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Frontiers in Biology

ISSN 1674-7984

ISSN 1674-7992(Online)

CN 11-5892/Q

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Front Biol    2011, Vol. 06 Issue (01) : 76-81    https://doi.org/10.1007/s11515-011-0980-6
REVIEW
3C-based methods to detect long-range chromatin interactions
Gang WEI(), Keji ZHAO()
Laboratory of Molecular Immunology, National Heart, Lung and Blood Institute, NIH, 9000 Rockville Pike, Bethesda, MD 20892, USA
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Abstract

Transcriptional regulatory regions are often located several thousand bases from the gene that they control. To function, the chromatin strand forms loops to juxtapose distal regions with the promoter. These long-range chromatin interactions have profound influences on the regulation of gene expression and mapping these interactions is currently a subject of intensive investigation. Chromosome conformation capture (3C) technology and its derivatives have been widely used to detect chromatin interactions and greatly contributed to understanding of the relationship between genome organization and genome function. Here we review these 3C-based methods for the study of long-range chromatin interactions and recent exciting findings obtained by using these technologies.
Keywords chromatin interactions      3C      gene regulation      next-generation sequencing     
Corresponding Author(s): WEI Gang,Email:weig2@mail.nih.gov; ZHAO Keji,Email:zhaok@nhlbi.nih.gov   
Issue Date: 01 February 2011
 Cite this article:   
Gang WEI,Keji ZHAO. 3C-based methods to detect long-range chromatin interactions[J]. Front Biol, 2011, 06(01): 76-81.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-011-0980-6
https://academic.hep.com.cn/fib/EN/Y2011/V06/I01/76
Fig.1  Schematic representation of the 3C-based methods. First, cells are fixed by formaldehyde treatment and interacted chromatin fragments that bind to the same protein complex are in close proximity. After digestion with a certain kind of restriction enzyme, chromatin fragments are re-ligated under diluted condition so that most of ligation reactions occur intramolecularly. The ligation products are subsequently purified, generating the conventional 3C-library. For 5C method, the conventional 3C library are denatured and annealed with a set of 5C primers so that multiple regions can be amplified with universal primers simultaneously. For 4C method, the conventional 3C library is further digestion with a second restriction enzyme and re-ligated intromolecularly, resulting in the circular ligation products, in which unknown regions that interacted with a pre-selected locus can be amplified by inverse PCR and identified by subsequent microarray analysis or deep sequencing. For Hi-C method, the digested chromatin fragments are filled in biotinylated dNTP before ligation so that the ligation products can be enriched by streptavidin beads for deep sequencing. In the case of ChIA-PET, the crosslinked chromatin is first fragmented by sonication instead of restriction enzyme digestion and chromatin immunoprecipitation is carried out to enrich the chromatin fragments that are associated with a specific protein. Biotin labeled half-linkers is ligated with the chromatin fragments and second ligation reactions occur intromolecularly through the cohesive ends of half linkers. The ligation products are subsequently digested by I and the DNA fragments containing linkers are enriched by streptavidin beads selection for deep sequencing.
Fig.1  Schematic representation of the 3C-based methods. First, cells are fixed by formaldehyde treatment and interacted chromatin fragments that bind to the same protein complex are in close proximity. After digestion with a certain kind of restriction enzyme, chromatin fragments are re-ligated under diluted condition so that most of ligation reactions occur intramolecularly. The ligation products are subsequently purified, generating the conventional 3C-library. For 5C method, the conventional 3C library are denatured and annealed with a set of 5C primers so that multiple regions can be amplified with universal primers simultaneously. For 4C method, the conventional 3C library is further digestion with a second restriction enzyme and re-ligated intromolecularly, resulting in the circular ligation products, in which unknown regions that interacted with a pre-selected locus can be amplified by inverse PCR and identified by subsequent microarray analysis or deep sequencing. For Hi-C method, the digested chromatin fragments are filled in biotinylated dNTP before ligation so that the ligation products can be enriched by streptavidin beads for deep sequencing. In the case of ChIA-PET, the crosslinked chromatin is first fragmented by sonication instead of restriction enzyme digestion and chromatin immunoprecipitation is carried out to enrich the chromatin fragments that are associated with a specific protein. Biotin labeled half-linkers is ligated with the chromatin fragments and second ligation reactions occur intromolecularly through the cohesive ends of half linkers. The ligation products are subsequently digested by I and the DNA fragments containing linkers are enriched by streptavidin beads selection for deep sequencing.
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