Chromosome conformation capture#Single-cell methods

Historically, microscopy was the primary method of investigating nuclear organization,{{cite journal | vauthors = Denker A, de Laat W | title = The second decade of 3C technologies: detailed insights into nuclear organization | journal = Genes & Development | volume = 30 | issue = 12 | pages = 1357–82 | date = June 2016 | pmid = 27340173 | pmc = 4926860 | doi = 10.1101/gad.281964.116 }} which can be dated back to 1590.{{cite web | title = Who invented the microscope? A complete Microscope History | url = http://www.history-of-the-microscope.org/history-of-the-microscope-who-invented-the-microscope.php | archive-url = https://web.archive.org/web/20180422180524/http://www.history-of-the-microscope.org/history-of-the-microscope-who-invented-the-microscope.php | archive-date = 22 April 2018 | url-status = dead | publisher = Vision Engineering Ltd }}

• In 1879, Walther Flemming coined the term chromatin.{{cn|date=September 2023}}
• In 1883, August Weismann connected chromatin with heredity.
• In 1884, Albrecht Kossel discovered histones.
• In 1888, Sutton and Boveri proposed the theory of continuity of chromatin during the cell cycle {{cite journal | vauthors = Martins LA | title = Did Sutton and Boveri propose the so-called Sutton-Boveri chromosome hypothesis? | journal = Genet. Mol. Biol. | date = 1999 | volume = 22 | issue = 2 | pages = 261–272 | doi = 10.1590/S1415-47571999000200022 | doi-access = free }}
• In 1889, Wilhelm von Waldemeyer created the term "chromosome".{{Cite web | url=http://public.oed.com/aspects-of-english/shapers-of-english/genes-and-genetics-the-language-of-scientific-discovery/ | title=Genes and genetics: The language of scientific discovery | date=2012-08-16 | work=Oxford English Dictionary | publisher=Oxford University Press | access-date=2017-12-07 | archive-date=2018-01-29 | archive-url=https://web.archive.org/web/20180129140501/http://public.oed.com/aspects-of-english/shapers-of-english/genes-and-genetics-the-language-of-scientific-discovery/ | url-status=dead }}
• In 1928, Emil Heitz coined the terms heterochromatin and euchromatin.{{Cite web | first = Mir | last = Harris | name-list-style = vanc | url=https://www.slideshare.net/hitheshck/heterochromatin-and-euchromatin-mains | title=Heterochromatin and euchromatin mains| date=2015-02-05}}
• In 1942, Conrad Waddington postulated the epigenetic landscapes.{{cite journal | vauthors = Deichmann U | title = Epigenetics: The origins and evolution of a fashionable topic | journal = Developmental Biology | volume = 416 | issue = 1 | pages = 249–254 | date = August 2016 | pmid = 27291929 | doi = 10.1016/j.ydbio.2016.06.005 | doi-access = free }}
• In 1948, Rollin Hotchkiss discovered DNA methylation.{{cite journal | vauthors = Lu H, Liu X, Deng Y, Qing H | title = DNA methylation, a hand behind neurodegenerative diseases | journal = Frontiers in Aging Neuroscience | volume = 5 | pages = 85 | date = December 2013 | pmid = 24367332 | pmc = 3851782 | doi = 10.3389/fnagi.2013.00085 | doi-access = free }}
• In 1953, Watson and Crick reported the double helix structure of DNA based on Rosalind Franklin's X-ray diffraction images.{{Cite web | url=https://profiles.nlm.nih.gov/SC/Views/Exhibit/narrative/doublehelix.html | title=The Francis Crick Papers: The Discovery of the Double Helix, 1951–1953}}{{Cite web |date=2016-06-01 |title=Francis Crick, Rosalind Franklin, James Watson, and Maurice Wilkins |url=https://www.sciencehistory.org/historical-profile/james-watson-francis-crick-maurice-wilkins-and-rosalind-franklin |access-date=2023-02-28 |website=Science History Institute |language=en}}
• In 1961, Mary Lyon postulated the principle of X-inactivation.
• In 1973/1974, chromatin fiber was discovered.
• In 1975, Pierre Chambon coined the term nucleosomes.
• In 1982, Chromosome territories were discovered.{{cite journal | vauthors = Cremer T, Cremer M | title = Chromosome territories | journal = Cold Spring Harbor Perspectives in Biology | volume = 2 | issue = 3 | pages = a003889 | date = March 2010 | pmid = 20300217 | pmc = 2829961 | doi = 10.1101/cshperspect.a003889 }}
• In 1984, John T. Lis innovated the Chromatin immunoprecipitation technique.
• In 1993, the Nuclear Ligation Assay was published, a method that could determine circularization frequencies of DNA in solution. This assay was used to show that estrogen induces an interaction between the prolactin gene promoter and a nearby enhancer.{{cite journal | vauthors = Cullen KE, Kladde MP, Seyfred MA | title = Interaction between transcription regulatory regions of prolactin chromatin | journal = Science | volume = 261 | issue = 5118 | pages = 203–6 | date = July 1993 | pmid = 8327891 | doi = 10.1126/science.8327891 | bibcode = 1993Sci...261..203C }}
• In 2002, Job Dekker introduced the new idea that dense matrices of interaction frequencies between loci could be used to infer the spatial organization of genomes. This idea was the basis for his development of the chromosome conformation capture (3C) assay, published in 2002 by Job Dekker and colleagues in the Kleckner lab at Harvard University.{{cite journal | vauthors = Dekker J, Rippe K, Dekker M, Kleckner N| title = Capturing chromosome conformation | journal = Science | volume = 295 | issue = 5558 | pages = 1306–11 | date = February 2002 | pmid = 11847345 | doi = 10.1126/science.1067799 | bibcode = 2002Sci...295.1306D | s2cid = 3561891 }}{{cite journal | vauthors = Osborne CS, Ewels PA, Young AN | title = Meet the neighbours: tools to dissect nuclear structure and function | journal = Briefings in Functional Genomics | volume = 10 | issue = 1 | pages = 11–7 | date = January 2011 | pmid = 21258046 | pmc = 3080762 | doi = 10.1093/bfgp/elq034 }}
