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

Abstract

A s high fetal hemoglobin levels ameliorate the underlying pathophysiological defects in sickle cell anemia and beta (β)-thalassemia, understanding the mechanisms that enforce silencing of fetal hemoglobin postnatally offers the promise of effective molecular therapy. Depletion of the Nucleosome Remodeling and Deacetylase complex member MBD2 causes a 10-20-fold increase in g-globin gene expression in adult β-globin locus yeast artificial chromosome transgenic mice. To determine the effect of MBD2 depletion in human erythroid cells, genome editing technology was utilized to knockout MBD2 in Human Umbilical cord Derived Erythroid Progenitor-2 cells resulting in g/g+β mRNA levels of approximately 50% and approximately 40% fetal hemoglobin by high performance liquid chromatography. In contrast, MBD3 knockout had no appreciable effect on g-globin expression. Knockdown of MBD2 in primary adult erythroid cells consistently increased g/g+β mRNA ratios by approximately 10-fold resulting in approximately 30-40% g/g+β mRNA levels and a corresponding increase in g-globin protein. MBD2 exerts its repressive effects through recruitment of the chromatin remodeler CHD4 via a coiledcoil domain, and the histone deacetylase core complex via an intrinsically disordered region. Enforced expression of wild-type MBD2 in MBD2 knockout cells caused a 5-fold decrease in g-globin mRNA while neither the coiled-coil mutant nor the intrinsically disordered region mutant MBD2 proteins had an inhibitory effect. Co-immunoprecipitation assays showed that the coiled-coil and intrinsically disorder region mutations disrupt complex formation by dissociating the CHD4 and the histone deacetylase core complex components, respectively. These results establish the MBD2 Nucleosome Remodeling and Deacetylase complex as a major silencer of fetal hemoglobin in human erythroid cells and point to the coiled-coil and intrinsically disordered region of MBD2 as potential therapeutic targets.

Direct Generation of Immortalized Erythroid Progenitor Cell Lines from Peripheral Blood Mononuclear Cells

“…As iEPCs can be maintained in culture as early erythroid progenitors and can be induced to differentiate to the cells of late stages of erythropoiesis, they have also been extensively used for studying the mechanisms of human erythropoiesis [ 63 , 64 ] and for identifying genetic factors involved in globin gene switching [ 26 , 28 , 65 , 66 , 67 ] by genetic manipulations of these cells by RNAi [ 20 , 67 , 68 ] and gene editing [ 23 , 26 , 28 , 69 , 70 ]. Genetic mutations were recently introduced in the genes associated with erythroid diseases to generate disease models [ 71 , 72 ].…”

ZNF410 represses fetal globin by devoted control of CHD4/NuRD

“…However ZBTB7A is required for terminal erythropoiesis and germinal center B cell maturation and plays important roles in Tlymphocytes, osteoclasts and HSCs 28 . A specific NuRD subcomplex including CHD4, GATAD2A, MBD2, MTA2 and HDAC2 is required for HbF silencing 6,29 . Targeting NuRD including key protein-protein interactions appears promising but would need to navigate the numerous gene expression programs that depend on this chromatin complex.…”

Regulating the Regulators: The Role of Histone Deacetylase 1 (HDAC1) in Erythropoiesis

“…The knock down of Mi-2β, the chromatin remodeler within NuRD corepressor complex, relieves γ-globin gene silencing in β-YAC (Yeast artificial chromosome) transgenic murine erythroid cells and in CD34(+) progenitor-derived human primary adult erythroid cells [ 167 ]. Depletion of MBD2, a component of the NuRD complex, increased γ to β mRNA ratios in adult erythroid cells [ 168 ]. γ-globin expression is also regulated by HDACs through chromatin modification, and several transcription factors that regulate HbF are associated with these HDAC proteins [ 166 ].…”