PLX109446

GSE121992: Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells

• Organsim human
• Type RNASEQ
• Target gene
• Project ARCHS4

As high fetal hemoglobin (HbF) levels ameliorate the underlying pathophysiologic defects in sickle cell anemia and -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 -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 /+ 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 /+ mRNA ratios by ~10-fold resulting in ~30-40% /+ 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. SOURCE: Gordon,D,Ginder (gdginder@vcu.edu) - 134 Goodwin Research Laboratory Virginia Commonwealth University