• In 2003, the Human Genome Project was finished.
• In 2006, Marieke Simonis invented 4C,{{cite journal | vauthors = Simonis M, Klous P, Splinter E, Moshkin Y, Willemsen R, de Wit E, van Steensel B, de Laat W | title = Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C) | journal = Nature Genetics | volume = 38 | issue = 11 | pages = 1348–54 | date = November 2006 | pmid = 17033623 | doi = 10.1038/ng1896 | s2cid = 22787572 }} Dostie, in the Dekker lab, invented 5C.{{cite journal | vauthors = Dostie J, Richmond TA, Arnaout RA, Selzer RR, Lee WL, Honan TA, Rubio ED, Krumm A, Lamb J, Nusbaum C, Green RD, Dekker J | display-authors = 6 | title = Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements | journal = Genome Research | volume = 16 | issue = 10 | pages = 1299–309 | date = October 2006 | pmid = 16954542 | pmc = 1581439 | doi = 10.1101/gr.5571506 }}
• In 2007, B. Franklin Pugh innovated ChIP-seq technique.{{cite journal | vauthors = Albert I, Mavrich TN, Tomsho LP, Qi J, Zanton SJ, Schuster SC, Pugh BF | title = Translational and rotational settings of H2A.Z nucleosomes across the Saccharomyces cerevisiae genome | journal = Nature | volume = 446 | issue = 7135 | pages = 572–6 | date = March 2007 | pmid = 17392789 | doi = 10.1038/nature05632 | bibcode = 2007Natur.446..572A | s2cid = 4416890 }}
• In 2009, Erez Lieberman Aiden and Job Dekker invented Hi-C,{{cite journal | vauthors = Lieberman-Aiden E, van Berkum NL, Williams L, Imakaev M, Ragoczy T, Telling A, Amit I, Lajoie BR, Sabo PJ, Dorschner MO, Sandstrom R, Bernstein B, Bender MA, Groudine M, Gnirke A, Stamatoyannopoulos J, Mirny LA, Lander ES, Dekker J | display-authors = 6 | title = Comprehensive mapping of long-range interactions reveals folding principles of the human genome | journal = Science | volume = 326 | issue = 5950 | pages = 289–93 | date = October 2009 | pmid = 19815776 | pmc = 2858594 | doi = 10.1126/science.1181369 | bibcode = 2009Sci...326..289L }} Melissa J. Fullwood and Yijun Ruan invented ChIA-PET.{{cite journal | vauthors = Fullwood MJ, Liu MH, Pan YF, Liu J, Xu H, Mohamed YB, Orlov YL, Velkov S, Ho A, Mei PH, Chew EG, Huang PY, Welboren WJ, Han Y, Ooi HS, Ariyaratne PN, Vega VB, Luo Y, Tan PY, Choy PY, Wansa KD, Zhao B, Lim KS, Leow SC, Yow JS, Joseph R, Li H, Desai KV, Thomsen JS, Lee YK, Karuturi RK, Herve T, Bourque G, Stunnenberg HG, Ruan X, Cacheux-Rataboul V, Sung WK, Liu ET, Wei CL, Cheung E, Ruan Y | display-authors = 6 | title = An oestrogen-receptor-alpha-bound human chromatin interactome | journal = Nature | volume = 462 | issue = 7269 | pages = 58–64 | date = November 2009 | pmid = 19890323 | pmc = 2774924 | doi = 10.1038/nature08497 | bibcode = 2009Natur.462...58F }}
• In 2012, The Ren group, and the groups led by Edith Heard and Job Dekker discovered Topologically Associating Domains (TADs) in mammals.{{cite journal | vauthors = Dixon JR, Selvaraj S, Yue F, Kim A, Li Y, Shen Y, Hu M, Liu JS, Ren B | title = Topological domains in mammalian genomes identified by analysis of chromatin interactions | journal = Nature | volume = 485 | issue = 7398 | pages = 376–80 | date = April 2012 | pmid = 22495300 | pmc = 3356448 | doi = 10.1038/nature11082 | bibcode = 2012Natur.485..376D }}{{cite journal | vauthors = Nora EP, Lajoie BR, Schulz EG, Giorgetti L, Okamoto I, Servant N, Piolot T, van Berkum NL, Meisig J, Sedat J, Gribnau J, Barillot E, Blüthgen N, Dekker J, Heard E | title = Spatial partitioning of the regulatory landscape of the X-inactivation centre | journal = Nature | volume = 485 | issue = 7398 | pages = 381–5 | date = April 2012 | pmid = 22495304 | pmc = 3555144 | doi = 10.1038/nature11049 | bibcode = 2012Natur.485..381N }}
• In 2013, Takashi Nagano and Peter Fraser introduced in-nuclei ligation for Hi-C and single-cell Hi-C.{{Cite journal|last1=Nagano|first1=Takashi|last2=Lubling|first2=Yaniv|last3=Stevens|first3=Tim J.|last4=Schoenfelder|first4=Stefan|last5=Yaffe|first5=Eitan|last6=Dean|first6=Wendy|last7=Laue|first7=Ernest D.|last8=Tanay|first8=Amos|last9=Fraser|first9=Peter|date=October 2013|title=Single-cell Hi-C reveals cell-to-cell variability in chromosome structure|journal=Nature|volume=502|issue=7469|pages=59–64|doi=10.1038/nature12593|pmc=3869051|pmid=24067610|bibcode=2013Natur.502...59N}}
• In 2014, Suhas Rao, Miriam Huntley, et al. developed in-situ Hi-C and the use of 4-cutter restriction enzymes, and released the first high-resolution datasets down to kilobase resolution for several human cell lines. They also identified the first clear evidence of CTCF-Cohesin looping in Hi-C maps and identified the convergent CTCF motif rule underlying these loops.{{Cite journal|last1=Rao|first1=Suhas|last2=Huntley|first2=Miriam|date=December 2014|title=A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping|journal=Cell|volume=159|issue=7|pages=1665–1680|doi=10.1016/j.cell.2014.11.021|pmc=5635824|pmid=25497547}}
• In 2018 S Schoenfelder et el established a comprehensive promoter capture Hi-C method to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types in a non-biased [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102006/ interactions in dozens of human and mouse cell types in a non-biased wa].

