Navigating the “Cs” Part 1: an Introduction to Chromatin Conformation Capture Techniques

Interest in the three-dimensional structure of chromatin has exploded over the past few years—and for good reason!  We now know that DNA isn’t randomly piled into the nucleus like a bowl of spaghetti, but arranged in functional loops and domains, more like a city blueprint.

This is particularly exciting to (epi)genomic scientists, because 3D chromatin architecture can help us understand the impact of noncoding single nucleotide polymorphisms (SNPs), DNA modifications, and altered protein binding on the expression of distal, sometimes inter-chromosomal genes

The first chromatin conformation capture (3C) protocol was published in 2002.¹ Since that time, the explosion of new chromatin capture techniques has been astounding. If you’re planning a 3C experiment of your own, it’s important to understand the similarities and differences between the major chromatin capture protocols.

Essentially, all 3C protocols are based on the idea that distal, functionally related segments of DNA are more likely to physically interact with each other. 3C protocols use cross-linking, usually with 1% formaldehyde, to preserve the physical interactions between sections of DNA and their associated proteins. Next, the chromatin is digested at predicted locations with a restriction endonuclease. Then, the digested chromatin is diluted and re-ligated.  After reversal of cross-linking and protein digestion, the result is a library of hybrid DNA where fragments that were in close 3D proximity with each other are, most likely, ligated together.

While most techniques begin with a variation of this standard protocol, they differ in terms of the scope of their output and the answers that they can provide.

Chromatin Conformation Capture (3C)

The original 3C method investigated an interaction between two predicted sections of DNA. These ‘one-to-one’ hypotheses are where 3C shines. This technique is best used when you want to know if two distal loci are interacting in a 3D nucleus. The original 3C uses carefully designed PCR primers to determine the interaction frequency between two specific segments of DNA.

Circular Chromatin Conformation Capture (4C)

Where 3C can test whether two genomic loci interact, 4C experiments aim to find multiple loci that interact with a single anchor point.² Inverse primers are designed for a single fragment such that PCR will circularize and capture all other fragments that are ligated to the anchor. This medium-throughput approach often ends in sequencing, or running the resulting libraries on array platforms to determine the interaction profile of a single site of it interest.

Carbon Copy Chromatin Conformation Capture (5C) and HiC

Recently, 3C technology has been adapted to accommodate higher throughput experiments. For example, 5C was designed using multiplexed primers and next-generation sequencing (NGS) to determine many interaction partners within a large, pre-determined genomic region.³ This type of experiment is most useful when looking to characterize the overall architecture of a potential regulatory region.

HiC functions at the level of the entire genome, aiming to sequence and detect all possible 3D interactions within the sample.⁴ While lower throughput techniques can be quite labor intensive in the wet-lab, the bulk of the effort with Hi-C occurs at the level of bioinformatics and interpretation. These experiments are most often used to examine the overall changing chromatin landscape during a cell’s development or after a specific treatment.

In addition to this list, an increasing number of hybrid techniques that use “C” methods at their core.  For example, “Capture-HiC” uses DNA hybridization probes to pull down a selected panel of target loci from a Hi-C library,⁵. Capture-HiC is particularly good at examining interactions that may be of higher importance. Similarly, “Chromatin Interaction Analysis by Paired-End Sequencing” (ChIA-PET) uses a hybrid chromatin immunoprecipitation/3C approach to investigate DNA-Protein-DNA interactions.⁶ Besides having a cute acronym, ChIA-PET is particularly helpful when investigating how a protein of interest impacts chromatin architecture.

Each approach to capture chromatin conformation is most useful to answer specific types of questions about nuclear architecture.  Once you’ve settled on the most appropriate technique for your experiment, you can begin to really dive into the details.

References

1. Dekker J et al. (2002) Capturing Chromatin Conformation. Science. 295(5558):1306–11.
2. Zhao Z et al. (2006) Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat Genet. 38(11):1341–7.
3. Dotsie et al. (2007) Chromosome conformation capture carbon copy technology. Curr Protoc Mol Biol. Chapter 21: Unit 21.14.
4. Lieberman-Aiden E et al. (2009) Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 326(5950):289–93.
5. Jager R et al. (2015) Capture Hi-C identifies the chromatin interactome of colorectal cancer risk loci. Nat Commun. 6:6178.
6. Fullwood M et al. (2009) An oestrogen-receptor-alpha-bound human chromatin interactome. Nature. 462(7269):58–64.