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Editorials
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Figure 1. Repression of the g-globin gene by MBD2. (A) Contrasting effect of deletions of MBD2 and MBD3 on fetal hemoglobin (HbF). (B) Amino acid substitutions within the intrinsically disordered region (IDR) and coiled-coiled (CC) domains of MBD2 disrupt interactions with components of the NURD co-repressor and fail to repress HbF in MBD2 knockout cells.
in chicken erythrocytes.18 Both MBD2 and/or MBD3 were identified as components of co-repressor complexes recruited by Bcl11A, ZBTB7A, and TR2/TR4.13,14 Although MBD2 and MBD3 were initially thought to be part of the same NURD complex, studies performed in mouse deletion models showed that these proteins have discrete physiological roles as MBD3-/- mice died in early embryogenesis while MBD2-/- mice remained viable and fertile.7 Subsequent biochemical analyses demonstrated that MBD2 and MBD3 were present in separate NURD complexes19 thus leading to the question of whether MBD2-NURD and MBD3-NURD have distinct functions in g-globin repression. In this issue of the Journal, Yu et al.6 has elegantly and clearly answered this question by analy- sis of g-globin expression in biallelic CRISPR/Cas9 knock- outs (KO) of MBD2 and MBD3 in the HUDEP-2 human erythroid cell line. HbF expression was reactivated to high levels in MBD2 KO cells but not in MBD3 KO cells (Figure 1A). Partial knockdown (KD) of MBD2 by lentivirus-deliv- ered siRNA increased g-globin expression substantially in both HUDEP-2 and primary human erythroid cell cul- tures. Importantly, no effect on expression of the known g-globin repressors including Bcl11A and ZBTB7A was observed in MBD2 KO cells. Erythroid differentiation was not blocked or substantially altered in MBD2 KO HUDEP- 2 cells or in MBD2 KD primary erythroid cell cultures. These experiments provide strong and definitive evidence that MBD2 and not MBD3 is responsible for g-globin repression in adult human erythroid cells. Recent results of a CRISPR/Cas9 mutagenesis screening have confirmed a specific role for MBD2 in g-globin silencing.20 In addi- tional experiments, the role of specific regions of MBD2 previously shown to function as important sites facilitat- ing protein-protein interactions within co-repressor com-
plexes were analyzed for their mechanistic roles in g-glo- bin repression (Figure 1B). The effect of amino acid substi- tutions within the CC domain of MBD2 that mediate interactions with GATAD2A and subsequent recruitment of the chromatin-modifying protein CH4D, and also with- in the IDR necessary for interaction of MBD2 with an HDAC core complex, were analyzed for their ability to repress g-globin expression in g-globin expressing MBD2 KO cells by forced expression of wild-type or mutant MBD2 delivered via lentiviral vectors. Wild-type MBD2 decreased g-globin expression but MBD2 containing site- specific mutations in the CC domain or IDR did not, indi- cating that protein-protein interactions facilitated by these regions were critical for g-globin repression.
Even though many important questions remain regard- ing the exact role of MBD2 in g-globin silencing, the essential work of identifying and developing small mole- cule pharmacological agents that target the CC domain and IDR and block the specific contacts mediating the critical functional interactions with other co-repressor proteins can now begin. Because the overall phenotypic effects observed in MBD2 KO mice are minor, it is rea- sonable to predict that drugs specifically targeting MBD2 would have minimal side effects in patients and thus offer great potential for future therapy for the hemoglo- binopathies.
References
1. Piel FB, Steinberg MH, Rees DC. Sickle Cell Disease. N Engl J Med. 2017;376(16):1561-1573.
2. Modell B, Darlison M. Global epidemiology of haemoglobin disor- ders and derived service indicators. Bull World health Organ. 2008;86(6):480-487.
3. Ginder GD. Epigenetic regulation of fetal globin gene expression in
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haematologica | 2019; 104(12)