All 3C methods start with a similar set of steps, performed on a sample of cells.

File:Chromosome conformation techniques.jpg

First, the cell genomes are cross-linked with formaldehyde,{{cite book | vauthors = Gavrilov A, Eivazova E, Priozhkova I, Lipinski M, Razin S, Vassetzky Y | chapter = Chromosome Conformation Capture (From 3C to 5C) and Its ChIP-Based Modification | volume = 567 | pages = 171–88 | year = 2009 | pmid = 19588093 | doi = 10.1007/978-1-60327-414-2_12 | isbn = 978-1-60327-413-5 | department = review | series = Methods in Molecular Biology | title = Chromatin Immunoprecipitation Assays }} which introduces bonds that "freeze" interactions between genomic loci. Treatment of cells with 1-3% formaldehyde, for 10-30min at room temperature is most common, however, standardization for preventing high protein-DNA cross linking is necessary, as this may negatively affect the efficiency of restriction digestion in the subsequent step.{{cite journal | vauthors = Naumova N, Smith EM, Zhan Y, Dekker J | title = Analysis of long-range chromatin interactions using Chromosome Conformation Capture | journal = Methods | volume = 58 | issue = 3 | pages = 192–203 | date = November 2012 | pmid = 22903059 | pmc = 3874837 | doi = 10.1016/j.ymeth.2012.07.022 }} The genome is then cut into fragments with a restriction endonuclease. The size of restriction fragments determines the resolution of interaction mapping. Restriction enzymes (REs) that make cuts on 6bp recognition sequences, such as EcoR1 or HindIII, are used for this purpose, as they cut the genome once every 4000bp, giving ~ 1 million fragments in the human genome.{{cite journal | vauthors = Belton JM, Dekker J | title = Chromosome Conformation Capture (3C) in Budding Yeast | journal = Cold Spring Harbor Protocols | volume = 2015 | issue = 6 | pages = 580–6 | date = June 2015 | pmid = 26034304 | doi = 10.1101/pdb.prot085175 | doi-access = free }} For more precise interaction mapping, a 4bp recognizing RE may also be used. The next step is, proximity based ligation. This takes place at low DNA concentrations or within intact, permeabilized nuclei in the presence of T4 DNA ligase,{{cite journal | vauthors = Gavrilov AA, Golov AK, Razin SV | title = Actual ligation frequencies in the chromosome conformation capture procedure | journal = PLOS ONE | volume = 8 | issue = 3 | pages = e60403 | date = 2013-03-26 | pmid = 23555968 | pmc = 3608588 | doi = 10.1371/journal.pone.0060403 | bibcode = 2013PLoSO...860403G | doi-access = free }} such that ligation between cross-linked interacting fragments is favored over ligation between fragments that are not cross-linked. Subsequently, interacting loci are quantified by amplifying ligated junctions by PCR methods.

The chromosome conformation capture (3C) experiment quantifies interactions between a single pair of genomic loci. For example, 3C can be used to test a candidate promoter-enhancer interaction. Ligated fragments are detected using PCR with known primers. That is why this technique requires the prior knowledge of the interacting regions.

Chromosome conformation capture-on-chip (4C) (also known as circular chromosome conformation capture) captures interactions between one locus and all other genomic loci. It involves a second ligation step, to create self-circularized DNA fragments, which are used to perform inverse PCR. Inverse PCR allows the known sequence to be used to amplify the unknown sequence ligated to it.{{cite journal |last1=Zhao |first1=Zhihu |last2=Tavoosidana |first2=Gholamreza |last3=Sjolinder |first3=Mikael |last4=Gondor |first4=Anita |last5=Mariano |first5=Piero |last6=Wang |first6=Sha |last7=Kanduri |first7=Chandrasekhar |last8=Lezcano |first8=Magda |last9=Sandhu |first9=Kuljeet Singh |last10=Singh |first10=Umashankar |last11=Pant |first11=Vinod |last12=Tiwari |first12=Vijay |last13=Kurukuti |first13=Sreenivasulu |last14=Ohlsson |first14=Rolf |title=Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. |journal=Nature Genetics |date=2006 |volume=38 |issue=11 |pages=1341–7 |pmid=17033624 |doi=10.1038/ng1891 |s2cid=2660843 }} In contrast to 3C and 5C, the 4C technique does not require the prior knowledge of both interacting chromosomal regions. Results obtained using 4C are highly reproducible with most of the interactions that are detected between regions proximal to one another. On a single microarray, approximately a million interactions can be analyzed. {{citation needed|date=June 2016}}

