The eukaryotic genome consists of densely packed molecules (i.e., linear chromosomes) that fill the microscopic space of the cell nucleus called nucleoplasm. The Chromatin Conformation Capture 3C assay is a useful technique to understand the functional significance of the tridimensional structure of the genome and chromatin rearrangements. This assay can examine how compaction level dictates chromatin accessibility of transcription factors to modulate gene expression in specific cell types.
Image Credit: Billion Photos/Shutterstock.com
Organization And Function of the Eukaryotic Genome
In eukaryotic organisms, the genome, i.e., the complete set of genetic information of an organism, is a dynamic structure that undergoes conformational changes capable of altering gene expression patterns during cellular processes. Moreover, chromatin can be defined as a macromolecular complex that involves both the DNA sequence and associated histone proteins, and it is commonly divided into linear-long DNA molecules called chromosomes.
The tridimensional organization of the chromatin is not equal in all regions of the genome. There are relatively compacted regions known as heterochromatin domains, which hamper the accessibility to transcription factors, and there are also many transcriptionally active regions known as euchromatin. It has been observed that certain regions of the genome may suffer conformational changes to modulate gene expression during cellular processes such as differentiation. In embryonic stem cells and other cell types, well-defined higher-order chromatin structures have already been identified and functionally characterized.
The assessment of the genome's architecture is increasingly gaining interest in the scientific community due to the identification of local changes in chromatin structure that may be linked to complex cell phenotypes. These tridimensional modifications are associated with epigenetic mutations (either aberrant DNA methylation or histone modifications) and chromosome rearrangements in cells that display faulty gene expression patterns associated with complex genetic diseases such as cancer.
What is Chromatin Conformation Capture?
Chromosome-conformation-capture protocols include diverse cutting-edge assays that have already shown their potential in assessing long-range gene regulation and identifying chromosome rearrangements (deletions, insertions, translocations duplications) and determination of topologically associating domain boundaries.
In particular, the Chromatin Conformation Capture (3C) assay is a powerful technique to understand the spatial chromatin organization by analyzing pairwise proximity measurements between different loci. These pairwise dimensions allow us to diagram tridimensional (3D) models for a given genomic region, linkage groups, chromosomes, or even the entire genome.
What Does the C3 Assay Involve?
Chromatin Conformation Capture (C3) assay consists of several sequential steps, which include formaldehyde cross-linking to capture interactions between chromatin segments, isolation, digestion using restriction enzymes, and finally, ligation. The resulting 3C library involves a collection of restriction fragments ligated together, while pairwise chromatin interactions can help us to decipher the regulatory roles of cis-regulatory elements (e.g., promoters and enhancers).
The 3C library is also useful for high-resolution analyses involving interacting nucleotide sequences in a given cell population. It is also important to note that interacting chromatin regions detected by 3C or derivative protocols should ideally be validated by another technique to unequivocally confirm the presence of such interactions (e.g., the fluorescence in situ hybridization assay).
3C-based Assays
Some of the techniques derived from 3C include:
- Circular chromosome conformation capture (4C): The 4C's technique is used to identify genomic fragments containing sequences that interact with target genomic loci. Moreover, the protocol is also known to have the potential to decipher how epigenetic regulation may rely on interactions at multiple genomic loci. For example, the C4 assay has enabled the identification of interactions in the maternally inherited H19 locus involved in the epigenetic phenomenon of genomic imprinting.
- Chromosome conformation capture carbon copy (5C): it is a protocol that allows the identification of genomic interactions between restriction fragments for linkage groups usually smaller in size than one megabase (1 Mb).
- ChIP-loop: ChIP-Loop Assay combines Chromatin immunoprecipitation (ChIP) and the 3C protocol to detect long-range chromatin interactions between multiple loci mediated by a target protein.
- Hi-C: this technique is used to determine genome-wide chromatin interactions by combining the 3C approach and next-generation sequencing (NGS). Hi-C relies on DNA-protein cross-linking with formaldehyde, sample fragmentation, ligation, and digestion by restriction enzymes.
- Capture-C: it is a procedure that analyzes chromatin interactions by combining three different technologies: C3, NGS sequencing, and oligonucleotide capture technology. The combined strategy allows the isolation of target genomic regions by using specifically designed RNA in solution.
Chromatin Conformation Capture | Chromosome Conformation Capture Assay | Hi-C Method |
C3-based Methods: A Future Beyond
Chromosome conformation capture-based assays are the 'gold standard' in assessing spatial chromatin organization. These strategies allow us to recognize potential interactions between loci of a chromosome. Genome sequencing of the resulting nucleotide sequences is critical in accurately detecting interacting genomic regions. The use of derivative techniques of the C3 assay sheds new light and insights into such interactions.
In the near future, it is expected to incorporate in vivo real maps of the 3D genome architecture for single cells, which will trigger a revolution in the study of gene expression.
Sources:
- Akgol Oksuz, Betul, et al. "Systematic evaluation of chromosome conformation capture assays." Nature methods 18.9 (2021): 1046-1055. DOI: https://doi.org/10.1038/s41592-021-01248-7
- Downes, Damien J., et al. "High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale." Nature communications 12.1 (2021): 1-15. DOI: https://doi.org/10.1038/s41467-020-20809-6
- Dostie, Josée, et al. "Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements." Genome research 16.10 (2006): 1299-1309. DOI: 10.1101/gr.5571506
- Gavrilov, Alexey, et al. "Chromosome conformation capture (from 3C to 5C) and its ChIP-based modification." Chromatin Immunoprecipitation Assays (2009): 171-188. DOI: https://doi.org/10.1007/978-1-60327-414-2_12
- Goel, Viraat Y., and Anders S. Hansen. "The macro and micro of chromosome conformation capture." Wiley Interdisciplinary Reviews: Developmental Biology 10.6 (2021): e395. DOI: https://doi.org/10.1002/wdev.395
- McArthur, Evonne, and John A. Capra. "Topologically associating domain boundaries that are stable across diverse cell types are evolutionarily constrained and enriched for heritability." The American Journal of Human Genetics 108.2 (2021): 269-283. DOI: https://doi.org/10.1016/j.ajhg.2021.01.001
- Sati, Satish, and Giacomo Cavalli. "Chromosome conformation capture technologies and their impact in understanding genome function." Chromosoma 126.1 (2017): 33-44. DOI: https://doi.org/10.1007/s00412-016-0593-6
Further Reading