Identification and characterization of the DNA binding domain of CpG-binding protein

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