Student Wiki on methodology

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Long-range interaction and chromatin loops: from 3C to Hi-C

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(Rossella Ciliberti)

3C, chromosome conformation capture

The relevance of three-dimensional (3D) genome organization for transcriptional regulation and thereby for cellular fate at large is now widely accepted. In fact, the first decade of 3C methods rendered unprecedented insights into genome topology.

Following the introduction of the nuclear ligation assay, a method already employing some of the key principles of 3C technology, the 3C methodologies introduced a very different, complementary toolbox that allowed the study of DNA folding at higher resolution and in a more systematic manner. 

The strategy of 3C to discover genomic architecture is based on quantifying the frequencies of contacts between distal DNA segments in cell populations. In contrast to cytogenetic approaches, 3C-based genomics strategies yield incomparable information-rich data describing genome topology at the genome-wide level, enabling more systematic genome topology studies at a higher resolution and throughput and providing deep insights into genome architecture and its impact on genome function.

The principal steps of 3C and 3C-based experiments are theoretically similar and have following principal steps: 

  • crosslink chromatin using a fixative agent in solution, most often formaldehyde, to create covalent bonds between DNA fragments bridged by proteins; 
  • isolate and digest the chromatin using a restriction enzyme such as HindIII, BglII, EcoRI, AciI, or DpnII at a low concentration to create pairs of crosslinked DNA fragments that are distant in linear distance but close in space; 
  • re-ligate the sticky ends of crosslinked DNA fragments to form chimeric molecules; reverse the crosslinks to obtain 3C templates; 
  • interrogate the rearranged DNA fragments by PCR or sequencing technologies.
An external file that holds a picture, illustration, etc.  Object name is 13039_2018_368_Fig1_HTML.jpg                                                               3C technology was developed to detect ligation junctions by PCR followed by gel electrophoresis. The first 3C assay inferred the 3D conformation of yeast chromosome III and showed that it forms a contorted ring. Next, this method was adapted for mammalian systems. 3C technology confirmed the existence of chromatin loops, which confer spatial contact between DNA fragments such as regulatory DNA elements and their target genes or the start and end of a gene.

Also, no matter which detection and quantification methods are used, reliably measuring and correctly interpreting contact frequencies by 3C is inherently difficult.

4C, chromatin conformation capture-on-Chip

If 3C could be considered an one-to-one approach, 4C technology is an one-to-all approach: 4C allows for the genome-wide identification of regions contacting a sequence of interest or “viewpoint.” In contrast to 3C, it requires no a priori knowledge or hypotheses of candidate contacting regions. A major advantage is that contact frequencies formed between an anchor sequence and a sequence of interest are appreciated in the context of all contacts formed with the anchor. 

4C technology was developed by combining 3C with microarray or, more recently, next-generation sequencing (NGS) technologies. This method is able to assess chromatin interactions between one genomic locus of interest (referred to as bait or viewpoint) and all other genomic loci (one versus all) . In 4C experiments, small DNA circles are created by cleaving with a second restriction enzyme and re-ligating 3C DNA templates (the initial steps of the protocol follow those of 3C methodology, but, upon obtaining the 3C template, in 4C, a second round of digestion is performed followed by another ligation step, resulting in small DNA circles, of which some contain the viewpoint plus contacting sequences). Then, inverse PCR using bait-specific primers is applied to amplify any interacting fragments. Finally, the interacting fragments are evaluated using microarrays or NGS.

A potential disadvantage of the technique is its limited ability to account for PCR amplification biases. Captured fragments are amplified with different efficiencies because of differences in size and GC content.

In general, 4C technology is an excellent strategy to survey the DNA contact profile of specific genomic sites.

5C, chromosome conformation capture carbon copy

Another variant of 3C is 5C. It is analogous to 3C technology but is a “many versus many” method, allowing the simultaneous detection of millions of interactions through the use of thousands of primers in a single assay. The main difference between 3C and 5C is the strategy for primer design. 5C primers have a universal sequence (usually T7 and T3) appended to the 5′ ends. This change, combined with multiplex PCR amplification and sequencing, allows researchers to detect contact events within a particular locus. Thus, in contrast to 3C, 5C has a higher throughput and a lower bias. However, 5C is still limited in terms of the size of the genomic region that can be assayed because of the DNA sequence requirement of interested regions, as well as the quantitatively inestimable PCR duplication.

(ChIA-PET), chromatin interaction analysis by paired-end tag sequencing 

Another 3C-based technology is ChIA-PET, which combines chromatin immunoprecipitation (ChIP) with 3C-type analysis to study genome-wide long-range chromatin interactions bound by one specific protein. 

The key features of ChIA-PET technology are that the interaction sites are enriched by ChIP using a specific antibody after chromatin digestion, as in a ChIP experiment. Then, DNA sequences tethered together and to the protein of interest are connected through proximity ligation with oligonucleotide DNA linkers, the sequence of which contains restriction sites for digestion in the next step. After high-throughput sequencing and bioinformatics analysis, an interactome map of the specific protein binding sites is achieved. Thus, ChIA-PET has been applied efficiently to study sites bound with specific transcription factors. Another advantage is that ChIA-PET has relatively low levels of library complexity compared with other 3C techniques; therefore, interactions that are identified with an extremely low number of reads are usually considered significant. Recently, an improved method, HiChIP, was developed and it can improve over 10-fold of the yield of chromatin interacting reads but with 100-fold lower requirement than that of ChIA-PET.


The development of high-throughput sequencing technology promoted the emergence of a series of “all versus all” methods. Of these, Hi-C was the first to be developed that does not depend on specific primers and generates genome-wide contact maps. In Hi-C experiments, the first step is to generate contact segments as with 3C, but the procedure is slightly different from the 3C. After restriction enzyme digestion, the sticky ends are filled in with biotin-labeled nucleotides followed by blunt-end ligation. The expected contacting DNAs are sheared and then purified in a biotin pull-down experiment using streptavidin beads to ensure that only biotinylated junctions are selected for further high-throughput sequencing and computational analysis.

The strategy of Hi-C data analysis is thus different from above methods due to the massive parallel NGS data obtained. 

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