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X. Yu et al.
lae of SCD lead to a shortened and reduced quality of life. Current treatments for SCD are primarily supportive. Hydroxyurea and L-glutamine are the only standard agents available that reduce the frequency of sickle cell crises. β-thalassemia major resulting from insufficient β-globin production has a high prevalence worldwide3 and has limited treatment options, with most patients remaining transfusion-dependent throughout life. The only curative treatment for either SCD or β-thalassemia is stem cell transplantation,4 which carries significant risks and is not readily accessible in developing nations. Thus new treatment options are needed. Importantly, sufficient levels of fetal hemoglobin (HbF) ameliorate the underlying
mutually exclusive within the same NuRD complex.33 Previous to this report, the precise role of MBD3-NuRD on g-globin gene repression had been less clearly defined.34,35
Here we show that MBD2 is among the strongest repressors of HbF expression in the adult erythroid pheno- type HUDEP-2 cell line, as knockout (KO) of MBD2 results in ~50% g/g+β mRNA compared to <1% in con- trols, while KO of MBD3 in HUDEP-2 cells has no appre- ciable effect on g-globin gene expression. We also estab- lish the functional importance of the CC domain and the IDR of MBD2 for g-globin silencing in human erythroid cells. Together these data solidify MBD2’s role as a major silencer of HbF and suggest novel strategies for targeting MBD2-NuRD in the β-type hemoglobinopathies.
5,6 1,7 pathophysiological defects in β-thalassemia and SCD.
Studies aimed at a full understanding of the mechanisms that enforce silencing of HbF expression in adult erythroid cells offer the promise of effective targeted molecular ther- apy.
During development, humans undergo a progressive switch from embryonic e (Hb Gower-1, Hb Gower-2) to fetal g (HbF) and finally to adult β (HbA) and d (HbA2) type globin production. By adulthood, g-globin typically makes up approximately 1-2% of total β-like globin chains in hemoglobin.8 Numerous transcriptional and epigenetic regulators of g-globin expression have been shown to mediate g-globin gene silencing, including BCL11A, KLF1/EKLF, LRF/Pokemon, MBD2-NuRD, and LSD-1, among others.9-16 The zinc finger transcription factors BCL11A and LRF have been shown to independently exert especially strong silencing of the g-globin gene in an immortalized Human Umbilical cord Derived Erythroid Progenitor-2 (HUDEP-2) cell line that displays an adult erythroid phenotype.13,17
In addition to transcription factors, epigenetic mecha- nisms, including DNA methylation and histone modifica- tions,12,18-23 are of importance in developmental globin gene regulation. MBD2, a member of the methyl-CpG binding domain (MBD) protein family that includes MeCP2, MBD1, MBD2, MBD3, and MBD4, binds to DNA contain- ing methylated CpG rich sequences with high affinity and recruits other members of the Nucleosome Remodeling and Deacetylase (NuRD) co-repressor complex through specific protein-protein interactions.24-28 The NuRD co- repressor complex, classically made up of one or more of at least six core proteins, including MBD2/3, CHD3/4, HDAC1/2, MTA1/2/3, RBBP4/7, and GATAD2A/B is unique in containing both an ATPase chromatin remodel- ing complex and a histone deacetylase complex (HDCC).29-31 Previous work by our group has shown that depletion of MBD2 or disruption of NuRD complex com- ponents abrogates silencing of fetal hemoglobin in multi- ple mammalian erythroid model systems.9,27,32
MBD2 interacts with GATAD2A and in turn CHD4 through a C-terminal coiled-coil (CC) motif and enforced expression of a GATAD2A CC domain inhibitory peptide abrogates the interaction of MBD2 with GATAD2A/CHD4 and partially relieves g-globin gene silencing in β-YAC bearing murine CID cells.27 More recently we have shown the functional importance of an intrinsically disordered region (IDR) within MBD2 for recruitment of the HDAC core of the NuRD complex to silence a highly methylated tumor suppressor gene in breast cancer cells.25
Unlike MBD2, MBD3 shows greatly reduced selectivity for methylated DNA. Additionally, MBD2 and MBD3 are
Methods
+
+
Human CD34 cells were purified from deidentified apheresis
units discarded by the VCU Bone Marrow Transplant Unit, and therefore Institutional Review Board exempt. CD34+ cells were isolated using the EasySep Human CD34 Positive Selection Kit
9,27
(StemCell Technologies Inc.) as described previously. Erythroid
differentiation and maturation were monitored by measuring expression of the erythroid lineage markers CD235a and CD71 via flow cytometry after expansion and differentiation (Online Supplementary Methods).
HUDEP-2 cell culture and erythroid differentiation
HUDEP-2, an immortalized human erythroid progenitor cell line, was a kind gift from Dr. Yukio Nakamura.17 Expansion and differentiation protocols for HUDEP-2 cells have been previously described17 and are detailed in the Online Supplementary Methods.
Genome editing-mediated depletion of MBD2/MBD3 in HUDEP-2 cells
sgRNA sequences targeting MBD2 or MBD3 were cloned into a LentiCRISPR-AcGFP backbone, packaged, and transduced (MOI=40) into HUDEP-2 cells. sgRNA sequences and cloning pro- tocols are detailed in the Online Supplementary Methods. For stable depletion of MBD2 or MBD3 in HUDEP-2 cells, single cell colonies were isolated by limiting dilution and screened by west- ern blotting. After three weeks of clonal expansion, three bi-allelic MBD2KO clones and five bi-allelic MBD3KO clones were expanded and analyzed individually as well as in pools.
Lentiviral-mediated “Add-back” of MBD2 in MBD2 null cells
pLV203 vectors containing sequences encoding MBD2sgR, IDRmutsgR, or CCmutsgR were packaged as described previous- ly25,27 and used to infect MBD2KO HUDEP-2 cells. MBD2 mutant sequences are provided (Online Supplementary Appendix and Online Supplementary Figures S7-S10). Translationally silent mutations were introduced into the MBD2 expression constructs to confer resistance to MBD2 shRNA and CRISPR/Cas9 sgRNA. The assess- ment of exogenous MBD2 expression in HUDEP-2 MBD2KO cells was carried out by western immunoblotting five days post lentivi- ral infection as described.27
Hemoglobin high performance liquid chromatography
Hemolysates were prepared from scramble sgRNA or MBD2KO HUDEP-2 cells (4 × 107 cells) on day 7 of differentiation. High performance liquid chromatography (HPLC) analysis was
Isolation and maturation of human CD34 cells
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haematologica | 2019; 104(12)