Association of LRF with MBD3-NuRD Versus MBD2 NuRD Mediates Opposing Effects on Developmental Globin Gene Regulation

Sufficiently high levels of HbF have been shown to ameliorate the pathophysiologic effects of HbS in sickle cell anemia patients bearing the homozygous codon 6 GAG to GTG mutation in the beta globin gene (HBB).Leukemia Related Factor (LRF, also known as ZBTB7A), BCL11A, and the MBD2-NuRD complex are major mediators of HbF silencing in adult erythroid cells, and combined depletion of both LRF and BCL11A was shown to result in nearly 100% HbF in adult erythroid cells (Masuda, etal, Science 2016). Both LRF and BCL11A are associated with a NuRD complex. In the case of BCL11A we have shown that this is specifically MBD2a-NuRD, and that silencing results from positioning of a nucleosome over the proximal gamma globin gene (HBG) promoter and is at least partially dependent on promoter CpG methylation. (Shang etal, Proc.Natl.Acad.Sci. USA, 2023). To define the specific NuRD complex that participates in LRF mediated HBG silencing we have investigated the roles of both MBD2-NuRD and MBD3-NuRD.NOME SEQ assays performed in MBD2 KO HUDEP-2 cells, showed the loss of a nucleosome positioned over the -189 GATA-1 site binding, which has been shown to be critical for HBG expression, and at which GATA-1 binds when the -198 HBG promoter binding site for LRF is mutated ( Doerfler, etal, Nature Genet., 2021). This suggests that MBD2-NuRD associates with LRF to silence HBG expression, consistent with the lack of effect of MBD3 depletion on HBG expression.

When both MBD2 and MBD3 were knocked out in HUDEP-2 cells, over 90% HBG/ (HBG+HBB) RNA levels were observed, similar to the levels observed with knockout of both LRF and BCL11A. However, the increase in HBG/(HBG+HBB) RNA ratio and corresponding HbF/ (HbF+HbA) ratio were due primarily to a 4-5-fold decrease in HBB RNA and protein, as the level of HBG RNA was the same or slightly less than in MBD2 KO cells.Chromatin conformation capture (3C) assays showed that combined depletion of MBD2 and MBD3 resulted in significantly decreased interaction of the Locus Control Region (LCR) with the HBB gene, increased LCR interaction with the BGLT3 locus and a very large increase in interaction of the 3’ beta globin locus enhancer with the HBB gene. These results demonstrate that MBD3-NuRD plays a critical role in the interaction of the LCR with the HBB gene to maintain its high level of expression in adult erythroid cells.

Depletion of LRF in MBD2 KO HUDEP-2 cells resulted in the same >90% HBG/(HBG+HBB) RNA level, as well as the same decrease in HBB RNA as depletion of MBD3. LRF depletion alone in WT HUDEP-2 cells also resulted in a significant decrease in HBB expression without a significant increase in HBG expression. This contrasts with the effect of mutation of the -198 LRF binding site in the HBG promoter, which results in high levels of HBG expression without a significant decrease in HBB expression ( Antoniou, etal, Nature Comm.,2022). Immunoprecipitation assays showed that LRF interacts with either MBD2-NuRD or MBD3-NuRD. These results are consistent with the fact that the repressive action of LRF, like that of BCL11A, requires association specifically with MBD2-NuRD to position a nucleosome and block the binding of a positive acting transcription factor, in this case GATA-1. Accordingly, disruption of MBD2-NuRD results in high level HBG expression while disruption of MBD3-NuRD does not, as we and others have shown. Conversely, LRF in association with MBD3-NuRD is required for maximum expression of the HBB gene in adult erythroid HUDEP-2 cells. Since combined disruption of the BCL11A-MBD2-NuRD complex and the LRF-MBD3-NuRD complex results in nearly 100% Hb F levels, these findings have implications for the treatment of sickle cell anemia, in which maximizing HbF levels while lowering HbS levels should provide the optimum protection from RBC sickling and its pathophysiologic consequences.