Quantitative analysis of chromosome conformation capture assays (3C-qPCR)
Abstract
Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7-9 days.
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BackgroundMethodSelf-citationLong non-coding RNAs and chromatin modifiers
Marchese, F. P., & Huarte, M. (2013) · Epigenetics · doi:10.4161/epi.27472 · Cited by 179 · see citing works
Kouwenhoven, E. N., van Heeringen, S. J., Tena, J. J., Oti, M., Dutilh, B. E., Alonso, M. E., de la Calle‐Mustienes, E., Smeenk, L., Rinne, T., Parsaulian, L., et al. (2010) · PLoS Genetics · doi:10.1371/journal.pgen.1001065 · Cited by 179 · see citing works
“Chromosome Conformation Capture (3C) assay Chromosome Conformation Capture (3C) assay was performed as referred in Hagege et al., 2007 [56].”
MethodA gene loop containing the floral repressor FLC is disrupted in the early phase of vernalization
Crevillén, P., Sönmez, Ç., Wu, Z., & Dean, C. (2012) · The EMBO Journal · doi:10.1038/emboj.2012.324 · Cited by 174 · see citing works
“This technique relies on formaldehyde crosslinking to detect interacting chromatin fragments in intact cells (Dekker et al , 2002; Hagege et al , 2007; Louwers et al , 2009b).”
Control of embryonic stem cell lineage commitment by core promoter factor, TAF3
Liu, Z., Scannell, D. R., Eisen, M. B., & Tjian, R. (2011) · Cell · doi:10.1016/j.cell.2011.08.005 · Cited by 167 · see citing works
Wheat chromatin architecture is organized in genome territories and transcription factories
Concia, L., Veluchamy, A., Ramirez-Prado, J. S., Martín, A. C., Huang, Y., Perez, M., Domenichini, S., Rodriguez‐Granados, N. Y., Kim, S.-K., Blein, T., et al. (2020) · Genome biology · doi:10.1186/s13059-020-01998-1 · Cited by 166 · see citing works
Gómez-Marín, C., Tena, J. J., Acemel, R. D., López-Mayorga, M., Naranjo, S., de la Calle-Mustienes, E., Maeso, I., Beccari, L., Aneas, I., Vielmas, E., et al. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1505463112 · Cited by 159 · see citing works
Biology and Clinical Implications of the 19q13 Aggressive Prostate Cancer Susceptibility Locus
Gao, P. H., Xia, J., Sipeky, C., Dong, X., Zhang, Q., Yang, Y., Zhang, P., Cruz, S. P., Zhang, K., Zhu, J., et al. (2018) · Cell · doi:10.1016/j.cell.2018.06.003 · Cited by 157 · see citing works
“Quantitative analysis of chromosome conformation capture assays Quantitative analysis of chromosome conformation capture assays (3C-qPCR) were performed as described previously (Hagège et al., 2007).”
Schödel, J., Bardella, C., Sciesielski, L. K., Brown, J. M., Pugh, C. W., Buckle, V. J., Tomlinson, I. P. M., Ratcliffe, P. J., & Mole, D. R. (2012) · Nature Genetics · doi:10.1038/ng.2204 · Cited by 157 · see citing works
p53 Activates the Long Noncoding RNA Pvt1b to Inhibit Myc and Suppress Tumorigenesis
Olivero, C. E., Martínez‐Terroba, E., Zimmer, J. T., Liao, C., Tesfaye, E., Hooshdaran, N., Schofield, J. A., Bendor, J., Fang, D., Simon, M. D., et al. (2020) · Molecular Cell · doi:10.1016/j.molcel.2019.12.014 · Cited by 158 · see citing works
“Chromosome Conformation Capture (3C)—Chromosome conformation capture was performed as described previously with minor modifications (Hagege et al., 2007).”
Long-Range Chromatin Interactions Drive Mutant TERT Promoter Activation
Akıncılar, S. C., Khattar, E., Boon, P. L. S., Unal, B., Fullwood, M. J., & Tergaonkar, V. (2016) · Cancer Discovery · doi:10.1158/2159-8290.cd-16-0177 · Cited by 153 · see citing works
“Chromosome Conformation Capture Assay (3C): 3C was performed as described previously (41).”
MethodIrimia, M., Tena, J. J., Alexis, M. S., Fernandez-Miñan, A., Maeso, I., Bogdanović, O., de la Calle-Mustienes, E., Roy, S. W., Gómez-Skarmeta, J. L., & Fraser, H. B. (2012) · Genome Research · doi:10.1101/gr.139725.112 · Cited by 146 · see citing works
“3C assays were performed as referred in Hagège et al. (2007) and Tena et al. (2011), (see Supplemental File S1).”