Chromosome conformation capture carbon copy (5C) detects interactions between all restriction fragments within a given region, with this region's size typically no greater than a megabase. This is done by ligating universal primers to all fragments. However, 5C has relatively low coverage. The 5C technique overcomes the junctional problems at the intramolecular ligation step and is useful for constructing complex interactions of specific loci of interest. This approach is unsuitable for conducting genome-wide complex interactions since that will require millions of 5C primers to be used.{{citation needed|date=June 2016}}

{{main|Hi-C (genomic analysis technique)}}

Hi-C uses high-throughput sequencing to find the nucleotide sequence of fragments and uses paired end sequencing, which retrieves a short sequence from each end of each ligated fragment. As such, for a given ligated fragment, the two sequences obtained should represent two different restriction fragments that were ligated together in the proximity based ligation step. The pair of sequences are individually aligned to the genome, thus determining the fragments involved in that ligation event. Hence, all possible pairwise interactions between fragments are tested.

A number of methods use oligonucleotide capture to enrich 3C and Hi-C libraries for specific loci of interest.{{cite patent|country=US|number=10287621|status=patent}}{{cite journal | vauthors = Schmitt AD, Hu M, Ren B | title = Genome-wide mapping and analysis of chromosome architecture | journal = Nature Reviews Molecular Cell Biology | volume = 17 | issue = 12 | pages = 743–755 | date = December 2016 | pmid = 27580841 | pmc = 5763923 | doi = 10.1038/nrm.2016.104 }} These methods include Capture-C,{{cite journal | vauthors = Hughes JR, Roberts N, McGowan S, Hay D, Giannoulatou E, Lynch M, De Gobbi M, Taylor S, Gibbons R, Higgs DR | display-authors = 6 | title = Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment | journal = Nature Genetics | volume = 46 | issue = 2 | pages = 205–12 | date = February 2014 | pmid = 24413732 | doi = 10.1038/ng.2871 | hdl = 2318/144575 | s2cid = 205348099 | url = https://serval.unil.ch/notice/serval:BIB_F8992FC64167 | hdl-access = free }} NG Capture-C,{{cite journal | vauthors = Davies JO, Telenius JM, McGowan SJ, Roberts NA, Taylor S, Higgs DR, Hughes JR | title = Multiplexed analysis of chromosome conformation at vastly improved sensitivity | journal = Nature Methods | volume = 13 | issue = 1 | pages = 74–80 | date = January 2016 | pmid = 26595209 | pmc = 4724891 | doi = 10.1038/nmeth.3664 }} Capture-3C, HiCap,{{Cite journal|last1=Sahlén|first1=Pelin|last2=Abdullayev|first2=Ilgar|last3=Ramsköld|first3=Daniel|last4=Matskova|first4=Liudmila|last5=Rilakovic|first5=Nemanja|last6=Lötstedt|first6=Britta|last7=Albert|first7=Thomas J.|last8=Lundeberg|first8=Joakim|last9=Sandberg|first9=Rickard|date=2015-08-03|title=Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution|journal=Genome Biology|volume=16|issue=1 |pages=156|doi=10.1186/s13059-015-0727-9|issn=1474-760X|pmc=4557751|pmid=26313521 |doi-access=free }} Capture Hi-C.{{cite journal | vauthors = Jäger R, Migliorini G, Henrion M, Kandaswamy R, Speedy HE, Heindl A, Whiffin N, Carnicer MJ, Broome L, Dryden N, Nagano T, Schoenfelder S, Enge M, Yuan Y, Taipale J, Fraser P, Fletcher O, Houlston RS | title = Capture Hi-C identifies the chromatin interactome of colorectal cancer risk loci | journal = Nature Communications | volume = 6 | pages = 6178 | date = February 2015 | pmid = 25695508 | pmc = 4346635 | doi = 10.1038/ncomms7178 | bibcode = 2015NatCo...6.6178J }} and Micro Capture-C.{{cite journal | vauthors = Hua P, Badat M, Hanssen L, Hentges L, Crump N, Downes D, Jeziorska DM, Oudelaar AM, Schwessinger R, Taylor S, Milne TA, Hughes JR, Higgs DR, Davies, JO| title = Defining genome architecture at base-pair resolution. | journal = Nature | date = June 2021 | volume = 595 | issue = 7865 | pages = 125–129 | doi = 10.1038/s41586-021-03639-4| pmid = 34108683 | bibcode = 2021Natur.595..125H | s2cid = 235394147 | url = https://ora.ox.ac.uk/objects/uuid:048c686c-f256-45ad-82c2-552e88b644c4 }} These methods are able to produce higher resolution and sensitivity than 4C based methods,{{cite journal | vauthors = Davies JO, Oudelaar AM, Higgs DR, Hughes JR | title = How best to identify chromosomal interactions: a comparison of approaches | journal = Nature Methods | volume = 14 | issue = 2 | pages = 125–134 | date = January 2017 | pmid = 28139673 | doi = 10.1038/nmeth.4146 | s2cid = 4136037 | url = https://ora.ox.ac.uk/objects/uuid:c233180b-e4a2-4288-b022-95549eb90d84 | url-access = subscription }} Micro Capture-C provides the highest resolution of the available 3C techniques and it is possible to generate base pair resolution data.

