Review ArticleA genome-wide 3C-method for characterizing the three-dimensional architectures of genomes
Introduction
Genomes carry both genetic and epigenetic information and serve as a scaffold for reading and transmitting both types of inheritable information. Insight into the 3D organization of the genome, including the multilevel folding and positioning of chromosomes within the nucleus, is essential for understanding various genomic functions [1]. To date, two types of tools have been used to dissect chromosome structure: microscopy-based imaging technologies and more recently developed molecular and biochemical tools. DNA imaging technologies are based on electron microscopy and light microscopy (reviewed in [2], [3], [4]). Electron microscopes have been typically employed in studies using cell-free systems, whereas light microscopy-based techniques, such as DNA fluorescence in situ hybridization (FISH) [5] and live-cell imaging [6] have been applied to visualize the organization of chromosomes in the nuclei of single cells in situ. Although microscopy has provided important insights into the 3D architecture of chromosomes, including their dynamic nature and non-random organization, limitations in resolution and throughput have reduced microscopy’s utility in understanding genome structure-function relationships.
During the past decade, several biochemical methods have been developed for characterizing genome architecture (reviewed in [7], [8]). By measuring spatial proximity, these new techniques offer detailed molecular views of chromosome structure beyond the resolution limits of microscopy. One subset of techniques includes chromatin immunoprecipitation (ChIP) and DamID methods, which probe physical contacts between genomic loci of interest and nuclear landmarks such as the nuclear envelope or nucleolus, yielding important information about the position of genomic loci in nuclear space [9], [10], [11], [12]. Another set of molecular tools, including RNA-TRAP [13] and 3C-based methods [14], are able to measure the relative spatial proximity between individual genomic loci, providing insight into the local or global folding of chromosomes and into the relative positioning of individual chromosomes in relationship to one another.
The relative simplicity of 3C has led to its widespread adoption in studies of long-range chromatin interactions, making it and its derivatives the most commonly used tools for characterizing chromosome structure [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25]. 3C is based on the principle of proximity ligation. Briefly, under conditions of very low DNA concentrations (usually less than 0.8 μg/μl), ligation between two cross-linked chromatin fragments is strongly favored over random inter-molecular ligation between two unassociated chromatin fragments [14]. All of the restriction enzyme digestion-based 3C techniques share four experimental steps: (1) cells are fixed with formaldehyde, which cross-links chromatin interactions; (2) the cross-linked chromatin is digested with a restriction enzyme (RE1); (3) DNA ends are ligated under conditions that favor intra-molecular ligation (proximity ligation); and (4) cross-links are reversed and DNA is recovered. However, the various 3C derivatives differ in their downstream steps for detecting chromatin interactions.
We recently developed a genomic method for mapping all the chromatin interactions that occur within a genome in an unbiased manner [26]. Briefly, the method starts with construction of a 3C library, followed by digestion of the library with a second restriction enzyme (RE2). As in the 4C protocol, the resulting DNA fragments are circularized to form small DNA circles. The circular DNA is subsequently digested again with the primary 3C RE1 to linearize the DNA. The re-opened RE1 sites serve as anchoring sites for the interacting DNA fragments and are ligated with an adapter containing an EcoP15I restriction site. The anchoring sites are then marked with biotin through DNA circularization, and the DNA circles are cut by the enzyme EcoP15I to produce biotin-labeled paired-end tags of 25–27 bp. The resulting biotin-labeled paired-end tags, representing the interacting DNA fragments, are pulled down with streptavidin beads, and paired-end sequencing enables the detection of ligation junctions (Fig. 1).
Chromatin interaction libraries generated with our method consist of DNA molecules with uniform structure and size (Fig. 1B), unlike those constructed with other recently developed similar methods such as Hi-C [19], [21] and TCC [18]. This unique feature of our method provides a straightforward way to calculate the interaction frequency of each individual chromatin interaction. Therefore, our method can be very useful for characterizing the 3D architectures of relatively simple genomes at unprecedented resolution (kb) as well as for the identification of functionally relevant (statistically significant) long-range chromatin interactions between distant genomic elements (such as promoter-enhancer interactions) on a whole-genome scale. In principle, this method is applicable to all genomes. Here, we describe the step-by-step protocol for the haploid budding yeast genome.
Section snippets
Cross-linking of yeast cells with formaldehyde
To capture dynamic chromosomal contacts, it is necessary to covalently link the interacting protein–protein or protein–DNA partners together. There are several cross-linking agents available. Among them, formaldehyde is the most widely used because (1) formaldehyde is cell-permeable; (2) the cross-linking reaction mediated by formaldehyde is very efficient and readily controllable (usually temperature and reaction time are the two adjustable parameters); (3) formaldehyde-mediated cross-linking
Data analysis, interpretation and expected results
To characterize the genome topology and to uncover its potential functional implications, the completion of library construction and high throughput sequencing is just the first step of a long journey – biological insights cannot be obtained without sophisticated computational analysis. Here we outline the basic data analysis pipeline we have implemented for characterizing the haploid budding yeast genome [26].
Limitations and alternative methods
Despite the successful application of our method to characterize the budding yeast genome, transitioning to diploid mammalian genomes requires several technical issues to be considered. First, in a diploid mammalian genome, each chromosome has a homologous partner, and it is not clear whether the two homologous chromosomes interact with other chromosomes in the same way or not. Hence, it might be important to distinguish the chromatin interactions involving any given chromosome from those of
Material
Reagents can be found in Table 2.
Buffer
Spheroplast buffer
1 M sorbitol
100 mM potassium phosphate (PH 7.5)
10× T4 DNA ligase Buffer
500 mM Tris-HCl, PH 7.5
100 mM DTT
100 mM MgCl2
10 mM ATP
TE Buffer (PH 8.0)
10 mM Tris–HCl (PH 8.0)
1 mM EDTA
Primer
HindIII-ECoP 15I-Adaptor-F 5′/5Phos/AGC TCT GCT GTA C 3′
HindIII-ECoP 15I-Adaptor-R 5′/5Phos/ACA GCA G 3′
Eco RI-ECoP 15I-Adaptor-F 5′/5Phos/AAT TTC TGC TGT AC 3′
Eco RI-ECoP 15I-Adaptor-R 5′/5Phos/ACA GCA GA 3′
Biotin-internal adaptor-F 5′/5Phos/CGTACAT(Bio)CCGCCTTGGCCGT 3′
Acknowledgements
Supported by NIH grants P01GM081619 (CBA), P41RR0011823 (WSN), and the Howard Hughes Medical Institute (SF). We thank Ferhat Ay for his assistance in preparing Fig. 4.
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