A 5′-Flanking C/G Pair at the Core Region Enhances the Recognition and Binding of Kaiso to Methylated DNA

Methyl CpG binding proteins (MBPs) are transcription factors that recognize the methylated CpG sites in DNA and mediate the DNA methylation signal into various downstream cellular processes. The C2H2 zinc finger (ZF) protein, Kaiso, also an MBP, preferentially binds to two symmetrically methylated CpG sites in DNA sequences via C-terminal C2H2 ZF domains and mediates the transcription regulation process. Investigation of the molecular mechanism of the recognition of methylated DNA (meDNA) by Kaiso is important to understand how this protein reads and translates this methylation signal into downstream transcription outcomes. Despite previous studies in Kaiso-meDNA interactions, detailed structural investigations on the sequence-specific interaction of Kaiso with the meDNA sequence are still lacking. In this work, we used molecular modeling and molecular dynamics (MD) simulation-based computational approaches to investigate the recognition of various methylated DNA sequences by Kaiso. Our MD simulation results show that the Kaiso-meDNA interaction is sequence specific. The recognition of meDNA by Kaiso is enhanced in the MeECad sequence compared to the MeCG2 sequence. Compared to the 5′-flanking T/A pair in MeCG2, both MeCG2_mutCG and MeECad sequences show that a C/G base pair allows GLU535 of Kaiso to preferably recognize and bind the core mCpG site. The core mCGmCG site is crucial for the recognition process and formation of a stable complex. Our results reveal that the 5′-flanking nucleotides are also important for the enhanced binding and recognition of methylated sites.

甲基CpG结合蛋白（Methyl CpG binding proteins, MBPs）是一类能够识别DNA中甲基化CpG位点，并将DNA甲基化信号传导至各类下游细胞过程的转录因子。C2H2型锌指蛋白（C2H2 zinc finger protein, ZF）凯索（Kaiso）同样属于MBPs，其通过C端C2H2型锌指结构域，优先结合DNA序列中的两处对称甲基化CpG位点，介导转录调控过程。解析凯索识别甲基化DNA（methylated DNA, meDNA）的分子机制，对于理解该蛋白如何读取并将甲基化信号转化为下游转录结果具有重要意义。尽管目前已有关于凯索与meDNA相互作用的相关研究，但针对凯索与meDNA序列的序列特异性相互作用的详细结构研究仍较为匮乏。本研究采用分子建模与基于分子动力学（molecular dynamics, MD）模拟的计算方法，探究凯索对各类甲基化DNA序列的识别过程。MD模拟结果表明，凯索与meDNA的相互作用具有序列特异性。相较于MeCG2序列，凯索对MeECad序列中meDNA的识别作用更强。与MeCG2序列的5'侧翼T/A碱基对相比，MeCG2_mutCG与MeECad序列均显示，C/G碱基对可使凯索的谷氨酸535（GLU535）更好地识别并结合核心mCpG位点。核心mCGmCG位点对于识别过程与稳定复合物的形成至关重要。本研究结果揭示，5'侧翼核苷酸对于增强甲基化位点的结合与识别同样具有重要作用。