MethodArloth, J., Bogdan, R., Weber, P., Frishman, G., Menke, A., Wagner, K. V., Balsevich, G., Schmidt, M. V., Karbalai, N., Czamara, D., et al. (2015) · Neuron · doi:10.1016/j.neuron.2015.05.034 · Cited by 142 · see citing works
Sekimata, M., Pérez‐Melgosa, M., Miller, S. A., Weinmann, A. S., Sabo, P. J., Sandstrom, R. S., Dorschner, M. O., Stamatoyannopoulos, J. A., & Wilson, C. B. (2009) · Immunity · doi:10.1016/j.immuni.2009.08.021 · Cited by 148 · see citing works
“Ligation products were quantified in triplicate by quantitative TaqMan real-time PCR as described (Hagege et al., 2007).”
MethodRamamoorthy, S., & Cidlowski, J. A. (2013) · Molecular and Cellular Biology · doi:10.1128/mcb.01151-12 · Cited by 146 · see citing works
“formed with A549 cells as described previously (35), with minor modifi-”
BackgroundConstructing 3D interaction maps from 1D epigenomes
Zhu, Y., Chen, Z., Zhang, K., Wang, M., Medovoy, D., Whitaker, J. W., Ding, B., Li, N., Zheng, L., & Wang, W. (2016) · Nature Communications · doi:10.1038/ncomms10812 · Cited by 139 · see citing works
“3C (chromosome conformation capture) libraries from IMR-90 cells were generated according to standard protocols as described previously in refs 24,62,63.”
Two Forms of Loops Generate the Chromatin Conformation of the Immunoglobulin Heavy-Chain Gene Locus
Guo, C., Gerasimova, T. I., Hao, H., Иванова, И., Chakraborty, T., Selimyan, R., Oltz, E. M., & Sen, R. (2011) · Cell · doi:10.1016/j.cell.2011.08.049 · Cited by 148 · see citing works
“3C ligation products were measured by the Taqman quantitative PCR technology (Hagege et al., 2007).”
Tissue- and Expression Level–Specific Chromatin Looping at Maizeb1Epialleles
Louwers, M., Bader, R., Haring, M., DRIEL, R. V., de Laat, W. L., & Stam, M. E. (2009) · The Plant Cell · doi:10.1105/tpc.108.064329 · Cited by 144 · see citing works
Stadhouders, R., Kolovos, P., Brouwer, R. W. W., Zuin, J., van den Heuvel, A., Kockx, C. E. M., Palstra, R.-J. T. S., Wendt, K. S., Grosveld, F. G., van IJcken, W. F. J., et al. (2013) · Nature Protocols · doi:10.1038/nprot.2013.018 · Cited by 141 · see citing works
FRIGIDA establishes a local chromosomal environment for FLOWERING LOCUS C mRNA production
Li, Z., Jiang, D., & He, Y. (2018) · Nature Plants · doi:10.1038/s41477-018-0250-6 · Cited by 143 · see citing works
Li, J.-H., Shou, J., Guo, Y., Tang, Y.-X., Wu, Y.-H., Jia, Z.-L., Zhai, Y.-N., Chen, Z.-F., Xu, Q., & Wu, Q. (2015) · Journal of Molecular Cell Biology · doi:10.1093/jmcb/mjv016 · Cited by 139 · see citing works
“Frequencies of inversions and duplications by CRISPR We estimated the frequencies of inversion and duplication events by a quantification method to correct differences in PCR efficiencies of primer pairs, similar to that of the chromosome conformation capture method (Hagège et al., 2007; Guo et al., 2012).”
MethodBET Inhibitors Suppress ALDH Activity by Targeting ALDH1A1 Super-Enhancer in Ovarian Cancer
Yokoyama, Y., Zhu, H., Lee, J., Kossenkov, A. V., Wu, S. Y., Wickramasinghe, J. M., Yin, X., Palozola, K. C., Gardini, A., Showe, L. C., et al. (2016) · Cancer Research · doi:10.1158/0008-5472.can-16-0854 · Cited by 142 · see citing works
“Quantitative PCR was performed by using Quantitect Probe PCR Master Mix (Qiagen) with custom probe and primers as described previously (18).”
