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Hi-C: A Method to Study the Three-dimensional Architecture of Genomes
- Source :
- MyJoVE Corporation, Journal of Visualized Experiments : JoVE
- Publication Year :
- 2010
- Publisher :
- MyJoVE Corporation, 2010.
-
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.
- Subjects :
- General Chemical Engineering
Issue 39
Computational biology
Biology
Genome
Chromosomes
General Biochemistry, Genetics and Molecular Biology
Chromosome conformation capture
03 medical and health sciences
0302 clinical medicine
Illumina Paired End sequencing
polymer physics
Enhancer
Chromosome Positioning
ChIA-PET
030304 developmental biology
Genomic organization
chromatin structure
Genetics
0303 health sciences
General Immunology and Microbiology
General Neuroscience
Chromosome
DNA
Genomics
Chromatin
Cellular Biology
Nucleic Acid Conformation
Human genome
030217 neurology & neurosurgery
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- MyJoVE Corporation, Journal of Visualized Experiments : JoVE
- Accession number :
- edsair.doi.dedup.....87e908ac020eec9eb25fb1a9f96febb4