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

Abstract

As 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 γ-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 γ/γ+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 γ-globin expression. Knockdown of MBD2 in primary adult erythroid cells consistently increased γ/γ+β mRNA ratios by approximately 10-fold resulting in approximately 30-40% γ/γ+β mRNA levels and a corresponding increase in γ-globin protein. MBD2 exerts its repressive effects through recruitment of the chromatin remodeler CHD4 via a coiled-coil 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 γ-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.

Figure 1.

CRISPR Cas9 mediated knockout of MBD2 in HUDEP-2 cells results in approximately 50% γ/γ+β RNA expression and proportionally increased the fold effect on fetal hemoglobin (HbF) by high performance liquid chromatography (HPLC). (A) Western blot showing complete depletion of MBD2 protein in three independent clonal MBD2KO HUDEP-2 cell lines. (B-D) Levels of globin gene mRNA expression by qualitative polymerase chain reaction in the three clonal MBD2KO and pooled MBD2KO HUDEP-2 cell lines compared to scrambled guide RNA (sgSCR) controls. (B) γ-globin mRNA as a percentage of total globin (γ/γ+β%) mRNA. (C) Relative γ-globin expression compared to sgSCR. (D) Relative β-globin expression compared to sgSCR (E) Approximately 40-fold increase in HbF as measured by HPLC in MBD2KO HUDEP-2 cells compared to scramble. Note that a monoallelic polymorphism in the γ-globin gene of HUDEP-2 cells results in two distinct HbF HPLC peaks, consistent with published data. (F) Western blot showing protein levels of established γ-globin gene silencers (LRF, BCL11A and KLF1) in MBD2KO HUDEP-2 cells compared to sgSCR control cells. Error bars represent ± Standard Deviation of three biological repeats. *P<0.05; **P<0.01; n.s.: P>0.05 between sample and scramble. Statistical testing was performed using analysis of variance followed by the Tukey’s honestly significant difference procedure post-hoc test.

Figure 2.

Lentiviral knockdown of MBD2 in HUDEP-2 cells results in progressively increased γ-globin gene expression. (A) Schema of HUDEP-2 cell expansion for four, seven, or ten days after MBD2 shRNA lentiviral transduction, prior to a 3-day differentiation period. mRNA and protein were harvested after the expansion and differentiation period for all samples. (B) Western blot showing qualitative degree of MBD2 protein knockdown at the day-4 time point. (C) Time-course plot of γ-globin mRNA as a percentage of total globin mRNA by qRT-PCR at the indicated time points. (D) Relative MBD2 mRNA expression normalized by cyclophilin A compared to the shSCR sample by quantitative real-time polymerase chain reaction at four, seven, and ten-day time points. Error bars represent ± Standard Deviation of three biological repeats. *P<0.05; **P<0.01; n.s.: P>0.05 between sample and scramble. Statistical testing was performed using the Student’s t-test.

Figure 3.

CRISPR/Cas9 mediated knockout of MBD3 in HUDEP-2 cells has no effect on γ-globin gene expression. (A) Western blot showing complete depletion of MBD3 protein in 5 out of 7 independent clonal MBD3KO HUDEP-2 cell lines, (B and C) Plots of γ-globin mRNA as a percentage of total globin mRNA by qRT-PCR in the pooled MBD2KO, five clonal MBD3KO, and pooled MBD3KO HUDEP-2 cell lines compared to scrambled guide RNA (sgSCR) controls. (B) y-axis is continuous from 0 to 100% γ/γ+β. (C) The same data with a break in the y-axis and zoomed in to more clearly show sgSCR and MBD3KO data points. Error bars represent ± Standard Deviation of three biological repeats. *P<0.05; **P<0.01; n.s.: P>0.05 between sample and scramble. Statistical testing was performed using analysis of variance followed by the Tukey’s honestly significant difference procedure post-hoc test.

Figure 4.

Enforced expression of wild-type (WT) MBD2 (MBD2sgR) but not MBD2 containing mutations in its IDR or coiled-coil domain suppresses gamma globin RNA expression in MBD2 knockout HUDEP-2 cells. (A) Schematic depicting domains mutated in MBD2 lentiviral expression vectors. Silent mutations in the GR domain (sgR) convey resistance to CRISPR/Cas9 cleavage. (B) Co-IP of exogenously expressed FLAG-tagged MBD2 mutant contructs in 293T cells using anti-FLAG shows differing abilities to pull down NuRD members CHD4, GATAD2A, MTA2, and HDAC2. (C) Western blot showing enforced expression levels of WT MBD2sgR, and CCmutsgR, and IDRmutsgR MBD2 mutants in MBD2KO HUDEP-2 cells compared to scramble control cell levels of MBD2. (D and E) MBD2 knockout HUDEP-2 cells with enforced expression of WT MBD2 (MBD2KO+MBD2sgR) but not IDR-mutant (IDRmutsgR) or CC-mutant (CCmutsgR), causes decreased γ/γ+β and relative γ-globin mRNA. Error bars represent ± Standard Deviation of three biological repeats. *P<0.05; **P<0.01; n.s.: P>0.05. Statistical testing was performed using analysis of variance followed by the Tukey’s honestly significant difference procedure post-hoc test.

Figure 5.

The R286E/L287A double mutation reduces the helical propensity of the intrinsically disordered region (IDR). (A) Wild-type (WT) and mutant spectra. An overlay of the 2D ¹⁵N-HSQC spectra is shown for the WT (red) and R286E/L287A (blue) MBD2-IDR. (B) ¹³C chemical shift changes. Bar graphs depict the differences in chemical shift of the carbonyl (C’) and for a-carbons (Cα) between WT and R286E/L287A MBD2-IDR (WT – mutant in ppm). Three regions that show helical propensity are indicated with blue ellipses and the site of mutation indicated with red squares. Positive chemical shift changes indicate that the mutant MBD2-IDR shows less helical propensity in the region surrounding the site of mutation. (C) The results suggest that the R286E/L287A mutation disrupts binding to the histone deacetylase core of NuRD by reducing inherent helical propensity.

Figure 6.

Lentiviral shRNA knockdown (Kd) of MBD2 in CD34 progenitor-derived primary human erythroid cells results in high level γ/γ+β RNA expression and γ-globin protein without affecting erythroid differentiation. (A) Relative Kd of MBD2 mRNA’. (B) approximately 10-fold increase in γ/γ+β mRNA in MBD2 kd primary erythroid cells, across two different levels of differentiation, compared to scrambled shRNA controls. (C) Increase in γ-globin protein without change in β-globin protein as measured by western blot using anti γ-globin and anti β-globin antibody. (D) Flow cytometry analysis showing equivalent erythroid differentiation profiles of scramble control (sc) and MBD2 Kd CD34⁺ cells at day 7. Error bars represent ± Standard Deviation of three biological repeats. *P<0.05; **P< 0.01; n.s.: P>0.05. Statistical testing was performed using the Student’s t-test.

Figure 7.

Working model of the functional importance of MBD2-NuRD interacting domains in fetal hemoglobin (HbF) regulation. Previous work by our group^,, and the findings presented here support a model in which the HDAC core complex (HDCC) members HDAC1/2, RBBP4-7, and MTA1/2 are recruited to MBD2-NuRD through an intrinsically disordered region of MBD2, while GATAD2A/B and CHD4 are recruited through a c-terminal coiled-coil motif of MBD2; independently decoupling either subcomplex results in an abrogation of MBD2-NuRD-mediated HbF silencing.