Regulome

Conventional Hi-C relies on freezing chromatin in its 3D conformation with a crosslinking agent (generally formaldehyde), followed by enzymatic DNA fragmentation. Historically performed with a restriction enzyme cutting frequently or rarely (such as HindIII), digestion has evolved toward the use of micrococcal endonucleases (Micro-C). This variant eliminates restriction site bias and offers nucleosome resolution, revealing micro-TADs and enhancer-promoter interactions with unprecedented precision.

After digestion, cohesive ends are filled in with biotinylated dNTPs and then proximity-ligated to preserve spatial information. Purification on streptavidin beads specifically isolates chimeric junctions before high-throughput sequencing is performed.

Technological innovations have transformed this methodology:

• Single-Cell Hi-C (scHi-C): Allows the 3D structure of the genome to be mapped cell by cell, revealing cell-to-cell variability in chromatin folding and spatiotemporal dynamics during differentiation or oncogenesis.
• Integration of Long-Read Sequencing (Nanopore, PacBio): Hi-C combined with long reads (Pore-C, Multi-contact 3C) overcomes the limitation of pairwise contact pairs. It identifies simultaneous multi-contact interactions within a single DNA molecule and facilitates the assembly of complex or polyploid genomes.
• Capture Hi-C (C-HiC): Combines RNA or DNA probe enrichment to specifically target regulatory regions or promoters at very high sequencing depth.

The integration of Hi-C with super-resolution imaging and computational modeling now offers a dynamic 4D view of the genome.