Abstract
DNA recognition by transcription activator-like effector (TALE) proteins is mediated by tandem repeats that specify nucleotides through repeat-variable diresidues. These repeat-variable diresidues form direct and sequence-specific contacts to DNA bases; hence, TALE–DNA interaction is sensitive to DNA chemical modifications. Here we conduct a thorough investigation, covering all theoretical repeat-variable diresidue combinations, for their recognition capabilities for 5-methylcytosine and 5-hydroxymethylcytosine, two important epigenetic markers in higher eukaryotes. We identify both specific and degenerate repeat-variable diresidues for 5-methylcytosine and 5-hydroxymethylcytosine. Utilizing these novel repeat-variable diresidues, we achieve methylation-dependent gene activation and genome editing in vivo; we also report base-resolution detection of 5hmC in an in vitro assay. Our work deciphers repeat-variable diresidues for 5-methylcytosine and 5-hydroxymethylcytosine, and provides tools for TALE-dependent epigenome recognition.
Highlights
DNA recognition by transcription activator-like effector (TALE) proteins is mediated by tandem repeats that specify nucleotides through repeat-variable diresidues
We showed in this study that DNA binding by TALE proteins is affected by DNA base modifications
The methyl and hydroxymethyl groups do not interfere with base-pairing; yet, their presence in the major groove of DNA duplex impact their interaction with TALE proteins
Summary
DNA recognition by transcription activator-like effector (TALE) proteins is mediated by tandem repeats that specify nucleotides through repeat-variable diresidues. These repeatvariable diresidues form direct and sequence-specific contacts to DNA bases; TALE–DNA interaction is sensitive to DNA chemical modifications. We conduct a thorough investigation, covering all theoretical repeat-variable diresidue combinations, for their recognition capabilities for 5-methylcytosine and 5-hydroxymethylcytosine, two important epigenetic markers in higher eukaryotes. In the crystal structures of TALE–DNA complex, the RVD loops contact the duplex major groove, in which the first residue stabilizes the proper loop conformation and the second residue makes a direct base-specific contact[35, 36] These observations illustrate the potential of TALEs in discriminating canonical and modified cytosine bases. The full potential of RVDs in recognizing 5mC and 5hmC has not been explored
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