Designer Receptors for Nucleotide Resolution Analysis of Genomic 5-Methylcytosine by Cellular Imaging.

We report programmable receptors for the imaging-based analysis of 5-methylcytosine (5mC) in user-defined DNA sequences of single cells. Using fluorescent transcription-activator-like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases via selective repeats, we imaged cellular SATIII DNA. This DNA is the origin of nuclear stress bodies (nSB), and exhibits aberrant methylation in several cancers. We achieve high nucleobase selectivity of natural repeats in imaging, and demonstrate universal nucleobase binding by an engineered repeat. We employ TALE pairs differing in only one such repeat in co-stains to detect 5mC in SATIII sequences with nucleotide resolution, independently from potential differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a function of 5mC in its recruitment as initial step of nSB formation. These studies open a new avenue for studying 5mC functions in chromatin regulation in situ - with nucleotide, locus and cell resolution.