High-Resolution Locus-Specific Mapping of Chromatin Contacts using Two-Photon Photoactivated DNA Cross-Linking

Abstract

Spatial organization of the genome remains an interesting and not fully understood area of nuclear architecture and gene regulation. Recently, there have been many technological and methodological developments towards this end from both imaging and genomic perspectives. Here we present a novel method to examine genomic contacts in vivo near a specific locus or nuclear region of interest. Using 3D localized two-photon excitation and a photoactivatable DNA crosslinker, the chromatin within a femtoliter volume around the region of interest can be labeled with an affinity tag. By carrying out this process on many cells using an automated microscope, we can then isolate the genomic DNA and pulldown the crosslinked DNA. While several DNA cross-linkers may be suitable for this method, we have initially focused on psoralen based compounds which are easily UV or two-photon photoactivated to form inter-strand DNA crosslinks preferentially at 5'-TpA dinucleotides in the genome. For photophysical optimization of the method, we use fluorescently labeled genomic loci where 200 copies of a transgene have been integrated at a single locus in U2OS cells. The DNA is extracted and purified using an affinity tag pull-down and the efficiency of crosslinking is quantified via qPCR. After the optimal photoactivation parameters are determined, the genomic locus will be irradiated and the crosslinked DNA extracted and sequenced as a proof-of-principle experiment. The long-term goal is to create a new method to bioinformatically map chromatin fragments at a nuclear region of interest, which can be performed before and after gene activation for any gene of interest.