Single-cell adaptations of these methods, such as ChIP-seq and Hi-C can be used to investigate the interactions occurring in individual cells.{{cite journal | vauthors = Nagano T, Lubling Y, Stevens TJ, Schoenfelder S, Yaffe E, Dean W, Laue ED, Tanay A, Fraser P | display-authors = 6 | title = Single-cell Hi-C reveals cell-to-cell variability in chromosome structure | journal = Nature | volume = 502 | issue = 7469 | pages = 59–64 | date = October 2013 | pmid = 24067610 | pmc = 3869051 | doi = 10.1038/nature12593 | bibcode = 2013Natur.502...59N }}{{cite journal | vauthors = Schwartzman O, Tanay A | title = Single-cell epigenomics: techniques and emerging applications | journal = Nature Reviews Genetics | volume = 16 | issue = 12 | pages = 716–26 | date = December 2015 | pmid = 26460349 | doi = 10.1038/nrg3980 | s2cid = 10326803 }}

A number of methods sequence multiple ligation junctions simultaneously to detect higher-order structures where multiple regions of chromatin may be interacting. These methods include Tri-C,{{Cite journal|last1=Oudelaar|first1=A. Marieke|last2=Davies|first2=James O. J.|last3=Hanssen|first3=Lars L. P.|last4=Telenius|first4=Jelena M.|last5=Schwessinger|first5=Ron|last6=Liu|first6=Yu|last7=Brown|first7=Jill M.|last8=Downes|first8=Damien J.|last9=Chiariello|first9=Andrea M.|last10=Bianco|first10=Simona|last11=Nicodemi|first11=Mario|date=2018|title=Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains|journal=Nature Genetics|language=en|volume=50|issue=12|pages=1744–1751|doi=10.1038/s41588-018-0253-2|pmid=30374068 |pmc=6265079 |issn=1546-1718}} 3way 4C/C-walks,{{Cite journal|last1=Olivares-Chauvet|first1=Pedro|last2=Mukamel|first2=Zohar|last3=Lifshitz|first3=Aviezer|last4=Schwartzman|first4=Omer|last5=Elkayam|first5=Noa Oded|last6=Lubling|first6=Yaniv|last7=Deikus|first7=Gintaras|last8=Sebra|first8=Robert P.|last9=Tanay|first9=Amos|date=2016|title=Capturing pairwise and multi-way chromosomal conformations using chromosomal walks|url=https://www.nature.com/articles/nature20158|journal=Nature|language=en|volume=540|issue=7632|pages=296–300|doi=10.1038/nature20158|pmid=27919068 |bibcode=2016Natur.540..296O |s2cid=786054 |issn=1476-4687|url-access=subscription}} and multi-contact 4C (MC-4C).{{Cite journal|last1=Allahyar|first1=Amin|last2=Vermeulen|first2=Carlo|last3=Bouwman|first3=Britta A. M.|last4=Krijger|first4=Peter H. L.|last5=Verstegen|first5=Marjon J. A. M.|last6=Geeven|first6=Geert|last7=van Kranenburg|first7=Melissa|last8=Pieterse|first8=Mark|last9=Straver|first9=Roy|last10=Haarhuis|first10=Judith H. I.|last11=Jalink|first11=Kees|date=2018|title=Enhancer hubs and loop collisions identified from single-allele topologies|url=https://www.nature.com/articles/s41588-018-0161-5|journal=Nature Genetics|language=en|volume=50|issue=8|pages=1151–1160|doi=10.1038/s41588-018-0161-5|pmid=29988121 |s2cid=49667747 |issn=1546-1718}}

ChIP-loop combines 3C with ChIP-seq to detect interactions between two loci of interest mediated by a protein of interest.{{cite journal | vauthors = Horike S, Cai S, Miyano M, Cheng JF, Kohwi-Shigematsu T | title = Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome | journal = Nature Genetics | volume = 37 | issue = 1 | pages = 31–40 | date = January 2005 | pmid = 15608638 | doi = 10.1038/ng1491 | s2cid = 2884412 }} The ChIP-loop may be useful in identifying long-range cis-interactions and trans interaction mediated through proteins since frequent DNA collisions will not occur. {{citation needed|date=June 2016}}

ChIA-PET combines Hi-C with ChIP-seq to detect all interactions mediated by a protein of interest. HiChIP was designed to allow similar analysis as ChIA-PET with less input material.{{cite journal | vauthors = Mumbach MR, Rubin AJ, Flynn RA, Dai C, Khavari PA, Greenleaf WJ, Chang HY | title = HiChIP: efficient and sensitive analysis of protein-directed genome architecture | journal = Nature Methods | volume = 13 | issue = 11 | pages = 919–922 | date = November 2016 | pmid = 27643841 | pmc = 5501173 | doi = 10.1038/nmeth.3999 }}

3C methods have led to a number of biological insights, including the discovery of new structural features of chromosomes, the cataloguing of chromatin loops, and increased understanding of transcriptional regulation mechanisms (the disruption of which can lead to disease).

