MBD2a-NuRD Binds to the γ-Globin Gene Promoter and Uniquely Forms a Complex Required for Bcl11a Mediated Silencing of Hb F Expression

A mechanistic understanding of γ-globin silencing is of clinical importance because reversal of silencing in adult erythroid cells results in high level expression Hb F which is therapeutic in sickle cell anemia and beta thalassemia. We describe here studies to elucidate the mechanism by which MBD2a-NuRD is an obligate partner of Bcl11a in silencing of HbF.

We have previously shown that depletion of MBD2 or disruption of the MBD2-NuRD chromatin remodeling complex, but not MBD3-NuRD complex results in high levels of Hb F despite a concomitant 2-3 fold increase in expression of Bcl11a, a major silencing transcription factor (Yu etal. Haematologica, 2019). At the same time, Bcl11a can interact with both the MBD3 and MBD2 NuRD complexes.

To elucidate the specific role of MBD2-NuRD, Chromatin Immunoprecipitation-qPCR(ChIP-qPCR) assays carried out in both adult erythroid phenotype HUDEP-2 and primary progenitor derived adult erythroid cells showed binding at the γ-globin gene promoter. To clarify the role of MBD2-NuRD in establishing closed chromatin configuration at the γ-globin gene loci, we carried out ATAC seq assays which showed that MBD2 knockout resulted in the same pattern of chromatin opening as reported for Bcl11a knockout in HUDEP-2 cells, consistent with our previous data showing ~40-50% Hb F in MBD2 knockout cells.

In order to examine the effect of MBD2-NuRD on nucleosome positioning over the γ-globin gene promoter, NOME-SEQ assays were performed in HUDEP 2 cells which showed a nucleosome positioned at -110 to + 36 relative to the transcription start site (TSS) in MBD +/+ cells but not MBD2 KO HUDEP-2 cells, such that the NFY-A transcriptional activator binding site but not the Bcl11a repressor binding site is occluded.

MBD2 exists in two isoforms in erythroid cells, resulting from alternative translation initiation sites. MBD2a contains an arginine rich (GR) domain which is absent in MBD2b and MBD3. Addback of MBD2a but not MBD2b in MBD2 KO HUDEP-2 cells resulted in silencing of γ-globin gene expression. Editing of the endogenous MBD2 gene such that predominantly MBD2b was expressed relieved silencing.

To understand how the GR domain contributes to silencing we carried out unbiased proteomic analysis of MBD2a vs MBD2b NuRD complexes, which identified only 3 proteins uniquely associated with MBD2a. One of these, PRMT5, has been implicated in silencing of HbF. Knockdown of PRMT5 resulted in a`from <1% to ~15% γ-globin RNA in parental HUDEP-2 cells and a 4-5 fold increase in primary human progenitor derived erythroid cells, but caused no significant further increase in MBD2 KO HUDEP-2 cells. ChIP qPCR assays showed that PRMT5 and its obligate binding partner MEP50 occupy the γ- promoter in parental HUDEP-2 cells and primary erythroid cells but not in MBD2 KO HUDEP-2 cells. Furthermore, knockdown of PRMT5 resulted in frequent displacement of the nucleosome positioned at -110 to + 36 relative to the TSS.

In order to determine the role that the GR domain might have in occupancy of MBD2a-NuRD at the γ-promoter, in vitro binding studies where done, which showed a requirement for both CpG methylation and the GR domain for high affinity binding of the methyl cytosine binding domain of MBD2 to the proximal γ-globin gene promoter sequences.

We conclude that MBD2a-NuRD is required for silencing of Hb F due to its ability to localize at the methylated γ-globin gene promoter in conjunction with Bcl11a and recruit PRMT5, thereby stably positioning a nucleosome that blocks binding of the transcriptional activator NFY-A. These results reinforce the concept that targeted disruption of this specific complex has therapeutic potential to increase HbF in sickle cell anemia and β thalassemia.

No relevant conflicts of interest to declare.