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.
Lee
J H
JH
Herman B Wells Center for Pediatric Research, Section of Pediatric Hematology/Oncology and the Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Voo
K S
KS
Skalnik
D G
DG
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D018899:CpG Islands; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation; D015032:Zinc / Q000494:pharmacology
D008024:Ligands; D009841:Oligonucleotides; D006639:Histidine; D004247:DNA; D015032:Zinc; D003545:Cysteine; D000409:Alanine