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 C-clamp type zinc finger domain. The structure of the C-clamp domain from human GEF family protein HDBP1 (C-clampHDBP1) 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···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 ε-amino groups of Lys379 and Lys380 from C-clampHDBP1. Consequently, methylation of the CpG dinucleotide almost abolishes the binding. Homology modeling reveals that the C-clamp domain from human TCF1E (C-clampTCF1E) binds DNA through essentially the same mechanism, with a similar “Arg···Arg-Lys-Lys” DNA recognition motif. The substitution of tryptophan by arginine makes C-clampHDBP1 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.CpG位点的DNA甲基化是表观遗传调控（epigenetic regulation）的核心标记之一，但转录因子如何受CpG甲基化调控的分子机制仍未得到充分阐明。本研究报道了TCF（T细胞因子，T-cell factor）与GEF（葡萄糖转运蛋白4增强因子，glucose transporter 4 enhancer factor）家族蛋白通过C-钳型锌指结构域（C-clamp type zinc finger domain）选择性靶向未甲基化DNA序列的分子机制。我们利用核磁共振波谱法（NMR spectroscopy）解析了人类GEF家族蛋白HDBP1的C-钳结构域（C-clampHDBP1）与DNA结合的复合物结构，该结构呈现独特的锌指折叠构象，并通过插入DNA大沟的“精氨酸···色氨酸-赖氨酸-赖氨酸”DNA识别基序，选择性结合RCCGG（R=A/G）DNA序列。CpG碱基对是该结合过程的核心，其与C-clampHDBP1中Trp378、Lys379的主链羰基，以及Lys379、Lys380的侧链ε-氨基形成了多重氢键相互作用。因此，CpG二核苷酸（CpG dinucleotide）的甲基化几乎完全阻断了该结合过程。同源建模（homology modeling）结果显示，人类TCF1E的C-钳结构域（C-clampTCF1E）通过几乎完全相同的机制结合DNA，其具有类似的“精氨酸···精氨酸-赖氨酸-赖氨酸”DNA识别基序。将C-clampHDBP1中的色氨酸替换为精氨酸后，该结构域会偏好结合RCCGC DNA序列。从果蝇到人类的TCF与GEF家族蛋白中，这两种标志性DNA识别基序分别保持高度保守。通过两个连续主链羰基识别CpG二核苷酸的机制，与CXXC型未甲基化CpG DNA结合结构域（CXXC type unmethylated CpG DNA binding domains）的识别机制一致，这表明未甲基化CpG结合蛋白共享一套共同的分子识别机制。