3C methods have demonstrated the importance of spatial proximity of regulatory elements to the genes that they regulate. For example, in tissues that express globin genes, the β-globin locus control region forms a loop with these genes. This loop is not found in tissues where the gene is not expressed.{{cite journal | vauthors = Tolhuis B, Palstra RJ, Splinter E, Grosveld F, de Laat W | title = Looping and interaction between hypersensitive sites in the active beta-globin locus | journal = Molecular Cell | volume = 10 | issue = 6 | pages = 1453–65 | date = December 2002 | pmid = 12504019 | doi = 10.1016/S1097-2765(02)00781-5 | doi-access = free }} This technology has further aided the genetic and epigenetic study of chromosomes both in model organisms and in humans.{{citation needed lead|date=April 2016}}

These methods have revealed large-scale organization of the genome into topologically associating domains (TADs), which correlate with epigenetic markers. Some TADs are transcriptionally active, while others are repressed.{{cite journal | vauthors = Cavalli G, Misteli T | title = Functional implications of genome topology | journal = Nature Structural & Molecular Biology | volume = 20 | issue = 3 | pages = 290–9 | date = March 2013 | pmid = 23463314 | pmc = 6320674 | doi = 10.1038/nsmb.2474 }} Many TADs have been found in D. melanogaster, mouse and human.{{cite journal | vauthors = Dekker J, Marti-Renom MA, Mirny LA | title = Exploring the three-dimensional organization of genomes: interpreting chromatin interaction data | journal = Nature Reviews Genetics | volume = 14 | issue = 6 | pages = 390–403 | date = June 2013 | pmid = 23657480 | pmc = 3874835 | doi = 10.1038/nrg3454 }} Moreover, CTCF and cohesin play important roles in determining TADs and enhancer-promoter interactions. The result shows that the orientation of CTCF binding motifs in an enhancer-promoter loop should be facing to each other in order for the enhancer to find its correct target.{{cite journal | vauthors = Guo Y, Xu Q, Canzio D, Shou J, Li J, Gorkin DU, Jung I, Wu H, Zhai Y, Tang Y, Lu Y, Wu Y, Jia Z, Li W, Zhang MQ, Ren B, Krainer AR, Maniatis T, Wu Q | display-authors = 6 | title = CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function | journal = Cell | volume = 162 | issue = 4 | pages = 900–10 | date = August 2015 | pmid = 26276636 | pmc = 4642453 | doi = 10.1016/j.cell.2015.07.038 }}

There are several diseases caused by defects in promoter-enhancer interactions, which are reviewed in this paper.{{cite journal | vauthors = Krijger PH, de Laat W | title = Regulation of disease-associated gene expression in the 3D genome | journal = Nature Reviews Molecular Cell Biology | volume = 17 | issue = 12 | pages = 771–782 | date = December 2016 | pmid = 27826147 | doi = 10.1038/nrm.2016.138 | s2cid = 11484886 }}

Beta thalassemia is a certain type of blood disorder caused by a deletion of LCR enhancer element.{{cite journal | vauthors = Fritsch EF, Lawn RM, Maniatis T | title = Characterisation of deletions which affect the expression of fetal globin genes in man | journal = Nature | volume = 279 | issue = 5714 | pages = 598–603 | date = June 1979 | pmid = 450109 | doi = 10.1038/279598a0| bibcode = 1979Natur.279..598F | s2cid = 4243029 }}{{cite journal | vauthors = Van der Ploeg LH, Konings A, Oort M, Roos D, Bernini L, Flavell RA | title = gamma-beta-Thalassaemia studies showing that deletion of the gamma- and delta-genes influences beta-globin gene expression in man | journal = Nature | volume = 283 | issue = 5748 | pages = 637–42 | date = February 1980 | pmid = 6153459 | doi = 10.1038/283637a0| bibcode = 1980Natur.283..637V | s2cid = 4371542 }}

Holoprosencephaly is cephalic disorder caused by a mutation in the SBE2 enhancer element, which in turn weakened the production of SHH gene.{{cite journal | vauthors = Jeong Y, El-Jaick K, Roessler E, Muenke M, Epstein DJ | title = A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers | journal = Development | volume = 133 | issue = 4 | pages = 761–72 | date = February 2006 | pmid = 16407397 | doi = 10.1242/dev.02239 | doi-access = free }}

PPD2 (polydactyly of a triphalangeal thumb) is caused by a mutation of ZRS enhancer, which in turn strengthened the production of SHH gene.{{cite journal | vauthors = Lettice LA, Heaney SJ, Purdie LA, Li L, de Beer P, Oostra BA, Goode D, Elgar G, Hill RE, de Graaff E | display-authors = 6 | title = A long-range Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly | journal = Human Molecular Genetics | volume = 12 | issue = 14 | pages = 1725–35 | date = July 2003 | pmid = 12837695 | doi = 10.1093/hmg/ddg180| doi-access = free }}{{cite journal | vauthors = Wieczorek D, Pawlik B, Li Y, Akarsu NA, Caliebe A, May KJ, Schweiger B, Vargas FR, Balci S, Gillessen-Kaesbach G, Wollnik B | display-authors = 6 | title = A specific mutation in the distant sonic hedgehog (SHH) cis-regulator (ZRS) causes Werner mesomelic syndrome (WMS) while complete ZRS duplications underlie Haas type polysyndactyly and preaxial polydactyly (PPD) with or without triphalangeal thumb | journal = Human Mutation | volume = 31 | issue = 1 | pages = 81–9 | date = January 2010 | pmid = 19847792 | doi = 10.1002/humu.21142 | s2cid = 1715146 }}

