Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

Abstract

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

SORBS2transcription is activated by telomere position effect–over long distance upon telomere shortening in muscle cells from patients with facioscapulohumeral dystrophy

“…We then applied to this cellular model, the unbiased technique, Hi-C chromosome conformation capture (van Berkum et al 2010) that permits identification of chromosomal interactions over long distances. We modified the technique by introducing a second capture step that retrieved interactions involving the small genomic region around the 4q35 locus (0.15% of the genome) as illustrated (Supplemental Figs.…”

“…Samples both for Hi-C and 3C were prepared as described (van Berkum et al 2010). Hi-C was modified to enrich for the 4q35 region.…”

"Mirror reads" in Hi-C data

“…HindIII), biotinylation of fragments, DNA proximity ligation of blunt ends, precipitation with streptavidin and amplification of all ligated fragments. The resulting library is sequenced with any pair-end method [1]. Then the sequencing data are mapped to the genome of the studied organism.…”

“…the hiclib library 1 ). The major processing steps 1 http://bitbucket.org/mirnylab/hiclib are read mapping, annotation of restriction fragments, filtering and binning of data.…”

Structure determination of genomic domains by satisfaction of spatial restraints

“…Such experiments are thus suitable for generating complete and extensive loci interaction matrices for large genomic regions (up to a few mega base pairs). Finally, the Hi-C technology, which allows for an unbiased genome-wide analysis, was recently developed to overcome the need for predefining a set of target loci to investigate (van Berkum et al 2010). Hi-C's key step is the imprinting of ligated products (i.e., products of interacting loci) with a biotin marker that later on is precipitated with streptavidin beads.…”