Structural Analysis of a Methyl Cytosine-Binding Protein Complex That Regulates Expression of Fetal/Embryonic Globin Genes

DNA methylation is one of the major epigenetic mechanisms for regulating gene expression in eukaryotic organisms. Regions of increased CpG content (CpG islands) are often associated with gene promoters and can recruit regulatory complexes that specifically recognize methyl cytosine and down-regulate expression of the associated gene. One such regulatory complex, MeCPC, has recently been purified from a primary cell source and characterized as binding to the methylated ρ-globin promoter in chickens [Kransdorf et al. Blood2006; 108:2836–45]. This complex contains the chicken homologues of MBD2, MTA1, HDAC2, p66, and Mi2. Methylation of the ρ-globin promoter and binding of the MeCPC promotes the switch to β-globin during development while siRNA knock-down of cMBD2 leads to a 25-fold increase in ρ-globin expression. Likewise, knockout of MBD2 results in a ~20 fold upregulation of the human gamma globin gene in β.YAC transgenic mice [Rupon et al. PNAS2006; 103:6617–22]. These observations suggest that disruption of the MeCPC 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. In order to determine the molecular details of the interactions that form the MeCPC, we are pursuing atomic resolution structural analysis of MBD2 and its binding partners. The solution structure of the methyl binding domain from MBD2 bound to a methylated oligonucleotide from the ρ-globin promoter has been determined by NMR. MBD2 recognizes a central methylated CpG in this oligonucleotide through highly conserved arginine and tyrosine amino acids. Interestingly, MBD2 adopts a single orientation on the ρ-globin promoter sequence despite the symmetry of the CpG sequence. This observation indicates that interactions between MBD2 and the nucleotides surrounding the methylated CpG likely orient the protein on this promoter and may help explain why different methyl binding domain proteins are selectively recruited to different promoter regions and CpG islands. This work represents the first atomic resolution structure reported to date of MBD2 bound to a methylated promoter sequence identified as a target in a primary cell source. Additional structural studies reported here build on the observation that MBD2 directly interacts with p66α through two highly conserved regions in p66α and two regions in MBD2 [Brackertz et al. J. Biol. Chem.2002; 277:40958–66]. The coiled coil domains of each protein have been characterized by gel filtration chromatography and NMR spectroscopy. These studies demonstrate that a specific and stable interaction is formed between the coiled domains from each protein. Intermolecular NOEs have been measured by NMR which highlight the amino residues responsible for direct protein:protein contacts. Ultimately these structural studies will provide a molecular basis for the development of agents that will specifically disrupt the MeCPC complex and increase expression of embryonic and fetal hemoglobin in adult erythrocytes.