MethodWu, D., Sunkel, B. D., Chen, Z., Liu, X., Ye, Z., Li, Q., Grenade, C., Ke, J., Zhang, C., Chen, H., et al. (2014) · Nucleic Acids Research · doi:10.1093/nar/gkt1382 · Cited by 138 · see citing works
“We asked whether GATA2 and FoxA1 are required in regulatory loop formation for androgen-dependent AR target gene expression by performing 3C assays (59) at the ABCC4 locus in LNCaP cells.”
MethodRole of LDB1 in the transition from chromatin looping to transcription activation
Krivega, I., Dale, R. K., & Dean, A. (2014) · Genes & Development · doi:10.1101/gad.239749.114 · Cited by 125 · see citing works
Medvedovic, J., Ebert, A., Tagoh, H., Tamir, I., Schwickert, T. A., Novatchkova, M., Sun, Q., Veld, P. J. H. I. ’., Guo, C., Yoon, H. S., et al. (2013) · Immunity · doi:10.1016/j.immuni.2013.08.011 · Cited by 136 · see citing works
“3C-qPCR Analysis 3C templates of pro-B cells were prepared and subjected to TaqMan PCR analysis with primers listed in Table S2, as described (Hagège et al., 2007).”
MethodA critical assessment of topologically associating domain prediction tools
Dali, R., & Blanchette, M. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx145 · Cited by 139 · see citing works
Palstra, R.-J. T. S., Simonis, M., Klous, P., Brasset, É., Eijkelkamp, B. A., & de Laat, W. L. (2008) · PLoS ONE · doi:10.1371/journal.pone.0001661 · Cited by 134 · see citing works
“We applied an improved version of 3C, 3C-qPCR [8,29] to compare the conformation of the bglobin locus between E14.”
MethodSelf-citationMED25 connects enhancer–promoter looping and MYC2-dependent activation of jasmonate signalling
Wang, H., Li, S.-Y., Li, Y., Xu, Y.-R., Wang, Y.-H., Zhang, R., Sun, W.-J., Chen, Q., Wang, X.-J., Li, C.-Y., et al. (2019) · Nature Plants · doi:10.1038/s41477-019-0441-9 · Cited by 115 · see citing works
Deep conservation of wrist and digit enhancers in fish
Gehrke, A. R., Schneider, I., de la Calle‐Mustienes, E., Tena, J. J., Gómez-Marín, C., Chandran, M., Nakamura, T., Braasch, I., Postlethwait, J. H., Gómez-Skármeta, J. L., et al. (2014) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1420208112 · Cited by 133 · see citing works
Super-Enhancers at the Nanog Locus Differentially Regulate Neighboring Pluripotency-Associated Genes
Blinka, S. M., Reimer, M. H., Pulakanti, K., & Rao, S. (2016) · Cell Reports · doi:10.1016/j.celrep.2016.09.002 · Cited by 128 · see citing works
Andrey, G., Schöpflin, R., Jerković, I., Heinrich, V., Ibrahim, D. M., Paliou, C., Hochradel, M., Timmermann, B., Haas, S. A., Vingron, M., et al. (2016) · Genome Research · doi:10.1101/gr.213066.116 · Cited by 122 · see citing works
“The 3C library was prepared according to Hagege et al. (2007), i.e., crosslinking, cell lysis, DpnII digestion, ligation, and de-crosslinking.”
MethodPhospho‐MED1‐enhanced UBE2C locus looping drives castration‐resistant prostate cancer growth
Chen, Z., Zhang, C., Wu, D., Chen, H., Rorick, A. M., Zhang, X., & Wang, Q. (2011) · The EMBO Journal · doi:10.1038/emboj.2011.154 · Cited by 128 · see citing works
Wu, Q., Sun, L., Hu, X., Wang, X., Xu, F., Chen, B., Liang, X., Xia, J., Wang, P., Aibara, D., et al. (2021) · Journal of Clinical Investigation · doi:10.1172/jci142865 · Cited by 131 · see citing works
“A 3C assay was conducted as described previously (54).”
