3C

Chromosome conformation capture (3C) technology and its derivatives have been widely used to analyze the spatial organization of chromatin in a cell. These methods quantify the number of interactions between genomic loci that are nearby in 3-D space, but may be separated by many nucleotides in the linear genome.  It can be applied to investigate the nuclear juxtaposition of any two genomic regions, in cis or trans. Such interactions may result from biological functions, such as promoter-enhancer interactions, or from random polymer looping, where undirected physical motion of chromatin causes loci to collide. 3C provides information on 3D chromatin structures that occur in living cells.

The protocol involves formaldehyde cross-linking of chromatin fragments and proteins in proximity followed by chromatin isolation and digestion with a restriction enzyme. The specific enzymes used are chosen in order to free a known or predicted DNA-DNA interaction mediated by a protein complex. The freed fragments are then ligated into rings and the crosslinks are reversed and the DNA is purified. Proteins are removed through the exposition to high ionic strands or high temperature. Both end-point PCR and quantitative real-time PCR (qPCR) can be employed to quantify the abundance of purified DNA 3C ligation products. The abundance of these recombinant  fragments directly correlates to the interaction frequency of the two ligated regions. 

This basic principle can be combined with other technologies to increase scale or specificity of the DNA loops being interrogated.

The derivative techniques of 3C are: 

• Circularized Chromosome Conformation Capture (4C)​
  
• Carbon Copy Chromosome Confromation Capture (5C)
  
• ​Chromatin interaction analysis by paired-end tag sequencing (CHIA-PET)
  
• ChIP-loop​
  
• ​Hi-C
  

As told before, 4C means Circularized Chromosome Conformation Capture. The first part of the protocol is equal to the one for basic 3C, so it will be reported without modification from the previous entry (tanks to the author).

The protocol involves formaldehyde cross-linking of chromatin fragments and proteins in proximity followed by chromatin isolation and digestion with a restriction enzyme. The specific enzymes used are chosen in order to free a known or predicted DNA-DNA interaction mediated by a protein complex.The freed fragments are then ligated into rings and the crosslinks are reversedand the DNA is purified. Proteins are removed through the exposition to high ionic strands or high temperature.

Following, these ligated segments are treated with another RE (a 4 pb RE), to create sticky ends: so, the two extremities will be able to interact with each other providing circularization. PCR is then carried out with divergent primers, preferentially recongnising the contact points of the two fragments.