Identification and Characterization of the DNA Binding Domain of CpG-binding Protein*

CpG-binding protein is a transcriptional activator that exhibits a unique DNA binding specificity for unmethylated CpG motifs. CpG-binding protein contains a cysteine-rich CXXC domain that is conserved in DNA methyltransferase 1, methyl binding domain protein 1, and human trithorax. In vitro DNA binding assays reveal that CpG-binding protein contains a single DNA binding domain comprised of the CXXC domain and a short carboxyl extension. Specific mutation to alanine of individual conserved cysteine residues within the CXXC domain abolishes DNA binding activity. Denaturation/renaturation experiments in the presence of various metal cations demonstrate that the CXXC domain requires zinc for efficient DNA binding activity. Ligand selection of high affinity binding sites from a pool of degenerate oligonucleotides reveals that CpG-binding protein interacts with a variety of sequences that contains the CpG dinucleotide with a consensus binding site of (A/C)CpG(A/C). Mutation of the CpG motif(s) present within ligand-selected oligonucleotides ablates the interaction with CpG-binding protein, and mutation to thymine of the nucleotides flanking the CpG motifs reduces the affinity of CpG-binding protein. Hence, a CpG motif is necessary and sufficient to comprise a binding site for CpG-binding protein, although the immediate flanking sequence affects binding affinity.

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