Mapping of Nucleosomes and DNA-Bound Proteins in Living Cells with Ionizing Radiation

Abstract

The folding of DNA into chromatin plays a crucial role in regulating transcription, DNA replication and DNA repair. Current methods to probe chromatin structure have provided significant insight into the organization of nucleosomes, their modifications and the proteins that bind them, along the linear sequence of the genome. However, most methods do not provide information about the structure of the unperturbed condensed chromatin fiber in enzymatically inaccessible regions of the genome. Here, we describe a novel method, called RICC-seq, for probing chromatin structure using hydroxyl radicals generated by ionizing radiation as a small molecule probe that generates sparse and spatially correlated strand breaks that result in short DNA fragments. The lengths and mapping locations of these fragments can be used to place pairwise distance constraints on sequence loci in 3D space, which can indicate nucleosome positions, while the locations of cleavage events can be used for DNA footprinting. RICC-seq produces chromatin structure data with relatively even genome-wide coverage, due to the small size of the hydroxyl radical. Although RICC-seq is compatible with formaldehyde fixation, it can be applied to live cells, thus providing information about the native state of chromatin in the nucleus. It also relies on a fundamentally different mechanism for structure mapping than enzyme-based or proximity ligation-based assays, and can thus be used for orthogonal validation of existing epigenomic data. We show that it can reproduce aspects of the CTCF footprint observed with DNase-seq and can report on both nucleosome structure with base pair resolution and on nucleosome position. RICC-seq is thus a novel and complementary addition to the epignomicist's toolbox.