MBD2a–NuRD binds to the methylated γ-globin gene promoter and uniquely forms a complex required for silencing of HbF expression

Abstract

During human development, there is a switch in the erythroid compartment at birth that results in silencing of expression of fetal hemoglobin (HbF). Reversal of this silencing has been shown to be effective in overcoming the pathophysiologic defect in sickle cell anemia. Among the many transcription factors and epigenetic effectors that are known to mediate HbF silencing, two of the most potent are BCL11A and MBD2–NuRD. In this report, we present direct evidence that MBD2–NuRD occupies the γ-globin gene promoter in adult erythroid cells and positions a nucleosome there that results in a closed chromatin conformation that prevents binding of the transcriptional activator, NF-Y. We show that the specific isoform, MBD2a, is required for the formation and stable occupancy of this repressor complex that includes BCL11A, MBD2a–NuRD, and the arginine methyltransferase, PRMT5. The methyl cytosine binding preference and the arginine-rich (GR) domain of MBD2a are required for high affinity binding to methylated γ-globin gene proximal promoter DNA sequences. Mutation of the methyl cytosine–binding domain (MBD) of MBD2 results in a variable but consistent loss of γ-globin gene silencing, in support of the importance of promoter methylation. The GR domain of MBD2a is also required for recruitment of PRMT5, which in turn results in placement of the repressive chromatin mark H3K8me2s at the promoter. These findings support a unified model that integrates the respective roles of BCL11A, MBD2a–NuRD, PRMT5, and DNA methylation in HbF silencing.

Methyl-CpG binding domain protein 2 (Mbd2) drives breast cancer progression through the modulation of epithelial-to-mesenchymal transition

“…6c . Since MBD2a and MBD2b are two major isoforms expressed in somatic cells 62 , we subsequently rescued the expression of the MBD2 protein by reintroducing either the full-length canonical MBD2a isoform or the MBD2b isoform, which lacks the arginine-rich domain at the N-terminus. We observed that the canonical MBD2a isoform rescued the expression of EMT genes ( SPARC , SPP1 , and CDH2 ) in MBD2 -depleted cells as well as the invasive phenotype of MDA-MB-231 cells (Supplementary Fig.…”

Roles of Nuclear Orphan Receptors TR2 and TR4 during Hematopoiesis

“…The NuRD complex has been shown to interact with LSD1 and DNMT1 to facilitate chromatin remodeling [64,65]. Numerous NuRD complex components, including MBD2, Mi2B (CHD4), and GATAD2A, have been shown to repress γ-globin expression directly or indirectly by disrupting the NuRD complex [55,[66][67][68]. However, it is not clear whether interaction exists between these proteins and TR2 and TR4 as it pertains to γ-globin expression.…”

C2H2 Zinc Finger Transcription Factors Associated with Hemoglobinopathies

“…The first was members of the nucleosome remodeling and deacetylase (NuRD) complex 59 – 61 including CHD4, MTA2, GATAD2A, MBD2 and HDAC2 that are already known to regulate HbF. 62 – 65 We will only discuss the MBD2 component of NuRD complex briefly, because the complex has been extensively studied as both an epigenetic corepressor and a silencer of HbF expression. 62 , 64 – 68 The second group consisted of seven C2H2 ZF proteins, including some known for silencing the fetal γ-globin genes in adult human erythroid cells: ZBTB7A, 69 BCL11A, 70 , KLF1, 71 , ZNF410, 57 , 58 IKZF1, WIZ and ZNF644.…”

“…ZBTB7A and BCL11A repress γ-globin expression by independently coordinating with a unique NuRD repressor complex. 69 A recent study established that the MBD2a-specific NuRD complex occupies the γ-globin gene promoter in HUDEP-2 cells, where it positions a nucleosome that spans nt positions −110 to +36, immediately adjacent to the BCL11A binding site at −115, 65 thus ensuring a closed chromatin conformation conductive to transcriptional silencing. To achieve this nucleosome positioning, MBD2α likely binds to methylated CpG dinucleotides at −50 and/or −53 ( Figure 1B and see graphical abstract ) and recruits the NuRD core complex.…”

“…This hypothesis is based on the following: (1) G9a and DNMT1 directly interact, 145 (2) DNA methylation is intricately connected to the presence of H3K9 methylation, 146 and (3) DNA methylation is required for the MBD2-NuRD complex to occupy the γ-globin promoters. 65 Although G9a/GLP enzymatic inhibitors are being tested in preclinical models as potential anticancer agents, 147 – 153 UNC0638, the most commonly-used small molecule inhibitor of G9a, induces HbF expression in erythroid cells. 154 – 156 …”