Targeting a Methyl Cytosine-Binding Protein Complex to Augment Fetal/Embryonic Globin Expression.
Authors: Williams David C. , Gnanapragasam Merlin Nithya , Webb Heather D , Scarsdale J. Neel , Ginder Gordon D. 
Published: October 2019

Abstract
Abstract 974
The vertebrate β-type globin genes were among the first genes shown to be regulated, at least in part, by DNA methylation. The mechanism of transcriptional repression by DNA methylation is chiefly through binding of methyl cytosine binding domain (MBD) proteins and their associated co-repressor complexes. The chicken homolog to an MBD2 containing NuRD co-repressor complex (MeCPC) has previously been purified from primary erythroid cells and characterized as binding to the methylated ρ-globin promoter in erythroid cells of adult chickens in which the gene is silent [Kransdorf et al. Blood 2006; 108:2836-45]. Knockdown of MBD2 by siRNA in MEL cells stably transfected with a methylated ρ-globin gene construct leads to a greater than 10-fold increase in ρ-globin gene expression. Likewise, knockout of MBD2 results in a ∼20 fold upregulation of the human gamma globin gene in adult erythroid cells of βYAC transgenic mice [Rupon et al. PNAS 2006; 103:6617-22]. These observations suggest that disruption of the interaction of MBD2 with its co-repressor complex in adult erythropoiesis would increase fetal hemoglobin expression; a therapeutically beneficial effect for both sickle cell anemia and β-thalassemia. This possibility is further supported by the observation that DNA methylation inhibitors such as 5-azacitidine can increase the expression of γ-globin in patients. Based on these studies, we have pursued structural analysis of the interaction between MBD2 and other components from the MeCPC. We have shown that the individual coiled coil regions from MBD2 and a subunit of the NuRD complex, p66α, form a stable heterodimeric complex. Solving the structure of this coiled coil complex by NMR reveals that the interaction involves a combination of hydrophobic and ionic interactions typical of coiled coil complexes as well as a unique charge interaction involving a pair of highly conserved glutamates residues from p66α and arginine residues from MBD2. The key residues involved in binding are conserved across species, between p66α and p66β homologs, as well as between MBD2, MBD3, and the MBD3L1-L5 homologs. We have shown that the p66α coiled coil can stably bind to MBD3 in solution, indicating that similar tertiary interactions are involved in forming both MBD2 and MBD3 containing NuRD complexes. In order to explore this interaction as a potential therapeutic target, we hypothesized that over-expressing the p66α coiled coil region in tissue culture would disrupt the formation of a normal MeCPC and thereby block the function of MBD2. As predicted, expressing this region in both avian (MEL-ρ) and human (CID-βYAC) tissue culture models of globin gene regulation in adult erythroid cells induces embryonic and fetal β-type globin gene expression, respectively. Furthermore, knock-down of p66α induces fetal/embryonic globin gene expression to a similar degree as knock-down of MBD2. These studies suggest a model in which the p66α coiled peptide can bind MBD2 and block recruitment of native p66α to the NuRD complex, thereby acting in a dominant-negative manner to disrupt MBD2 function. We propose that a peptidomimetic of the p66α coiled coil region could be used therapeutically to augment fetal hemoglobin expression.

Disclosures:
No relevant conflicts of interest to declare.