While chromosomes have been captured in promiscuous situations before, a tiny spying device of the genomic variety has captured chromosomes in some of their most intimate conformations. By applying Oxford Nanopore Technologies (ONT) MinION portable sequencer to the study of chromosome contacts, the labs of Wouter de Laat and Jeroen de Ridder at Utrecht University (Netherlands) bring forth multi-contact 4C (MC-4C), which captures enhancer hubs and loop collisions at single-allele resolution. MC-4C takes advantage of the large DNA concatemers (>10 kb) created by 3C-based protocols; however, instead of further trimming these products for the analysis of single ligation junctions, MC-4C takes full advantage of them to assay multi-way contacts.
Here’s how this technique works:
- Chromatin is cross-linked in situ
- Chromatin is fragmented by restriction enzyme digestion (DpnII)
- The fragments are religated in a proximity-based manner andthe cross-links are reversed
- An in vitro Cas9 digestion of the sequence of interest, known as the viewpoint, is used to eliminate uninformative undigested products and reduce rolling circle amplification by targeting in between the inverse PCR primers
- Inverse PCR is then used to amplify the products containing the viewpoint
- The PCR products are then size selected and subjected to library preparation
- The libraries are sequencing on MinION
- An elaborate bioinformatic workflow is then used to preprocess the data, where PCR duplicates are removed, and also conduct downstream analysis
The talented team then applied MC-4C to the complex chromatin topologies of the mouse β-globin and Pcdhα loci, as they contain multiple gene promoters and enhancer elements that interact in a dynamic fashion to shape developmental gene expression patterns. In these experiments, the talented team observed that the individual enhancer elements come together to form an enhancer hub that can accommodate two genes at the same time. Finally, they also demonstrated that MC-4C can detect loop collisions. Overall, MC-4C represents an exciting new approach for studying multi-way chromosome contacts at single-allele resolution.