Reading Time and DNA Sequence Preference of TET3 CXXC Domain Revealed by Single‐Molecule Profiling†

Abstract

Comprehensive SummaryRecognition of CpG dinucleotide DNA in epigenetic information flow plays a pivotal role for cellular differentiation and development. The TET3 CXXC domain binds to CpG DNA, serving a basic epigenetic information reading mechanism. During the selective recognition of a CpG motif by a CXXC domain from crowded binding sites in a gene sequence, the protein‐DNA interactions are beyond CpG dinucleotide. However, the selective binding dynamics of CpG within a long DNA context by epigenetic enzymes have been rarely exploited, which is hard for ensemble methods to probe. Here, we used single‐molecule magnetic tweezers to quantitatively examine the dynamics of TET3's CXXC domain on a Hoxa9 promoter DNA. Our single‐molecule binding profile revealed that CXXC‐DNA interactions involve both CpG motifs and their flanking sequences. The residence time of TET3 CXXC differs by about 1000 times in five distinguished CpG clusters in the context of a CpG island. Moreover, we performed multi‐state hidden Markov modeling analysis on the zipping/unzipping dynamics of a CpG hairpin, discovering TET3 CXXC's preference on CpG motifs regarding the –2 to +2 flanking bases. Our results shed light on the selective binding dynamics of a CXXC on a gene sequence, facilitating studies on epigenetic information reading mechanisms.