Adenocarcinoma of the lung can be caused by a duplication of enhancer element for MYC gene.{{cite journal | vauthors = Zhang X, Choi PS, Francis JM, Imielinski M, Watanabe H, Cherniack AD, Meyerson M | title = Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers | journal = Nature Genetics | volume = 48 | issue = 2 | pages = 176–82 | date = February 2016 | pmid = 26656844 | pmc = 4857881 | doi = 10.1038/ng.3470 }}

T-cell acute lymphoblastic leukemia is caused by an introduction of a new enhancer.{{cite journal | vauthors = Mansour MR, Abraham BJ, Anders L, Berezovskaya A, Gutierrez A, Durbin AD, Etchin J, Lawton L, Sallan SE, Silverman LB, Loh ML, Hunger SP, Sanda T, Young RA, Look AT | display-authors = 6 | title = Oncogene regulation. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element | journal = Science | volume = 346 | issue = 6215 | pages = 1373–7 | date = December 2014 | pmid = 25394790 | pmc = 4720521 | doi = 10.1126/science.1259037 }}

File:Hi c visualization.gif

The different 3C-style experiments produce data with very different structures and statistical properties. As such, specific analysis packages exist for each experiment type.

Hi-C data is often used to analyze genome-wide chromatin organization, such as topologically associating domains (TADs), linearly contiguous regions of the genome that are associated in 3-D space. Several algorithms have been developed to identify TADs from Hi-C data.{{cite journal | vauthors = Dixon JR, Selvaraj S, Yue F, Kim A, Li Y, Shen Y, Hu M, Liu JS, Ren B | display-authors = 6 | title = Topological domains in mammalian genomes identified by analysis of chromatin interactions | journal = Nature | volume = 485 | issue = 7398 | pages = 376–80 | date = April 2012 | pmid = 22495300 | pmc = 3356448 | doi = 10.1038/nature11082 | bibcode = 2012Natur.485..376D }}

Hi-C and its subsequent analyses are evolving. Fit-Hi-C is a method based on a discrete binning approach with modifications of adding distance of interaction (initial spline fitting, aka spline-1) and refining the null model (spline-2). The result of Fit-Hi-C is a list of pairwise intra-chromosomal interactions with their p-values and q-values.{{cite journal | vauthors = Yardımcı GG, Noble WS | title = Software tools for visualizing Hi-C data | journal = Genome Biology | volume = 18 | issue = 1 | pages = 26 | date = February 2017 | pmid = 28159004 | pmc = 5290626 | doi = 10.1186/s13059-017-1161-y | doi-access = free }}

The 3-D organization of the genome can also be analyzed via eigendecomposition of the contact matrix. Each eigenvector corresponds to a set of loci, which are not necessarily linearly contiguous, that share structural features.{{cite journal | vauthors = Imakaev M, Fudenberg G, McCord RP, Naumova N, Goloborodko A, Lajoie BR, Dekker J, Mirny LA | display-authors = 6 | title = Iterative correction of Hi-C data reveals hallmarks of chromosome organization | journal = Nature Methods | volume = 9 | issue = 10 | pages = 999–1003 | date = October 2012 | pmid = 22941365 | pmc = 3816492 | doi = 10.1038/nmeth.2148 }}

A significant confounding factor in 3C technologies is the frequent non-specific interactions between genomic loci that occur due to random polymer behavior. An interaction between two loci must be confirmed as specific through statistical significance testing.

There are two major ways of normalizing raw Hi-C contact heat maps. The first way is to assume equal visibility, meaning there is an equal chance for each chromosomal position to have an interaction. Therefore, the true signal of a Hi-C contact map should be a balanced matrix (Balanced matrix has constant row sums and column sums). An example of algorithms that assumes equal visibility is Sinkhorn-Knopp algorithm, which scales the raw Hi-C contact map into a balanced matrix.

The other way is to assume there is a bias associated with each chromosomal position. The contact map value at each coordinate will be the true signal at that position times bias associated with the two contact positions. An example of algorithms that aim to solve this model of bias is iterative correction, which iteratively regressed out row and column bias from the raw Hi-C contact map. There are a number of software tools available for analysis of Hi-C data.{{cite journal | vauthors = Imakaev M, Fudenberg G, McCord RP, Naumova N, Goloborodko A, Lajoie BR, Dekker J, Mirny LA | title = Iterative correction of Hi-C data reveals hallmarks of chromosome organization | journal = Nature Methods | volume = 9 | issue = 10 | pages = 999–1003 | date = October 2012 | pmid = 22941365 | pmc = 3816492 | doi = 10.1038/nmeth.2148 }}

DNA motifs are specific short DNA sequences, often 8-20 nucleotides in length{{cite journal | vauthors = Zambelli F, Pesole G, Pavesi G | title = Motif discovery and transcription factor binding sites before and after the next-generation sequencing era | journal = Briefings in Bioinformatics | volume = 14 | issue = 2 | pages = 225–37 | date = March 2013 | pmid = 22517426 | pmc = 3603212 | doi = 10.1093/bib/bbs016 }} which are statistically overrepresented in a set of sequences with a common biological function. Currently, regulatory motifs on the long-range chromatin interactions have not been studied extensively. Several studies have focused on elucidating the impact of DNA motifs in promoter-enhancer interactions.

Bailey et al. has identified that ZNF143 motif in the promoter regions provides sequence specificity for promoter-enhancer interactions.Bailey, S. D., Zhang, X., Desai, K., Aid, M., Corradin, O., Cowper-Sal·lari, R., … Lupien, M. (2015). ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters. Nature Communications, 2, 6186. Retrieved from https://doi.org/10.1038/ncomms7186 Mutation of ZNF143 motif decreased the frequency of promoter-enhancer interactions suggesting that ZNF143 is a novel chromatin-looping factor.

