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The concept of chromatin contact mapping, or determining the three-dimensional structure conformation and interactions of chromatin domains, is now a reality because of Chromatin Conformation Capture (3C) and subsequent methods born out of that approach. Several new 3C-based techniques have emerged, each with particular strengths and applications, but the sheer variety creates a challenge when selecting the best method for a specific situation.
In 2002, Job Dekker and colleagues published 3C as a novel approach for determining 3D chromatin structures and interactions in vivo (Dekker et al., 2002).
3C analyzes excised DNA fragments generated from formaldehyde cross-linked, then restriction enzyme digested chromatin to find points where selected DNA regions are connected through a protein complex. The frequency and identity of these fragments are then determined by quantitive PCR (qPCR).
Not only is 3C an important technology on its own, but it has also become the foundation for a host of related techniques that have been developed to achieve greater scale, throughput or specificity. These techniques are discussed below with helpful hints for getting started.
4C enables identification of previously unknown DNA regions that interact with a locus of interest, which makes 4C especially well suited to discover novel interactions with a specific region that is being investigated (Dekker et al., 2006).
4C helpful hits:
This technique generates a library of any ligation products from DNA regions that associate with the target loci, which are then analyzed by next-generation sequencing.
5C is ideal when great detail about all the interactions in a given region is needed, for example when diagramming a detailed interaction matrix of a particular chromosome. However, 5C is not truly genome-wide, since each 5C primer must be designed individually, so it is best suited to particular regions (Dotsie and Dekker, 2007).
5C helpful hints:
Fig 1. Schematic of the 3C-based technologies
(Adapted from original graphic in Trends in Cell Biology)
ChIA-PET takes aspects of chromatin immunoprecipitation (ChIP) and 3C to analyze the interplay of distant DNA regions through a particular protein.
ChIA-PET is best used for discovery experiments involving a protein of interest and unknown DNA binding targets. Transcription factor binding sites, for example, are best studied with ChIA-PET since this technique requires the DNA to be bound by the transcription factor in vivo in order for the interaction to be called (Fullwood et al., 2009).
ChIA-PET helpful hits:
ChIP-loop is a mixture chromatin immunoprecipitation (ChIP) and 3C that employs antibodies targeted to proteins that are suspected to bind a DNA region of interest. ChIP-loop is ideal to find out if two known DNA regions interact via a protein of interest. It is well suited to confirmation of suspected interactions, but not discovery of novel ones (Horike et al., 2005).
ChIP-loop helpful hints:
Hi-C amplifies ligation products from the entire genome that interact with the desired DNA locus, and then assesses their frequencies by high-throughput sequencing. Hi-C is a great choice when broad coverage of the entire genome is required, but resolution is not of great concern. For example, mapping the genome-wide changes in chromosome structure in tumor cells (Lieberman-Aiden et al., 2009).
Hi-C helpful hints:
Capture-C uses a combination of 3C and oligonucleotide capture technology (OCT), together with high-throughput sequencing to study hundreds of loci at once. Capture-C is perfect when both high resolution and genomic-wide scale are required. For example, analyzing the functional effect of every disease-associated SNP in the genome on local chromatin structure (Hughes et al., 2014).
Capture-C helpful hints:
The abundance of available 3C related techniques can make it a chore to choose just one of the many options, but it also means that there is likely one that is ideally suited for any experimental scenario. The following table can help point out where each method really shines.
As the C methods continue to evolve, become more refined and their use expands, they will be a valuable tool in understanding of how chromatin structure, protein interactions and DNA sequence all work together to control gene expression for years to come.
|3C method||Unique benefit||Best application||Expert tips|
|4C||Detects interactions of unknown DNA regions.||Searching for novel associations with a particular region of interest.|
|5C||Specially designed primers allow for interaction analysis of a specific locus in great detail||Creating detailed interaction matrices and mapping 3D structures of a DNA region.|
|ChIA-PET||Captures distant DNA fragments associate through a specific protein.||Identifying DNA regions that interact with a protein and each other. For example: transcription factor binding sites.|
|ChIP-loop||Reduced background and improved specificity over standard 3C.||Leveraging antibody specificity to determine DNA loci interacting with a particular protein.|
|Hi-C||Harnesses advanced sequencing for genome-wide capability.||Forming a broad snapshot of chromatin structure across the entire genome, with moderate resolution.|
|Capture-C||Oligonucleotide capture technology (OCT) and sequencing make this approach both high-throughput and high resolution.||Characterizing hundreds of loci in one experiment, while still maintaining very high resolution.|
Chen X, Shi C, Yammine S, Göndör A, Rönnlund D, Fernandez-Woodbridge A, Sumida N, Widengren J and Ohlsson R (2014). Chromatin in situ proximity (ChrISP): single-cell analysis of chromatin proximities at a high resolution. Biotechniques, 56, 117-124.
Kolovos P, van de Werken HJ, Kepper N, Zuin J, Brouwer RW, Kockx CE, Wendt KS, van IJcken WF, Grosveld F and Knoch TA (2014). Targeted Chromatin Capture (T2C): a novel high resolution high-throughput method to detect genomic interactions and regulatory elements. Epigenetics Chromatin, 7, 10.
These two new methods, ChrISP and T2C, offer novel approaches to answer many of the same questions that the 3C variants have been designed to interrogate. It bears monitoring how well they perform in the hands of researchers in comparison to more established protocols.