Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells

As high fetal hemoglobin (HbF) levels ameliorate the underlying pathophysiologic defects in sickle cell anemia and ß-thalassemia, understanding the mechanisms that enforce silencing of HbF postnatally offers the promise of effective molecular therapy. Depletion of Methyl cytosine Binding Domain protein 2 (MBD2) causes a 10-20 fold increase in ?-globin gene expression in adult ß-YAC transgenic mice. To determine the effect of MBD2 depletion in human erythroid cells, CRISPR-Cas9 mediated knockout (KO) was carried out in Human Umbilical cord Derived Erythroid Progenitor-2 (HUDEP-2) erythroid cells resulting in ?/?+ß mRNA levels of ~50% and ~40% HbF by HPLC. In contrast, MBD3 KO had no appreciable effect on ?-globin mRNA. Knockdown (Kd) of MBD2 in primary adult erythroid cells consistently increased ?/?+ß mRNA ratios by ~10-fold resulting in ~30-40% ?/?+ß mRNA levels and a corresponding increase in ?-globin protein. MBD2 exerts its repressive effects through recruitment of the CHD4 chromatin remodeling component of NuRD through a coiled-coil (CC) domain, and the histone deacetylase (HDCC) component via an intrinsically disordered region (IDR). Enforced expression of wild-type MBD2 in MBD2KO HUDEP-2 cells caused a 5-fold decrease in ?-globin mRNA while neither the CC mutant nor the IDR mutant MBD2 proteins had an inhibitory effect. Consistently, co-immunoprecipitation assays showed that the CC and IDR domain mutations disrupt NuRD complex formation by dissociating the CHD4 and HDCC domains respectively. These results establish MBD2-NuRD as a major silencer of HbF in human erythroid cells and point to the CC and IDR domains of MBD2-NuRD as potential therapeutic targets. Overall design: RNA-seq was performed on scrambled gRNA control and MBD2 knockout HUDEP-2 cells before and after erythroid differentiation in order to investigate the effects of MBD2 depletion on gene expression in a human erythroid cell model system. 3 replicates were performed for each of these 4 conditions, for a total of 12 samples.