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What is chromatin conformation capture?
Chromatin conformation capture is a technique whereby the three-dimensional structure of chromatin — the condensed complex of DNA and protein that comprise chromosomes — is stabilised through chemical crosslinking to preserve spatial organisation within the nucleus (Sati and Cavalli, 2017). These nuclear structures may then be interrogated through a variety of approaches to enable us to understand DNA interactions, proximity in sequence space, and the three-dimensional structures of the chromatin within the nucleus.
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How does the Pore-C protocol work?
The Pore-C protocol has been developed based on research by Oxford Nanopore Technologies and published literature (Lieberman-Aiden et al., 2009; Comet et al., 2011; Belton et al., 2012; Gavrilov, Golov and Razin, 2013; Nagano et al., 2015; Belaghzal, Dekker and Gibcus, 2017; Ulahannan et al., 2019). The protocol employs an ‘in-nucleus’ chromatin conformation capture approach to stabilise chromatin interactions within the nucleus (Nagano et al., 2015). This method contains the full complement of chromosomes of each cell within its own crosslinked cage of cytoskeleton and other proteins. This approach is thought to preserve nuclear structures, enhancing recovery of interactions within each chromosome known as cis-chromosomal contacts, whilst reducing the frequency of interactions between chromosomes known as trans-chromosomal contacts.
This process is achieved through a series of chemical and biological manipulations over the course of several days (Figure 1):
Figure 1. The Pore-C protocol carries out these stages to manipulate and stabilise the chromatin interactions using in-nucleus chromatin conformation. -
Steps in the protocol
Day 1
Samples other than cell culture first require sample preparation as instructed in the appropriate Pore-C sample preparation protocol for the sample type. The three-dimensional interactions of DNA within the nucleus are stabilised by chemically crosslinking DNA and protein within the nucleus using formaldehyde.
For plant samples, crosslinking occurs in the presence of a pressure vacuum to allow the formaldehyde to infiltrate the plant tissue. Following this process, proximal interactions of the DNA are preserved for the remainder of the protocol.Note: This is the only point the protocol may be paused and the sample pellets snap-frozen in liquid nitrogen for use within a year.
Next, the nuclei are permeabilised to expose the crosslinked cytoskeleton cage and nuclear structures before the chromatin is then denatured using detergent and gentle heat. This process relaxes the chromatin but maintains proximal crosslinked interaction. Following chromatin denaturation, the DNA is accessible to restriction digestion. A suitably selected restriction enzyme is added to samples and passively diffuses through the crosslinked cytoskeleton cage and nuclear structures to digest the genome at compatible recognition sites.
Over the course of an overnight incubation, this process gradually creates clusters of DNA fragments, or monomers, held in proximity by crosslinks between DNA and the cytoskeleton. This preserves the original interactions that were stabilised at the time of crosslinking.
Day 2
After the overnight restriction digestion, the restriction enzymes must be inactivated. Depending on the choice of restriction enzyme, this may be achieved with either heat or chemical denaturation. This step prevents the enzymes re-digesting ligated products later in the protocol.
Following the overnight restriction digestion, the clusters of crosslinked DNA are ligated together in proximity. DNA ligase is added to the sample and passively diffuses through the crosslinked cytoskeleton cage to ligate the cohesive ends of proximal monomers into chimeric Pore-C polymers.
Once the ligation is complete, the ligated products can be released from crosslinked cytoskeleton cages. An overnight proteinase K digestion is used to degrade all the protein structures in the samples, releasing the chimeric Pore-C polymers into solution as dsDNA.
Day 3
On the final day of the protocol, the Pore-C DNA extract can be isolated. Following the overnight proteinase K digestion, the chimeric Pore-C dsDNA polymers are in a mixed solution of polypeptide fragments and residual reaction buffers. A phenol:chloroform extraction is used to remove the peptides from the sample, followed by an ethanol precipitation to purify the DNA from the residual reaction buffers and phenol.
The final product is the Pore-C DNA extract, which is a pool of chimeric dsDNA polymers made of multiple ligated monomers. These were originally in proximity within cells at the time of crosslinking at the start of the protocol. By sequencing the junctions between these monomers, inferences can be made about DNA interactions, proximity in sequence space, and the three-dimensional structures of chromatin within the nucleus.