Detecting Protein-DNA Binding in Single Molecules using Antibody Guided Methylation

Abstract

Characterization of DNA binding sites for specific proteins is of fundamental importance in molecular biology. It is commonly addressed experimentally by chromatin immunoprecipitation and sequencing (ChIP-seq) of bulk samples (103-107cells). We have developed an alternative method that uses a Chromatin Antibody-mediated Methylating Protein (ChAMP) composed of a GpC methyltransferase fused to protein G. By tethering ChAMP to a primary antibody directed against the DNA-binding protein of interest, and selectively switching on its enzymatic activityin situ, we generated distinct and identifiable methylation patterns adjacent to the protein binding sites. This method is compatible with methods of single-cell methylation-detection and single molecule methylation identification. Indeed, as every binding event generates multiple nearby methylations, we were able to confidently detect protein binding in long single molecules.Abstract FigureGraphical abstract(i) ChAMP is added to fix and permeabilized cells, where it binds (ii) any antibody, and upon the addition of SAM, methylates nearby GpC sites, to be detected by sequencing (iii-iv).