of functional (cell culture), genetic and epigenetic approaches to determine where and how LSD1 regulates gene expression and fate specification in MEP, ErP, and MkP. Computational analyses will determine candidate gene targets, and potential genomic sites to force epigenetic modifications using different fusion proteins based on the catalytically inactive Cas9 endonuclease (dCas9). These results will establish the epigenetic mechanisms that govern erythroid lineage commitment mediated by LSD1. They will also shed light on the distinct lineage-specific epigenetic mechanisms that mediate and maintain fate specification in hematopoiesis.