For genome-scale motif analysis, in 2016, Wong et al. reported a list of 19,491 DNA motif pairs for K562 cell line on the promoter-enhancer interactions.K. Wong, Y. Li, and C. Peng, “Identification of coupling DNA motif pairs on long-range chromatin interactions in human,” vol. 32, no. September 2015, pp. 321–324, 2016. As a result, they proposed that motif pairing multiplicity (number of motifs that are paired with a given motif) is linked to interaction distance and regulatory region type. In the next year, Wong published another article reporting 18,879 motif pairs in 6 human cell lines.Ka-Chun Wong; MotifHyades: expectation maximization for de novo DNA motif pair discovery on paired sequences, Bioinformatics, Volume 33, Issue 19, 1 October 2017, Pages 3028–3035, https://doi.org/10.1093/bioinformatics/btx381 A novel contribution of this work is MotifHyades, a motif discovery tool that can be directly applied to paired sequences.

The 3C-based techniques can provide insights into the chromosomal rearrangements in the cancer genomes.{{cite journal | vauthors = Harewood L, Kishore K, Eldridge MD, Wingett S, Pearson D, Schoenfelder S, Collins VP, Fraser P | title = Hi-C as a tool for precise detection and characterisation of chromosomal rearrangements and copy number variation in human tumours | journal = Genome Biology | volume = 18 | issue = 1 | pages = 125 | date = June 2017 | pmid = 28655341 | pmc = 5488307 | doi = 10.1186/s13059-017-1253-8 | doi-access = free }} Moreover, they can show changes of spatial proximity for regulatory elements and their target genes, which bring deeper understanding of the structural and functional basis of the genome.{{cite journal | vauthors = Taberlay PC, Achinger-Kawecka J, Lun AT, Buske FA, Sabir K, Gould CM, Zotenko E, Bert SA, Giles KA, Bauer DC, Smyth GK, Stirzaker C, O'Donoghue SI, Clark SJ | display-authors = 6 | title = Three-dimensional disorganization of the cancer genome occurs coincident with long-range genetic and epigenetic alterations | journal = Genome Research | volume = 26 | issue = 6 | pages = 719–31 | date = June 2016 | pmid = 27053337 | pmc = 4889976 | doi = 10.1101/gr.201517.115 }}

{{Reflist|33em}}

{{refbegin|33em}}

• {{cite journal | vauthors = Barutcu AR, Fritz AJ, Zaidi SK, van Wijnen AJ, Lian JB, Stein JL, Nickerson JA, Imbalzano AN, Stein GS | title = C-ing the Genome: A Compendium of Chromosome Conformation Capture Methods to Study Higher-Order Chromatin Organization | journal = Journal of Cellular Physiology | volume = 231 | issue = 1 | pages = 31–5 | date = January 2016 | pmid = 26059817 | pmc = 4586368 | doi = 10.1002/jcp.25062 }}
• {{cite journal | vauthors = Marbouty M, Koszul R | title = Metagenome Analysis Exploiting High-Throughput Chromosome Conformation Capture (3C) Data | journal = Trends in Genetics | volume = 31 | issue = 12 | pages = 673–682 | date = December 2015 | pmid = 26608779 | pmc = 6831814 | doi = 10.1016/j.tig.2015.10.003 | department = review }}
• {{cite journal | vauthors = Dekker J | title = Two ways to fold the genome during the cell cycle: insights obtained with chromosome conformation capture | journal = Epigenetics & Chromatin | volume = 7 | issue = 1 | pages = 25 | date = 25 November 2014 | pmid = 25435919 | pmc = 4247682 | doi = 10.1186/1756-8935-7-25 | doi-access = free }}
• {{cite journal | vauthors = O'Sullivan JM, Hendy MD, Pichugina T, Wake GC, Langowski J | title = The statistical-mechanics of chromosome conformation capture | journal = Nucleus | volume = 4 | issue = 5 | pages = 390–8 | date = September–October 2013 | pmid = 24051548 | pmc = 3899129 | doi = 10.4161/nucl.26513 }}
• {{cite journal | vauthors = Umbarger MA | title = Chromosome conformation capture assays in bacteria | journal = Methods | volume = 58 | issue = 3 | pages = 212–20 | date = November 2012 | pmid = 22776362 | doi = 10.1016/j.ymeth.2012.06.017 | s2cid = 24234275 | department = review }}
• {{cite journal | vauthors = Parelho V, Merkenschlager M | title = Gene expression: growing up together may help genes go their separate ways | journal = European Journal of Human Genetics | volume = 13 | issue = 9 | pages = 993–4 | date = September 2005 | pmid = 15999115 | doi = 10.1038/sj.ejhg.5201464 | s2cid = 29714576 | department = news and commentary | doi-access = free }}
• {{cite web |title=Chromosome conformation capture |format=commercial method | last1 = Marvin | first1 = Marcus | last2 = Tan-Wong | first2 = Sue Mei | name-list-style = vanc |date=2016-04-23 | work= Abcam PLC |url=http://www.abcam.com/index.html?pageconfig=resource&rid=10010&pid=5 |access-date=23 April 2016}}

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• {{annotated link|Genetic testing}}

{{DEFAULTSORT:Chromosome Conformation Capture}}

Category:Biochemical separation processes

Category:Chromosomes

Category:Nuclear organization