The Role of the MBD2-NuRD Complex in Gamma-Globin Gene Silencing

PROJECT SUMMARY Epigenetic mechanisms are increasingly recognized as critical regulators of gene expression and other essential cellular functions in health and disease. Among these regulators, the MBD2-NuRD chromatin remodeling complex has been shown to be central to the silencing of fetal hemoglobin expression in adult erythropoiesis, as well as the survival and growth of cancer cells. As reactivation of fetal hemoglobin is of proven therapeutic benefit in sickle cell anemia, the central and long-term goal of this project is to discover small molecules and cyclic peptides that can disrupt protein-protein interactions in the MBD2-NuRD complex that will lead to the development of much-needed new therapy for the vast number of sickle cell anemia patients who will not benefit in the foreseeable future from gene therapy or stem cell transplantation. This goal will be pursued through the following specific aims: 1) To determine the cellular effects of mutations that disrupt the association of the GATAD2A CR-2 domain and the C-terminal domain (CTD) of CHD4 of the NuRD holo-complex and disrupt its interaction with MBD2, as well as effects mutations that disrupt the interaction between the MBD2 intrinsically disordered domain (IDR) and the histone deacetylase subcomplex (HDCC) of NuRD and the resultant effect of these mutations on fetal hemoglobin (HbF) expression. 2) To characterize in molecular detail the structural domains of these key interactions to facilitate discovery of small molecules that bind tightly to the CHD4-CTD pocket domain and disrupt its interaction with the GATAD2A CR-2 domain and those that bind tightly to the HDCC pocket domain to disrupt its interaction with the MBD2 IDR, thus in either case disrupting the NuRD holo- complex. The experimental approach employs a real-time and iterative feedback of a combination of detailed structural determination of the involved protein-protein interfaces, precise base-specific gene editing to disrupt these interactions in adult phenotype erythroid cells, comprehensive screening for discovery of small molecules that disrupt the interactions, and testing the effects of disruption of the interactions on Hb F expression and other cellular effects in erythroid cell culture assay systems and an animal model of primary human erythropoiesis. These experiments will advance the goal of developing effective new agents for treating sickle cell anemia and may also advance efforts to develop improved therapy for resistant cancers.