Genome-Wide Mapping of Chromatin Secondary Structure using Ionizing Radiation Coupled with Sequencing

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Chromatin packages two meters of human DNA into a five-micron nucleus while allowing regulated access to the genome. At the intermediate length scale of ∼1 kilobase, which spans nucleosome-nucleosome interactions, chromatin structure is expected to play a crucial role in regulating transcription, DNA replication and DNA repair. However, our structural understanding of this level of chromatin organization continues to lag behind our rapidly developing understanding of both single nucleosomes and higher-order, long-range interactions. We have developed a novel method, RICC-seq, for probing chromatin structure at this poorly understood length scale. RICC-seq uses hydroxyl radicals generated by ionizing radiation to create 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 basepair-resolved pairwise distance constraints on sequence loci in 3D space. In order to understand the detailed architecture underlying chromatin compaction and DNA accessibility, we have generated the first genome-wide RICC-seq maps of chromatin compaction in terminally differentiated fibroblasts, comparing chromatin structure between heterochromatic and euchromatic regions. These data reveal differential packaging geometries for heterochromatic regions with significantly more DNA contacts with specific chromatin post translational modifications. We envision that RICC-seq is a generalizable method for high-resolution understanding of condensed nucleic acids with potential applications to targets as diverse as folded RNA to viral genomes within capsids.