Selective Nonmethylated CpG DNA Recognition Mechanism of Cysteine Clamp Domains

Methylation of DNA at CpG sites is a major mark for epigenetic regulation, but how transcription factors are influenced by CpG methylation is not well understood. Here, we report the molecular mechanisms of how the TCF (T-cell factor) and GEF (glucose transporter 4 enhancer factor) families of proteins selectively target unmethylated DNA sequences with a Cclamp type zinc finger domain. The structure of the C-clamp domain from human GEF family protein HDBP1 (C-clamp(HDBP1)) in complex with DNA was determined using NMR spectroscopy, which adopts a unique zinc finger fold and selectively binds RCCGG (R = A/G) DNA sequences with an Arg center dot center dot center dot Trp-Lys-Lys DNA recognition motif inserted in the major groove. The CpG base pairs are central to the binding due to multiple hydrogen bonds formed with the backbone carbonyl groups of Trp378 and Lys379, as well as the side chain e-amino groups of Lys379 and Lys380 from C-clamp(HDBP1). Consequently, methylation of the CpG dinucleotide almost abolishes the binding. Homology modeling reveals that the C-clamp domain from human TCF1E (C-clamp(TCF1E)) binds DNA through essentially the same mechanism, with a similar Arg center dot center dot center dot Arg-Lys-Lys DNA recognition motif. The substitution of tryptophan by arginine makes C-clamp(HDBP1) prefer RCCGC DNA sequences. The two signature DNA recognition motifs are invariant in the GEF and TCF families of proteins, respectively, from fly to human. The recognition of the CpG dinucleotide through two consecutive backbone carbonyl groups is the same as that of the CXXC type unmethylated CpG DNA binding domains, suggesting a common mechanism shared by unmethylated CpG binding proteins.

Automated summary

The study reveals the molecular mechanisms of how TCF and GEF protein families selectively target unmethylated DNA sequences with a C-clamp type zinc finger domain. The structures of the C-clamp domains in HDBP1 and TCF1E were determined, showing a unique zinc finger fold and specific DNA recognition motifs. CpG base pairs play a central role in the binding mechanism, with multiple hydrogen bonds formed between specific residues in the C-clamp domain and the DNA backbone, highlighting the importance of unmethylated CpG binding proteins.