Investigating the mechanisms of polycomb group protein mediated fetal hemoglobin silencing

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

Project Summary/Abstract The fetal to adult hemoglobin switch is a developmental event in erythroid cells that reconfigures the beta globin locus from a chromatin environment permissive to transcription of fetal type globin to a chromatin environment that favors transcription of adult beta globin. There is tremendous interest in understanding the process of globin switching because genetic diseases affecting the beta globin locus including sickle cell disease and beta thalassemia can be effectively treated by increasing the amount of fetal hemoglobin (HbF) produced in adult erythroid cells. While there is an established role for sequence specific transcription factors in the fetal to adult globin switch, the contributions of epigenetic factors in the establishment and maintenance of globin switching are far less well characterized. Recent work demonstrated that polycomb group proteins (PcG) participate in fetal globin silencing. PcG proteins are a highly diverse group of epigenetic modifiers, and their collective activity is critical for modulating gene expression in a tissue and differentiation specific manner. Due to the inherent complexity of PcG proteins, it is possible that there are specific PcG subunit compositions in erythroid cells that account for PcG mediated HbF silencing. However, PcG proteins are also involved in silencing important developmental and cell cycle regulatory genes, and many PcG proteins are aberrantly expressed or mutated in a variety of malignancies. I hypothesized that by defining the PcG subunits involved in HbF silencing, it may be possible to selectively perturb their function but without the broader detrimental effects cell viability and differentiation. This in turn may present new pharmacologic targets for the treatment of sickle cell disease. To identify the PcG subunits and protein domains involved in HbF silencing, I performed a high density CRISPR-Cas9 screen targeting nearly all PAM sites in the polycomb repressive complex 2 (PRC2) as well as most domain encoding regions of polycomb repressive complex 1 (PRC1). In my first aim, I will use CRISPR to introduce targeted mutations in key candidate regions identified in the initial screen of PRC2 to decouple HbF silencing from defects in erythroid viability. In my second aim, I will dissect the contributions of a novel PRC1 component identified in the screen as a potential negative regulator or HbF in erythroid cells. The proposed study will further the knowledge of how PcG proteins modulate chromatin and silence genes in the erythroid lineage, as well as provide a basis for the rational targeting of specific polycomb complexes to re- activate HbF for the treatment of sickle cell disease and beta thalassemia. In the long term, I envision a career as independent physician scientist in an academic setting. Through the combined mentorship of my sponsor and the resources available to me at the University of Pennsylvania/Children’s Hospital of Philadelphia, I am in an excellent position to develop the necessary skills and knowledge to become a successful physician scientist.