MethodZheng, C., Zhang, B., Li, Y., Liu, K., Wei, W., Liang, S., Guo, H., Ma, K., Liu, Y., Wang, J., et al. (2023) · Advanced Science · doi:10.1002/advs.202206798 · Cited by 130 · see citing works
Chakraborty, S., Pramanik, J., & Mahata, B. (2021) · Genes and Immunity · doi:10.1038/s41435-021-00139-3 · Cited by 130 · see citing works
EBV Latency Types Adopt Alternative Chromatin Conformations
Tempera, I., Klichinsky, M., & Lieberman, P. M. (2011) · PLoS Pathogens · doi:10.1371/journal.ppat.1002180 · Cited by 128 · see citing works
Acemel, R. D., Tena, J. J., Irastorza-Azcárate, I., Marlétaz, F., Gómez-Marín, C., de la Calle‐Mustienes, E., Bertrand, S., Díaz, S. G., Aldea, D., Aury, J., et al. (2016) · Nature Genetics · doi:10.1038/ng.3497 · Cited by 128 · see citing works
PU.1 Level-Directed Chromatin Structure Remodeling at the Irf8 Gene Drives Dendritic Cell Commitment
Schönheit, J., Kuhl, C., Gebhardt, M. L., Klett, F. F., Riemke, P., Scheller, M., Huang, G., Naumann, R., Leutz, A., Stocking, C., et al. (2013) · Cell Reports · doi:10.1016/j.celrep.2013.04.007 · Cited by 127 · see citing works
MED23 Mutation Links Intellectual Disability to Dysregulation of Immediate Early Gene Expression
Hashimoto, S., Boissel, S., Zarhrate, M., Rio, M., Münnich, A., Egly, J., & Colleaux, L. (2011) · Science · doi:10.1126/science.1206638 · Cited by 125 · see citing works
Ryan, R. J. H., Drier, Y., Whitton, H. J., Cotton, M. J., Kaur, J., Issner, R., Gillespie, S. M., Epstein, C. B., Nardi, V., Sohani, A. R., et al. (2015) · Cancer Discovery · doi:10.1158/2159-8290.cd-15-0370 · Cited by 124 · see citing works
“The 3C analysis was performed on 10 million cultured cells or 20 to 40 mg of 25 micron–sectioned frozen tissue according to published protocols (50).”
MethodComet, I. V., Schuettengruber, B., Sexton, T., & Cavalli, G. (2011) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1002059108 · Cited by 124 · see citing works
Dong, M., Zhang, Y., Chen, M., Tan, Y., Min, J., He, X., Liu, F., Gu, J., Jiang, H., Zheng, L., et al. (2024) · Acta Pharmaceutica Sinica B · doi:10.1016/j.apsb.2024.03.008 · Cited by 126 · see citing works
Li, H., Müller, M., Bahechar, I. A., Kyrchanova, O., Ohno, K., Georgiev, P. G., & Pirrotta, V. (2010) · Molecular and Cellular Biology · doi:10.1128/mcb.00849-10 · Cited by 118 · see citing works
“as described previously (7, 14) with few modifications.”
MethodZhang, X., Sallari, R. C., Bailey, S. D., Moore, J. H., & Lupien, M. (2012) · Genome Research · doi:10.1101/gr.135665.111 · Cited by 121 · see citing works
“Chromosome conformation capture (3C) technology coupled to qPCR was performed according to a published protocol (Hagege et al. 2007).”
MethodInfluentialTakayama, K., Misawa, A., Suzuki, T., Takagi, K., Hayashizaki, Y., Fujimura, T., Homma, Y., Takahashi, S., Urano, T., & Inoue, S. (2015) · Nature Communications · doi:10.1038/ncomms9219 · Cited by 119 · see citing works
- Authors
- Hagège, H., Klous, P., Braem, C., Splinter, E., Dekker, J., Cathala, G., de Laat, W., & Forné, T.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- Erasmus MC (NL)
- University of Massachusetts Chan Medical School (US)
- Corresponding author
- Forné, Thierry
- Venue
- Nature Protocols
- Published
- 2007-07
- Volume
- 2
- Issue
- 7
- Pages
- 1722-1733
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2048002474
- Cited by
- 720
- References
- 24
- Citations by year
- 2026: 19 · 2025: 42 · 2024: 22 · 2023: 43 · 2022: 50 · 2021: 45 · 2020: 38 · 2019: 40 · 2018: 52 · 2017: 44 · 2016: 46 · 2015: 50 · 2014: 45 · 2013: 41 · 2012: 43
- FWCI
- 6.95
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- eScholarship@UMassChan (submittedVersion)
- HAL (submittedVersion)
- Funders
- Nederlandse Organisatie voor Wetenschappelijk Onderzoek
- Centre National de la Recherche Scientifique
- National Institutes of Health
- National Human Genome Research Institute (HG003143, R01HG003143)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Chromosomal and Genetic Variations
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
- Keywords
- chromosome conformation capture, TaqMan real-time PCR, genome folding, enhancers, insulators, gene expression regulation, formaldehyde crosslinking
- MeSH terms
- Animals, Chromatin, Chromosomes, Formaldehyde, Genes, Mammals, Models, Molecular, Templates, Genetic, Restriction Mapping, Polymerase Chain Reaction, DNA Primers
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