Application of Chromosome Conformation Capture (3C) to the Study of Human Genetic Disease

Abstract

Chromosome conformation capture (3C) is a powerful technique that allows for the generation of 3‐dimensional transcriptome organisational maps. 3C facilitates the identification and quantitative measurement of distant chromosomal regulatory elements with proximal gene promoter regions. This is achieved by fixing protein onto deoxyribonucleic acid (DNA) followed by restriction enzyme digestion or sonication to shear the DNA prior to re‐ligation of the fixed DNA region. PCR amplification of a chromosomal region of interest can then be performed and quantified. Next generation sequencing platforms on prepared 3C chromatin can provide information on whole genome regulatory networks. The technology and its modified derivatives provide high levels of resolution and throughput for determining nuclear organisation and allows for the identification of novel noncoding DNA regions that are associated with controlling gene expression. 3C is providing important insights into underlying mechanisms that may be responsible for various human diseases including cancer, muscular dystrophies, neurological and metabolic disorders.

Key Concepts:

  • Chromosome conformation capture (3C) is a key strategy used to determine distant chromosomal regulatory regions involved with controlling gene expression.

  • Single nucleotide polymorphic (SNP) DNA variations in noncoding region DNA can now be interrogated to determine a functional role in altering gene expression.

  • The 3‐dimensional chromosome looping architecture can be dissected to determine multiple chromosomal regions involved with regulating gene expression through transcription factory interaction.

  • Mutations in distant chromosomal regulatory regions may lead to uncontrolled repression of oncogenes allowing for increases in tumour growth and development.

  • Identifying noncoding chromosomal regions associated with nuclear adaptive mechanisms in response to DNA mutation may allow us to determine critical regions involved with maintaining cellular homoeostasis.

  • High‐throughput sequencing strategies are allowing for unparalleled identification of DNA variation in the human genome.

Keywords: chromosome conformation capture; gene regulation; SNP; cancer; human disease; mutation; malfunction; enhancers; repressors

Figure 1.

Chromosome conformation capture. (a) Linear chromosomal DNA containing protein‐binding regions of interest. (b) Looping of chromosome allows interaction of upstream and proximal promoter region proteins. Cross‐linking with formaldehyde fixes proteins in position. (c) Restriction enzyme digestion of cross‐linked DNA produces cohesive overlapping ends. (d) Ligation of protein cross‐linked DNA. (e) Reversal of cross‐linking to remove DNA bound proteins. (f) PCR to determine cross‐linking of upstream region with the proximal promoter region.

Figure 2.

The ratio of noncoding DNA to total genomic DNA (ncDNA/tgDNA) increases with species complexity. Figure is simplified from Taft et al. .

close

References

Barnett DH, Sheng S, Charn TH et al. (2008) Estrogen receptor regulation of carbonic anhydrase XII through a distal enhancer in breast cancer. Cancer Research 68: 3505–3515.

Bau D, Sanyal A, Lajoie BR et al. (2011) The three‐dimensional folding of the alpha‐globin gene domain reveals formation of chromatin globules. Nature Structural & Molecular Biology 18: 107–114.

Bodega B, Ramirez GD, Grasser F et al. (2009) Remodeling of the chromatin structure of the facioscapulohumeral muscular dystrophy (FSHD) locus and upregulation of FSHD‐related gene 1 (FRG1) expression during human myogenic differentiation. BMC Biology 7: 41.

Collins AL, Levenson JM, Vilaythong AP et al. (2004) Mild overexpression of MeCP2 causes a progressive neurological disorder in mice. Human Molecular Genetics 13: 2679–2689.

Davison LJ, Wallace C, Cooper JD et al. (2012) Long‐range DNA looping and gene expression analyses identify DEXI as an autoimmune disease candidate gene. Human Molecular Genetics 21: 322–333.

D'Haene B, Attanasio C, Beysen D et al. (2009) Disease‐causing 7.4 kb cis‐regulatory deletion disrupting conserved non‐coding sequences and their interaction with the FOXL2 promotor: implications for mutation screening. PLoS Genetics 5: e1000522.

Dekker J, Rippe K, Dekker M and Kleckner N (2002) Capturing chromosome conformation. Science 295: 1306–1311.

Deshane J, Kim J, Bolisetty S et al. (2010) Sp1 regulates chromatin looping between an intronic enhancer and distal promoter of the human heme oxygenase‐1 gene in renal cells. Journal of Biological Chemistry 285: 16476–16486.

Dhar SS, Ongwijitwat S and Wong‐Riley MTT (2009) Chromosome conformation capture of all 13 genomic loci in the transcriptional regulation of the multisubunit bigenomic cytochrome c oxidase in neurons. Journal of Biological Chemistry 284: 18644–18650.

Dostie J, Richmond TA, Arnaout RA et al. (2006) chromosome conformation capture carbon copy (5C): a massively parallel solution for mapping interactions between genomic elements. Genome Research 16: 1299–1309.

Fraser J, Rousseau M, Shenker S et al. (2009) Chromatin conformation signatures of cellular differentiation. Genome Biology 10: R37.

Fullwood MJ, Liu MH, Pan YF et al. (2009) An oestrogen‐receptor‐alpha‐bound human chromatin interactome. Nature 462: 58–64.

van Geel M, van Deutekom JC, van Staalduinen A et al. (2000) Identification of a novel beta‐tubulin subfamily with one member (TUBB4Q) located near the telomere of chromosome region 4q35. Cytogenetics and Cell Genetics 88: 316–321.

Gonzales ML and LaSalle JM (2010) The role of MeCP2 in brain development and neurodevelopmental disorders. Current Psychiatry Reports 12: 127–134.

de Greef JC, Lemmers RJ, Camano P et al. (2010) Clinical features of facioscapulohumeral muscular dystrophy 2. Neurology 75: 1548–1554.

Guan Y, Kuo WL, Stilwell JL et al. (2007) Amplification of PVT1 contributes to the pathophysiology of ovarian and breast cancer. Clinical Cancer Research 13: 5745–5755.

Handoko L, Xu H, Li G et al. (2011) CTCF‐mediated functional chromatin interactome in pluripotent cells. Nature Genetics 43: 630–638.

Hanel ML, Wuebbles RD and Jones PL (2009) Muscular dystrophy candidate gene FRG1 is critical for muscle development. Developmental Dynamics 238: 1502–1512.

Horike S, Cai S, Miyano M, Cheng JF and Kohwi‐Shigematsu T (2005) Loss of silent‐chromatin looping and impaired imprinting of DLX5 in Rett syndrome. Nature Genetics 37: 31–40.

Howard ML and Davidson EH (2004) cis‐Regulatory control circuits in development. Developmental Biology 271: 109–118.

Huang L, Yu D, Wu C et al. (2012) Copy number variation at 6q13 functions as a long‐range regulator and is associated with pancreatic cancer risk. Carcinogenesis 33: 94–100.

Hwang S, Kwak SH, Bhak J et al. (2011) Gene expression pattern in transmitochondrial cytoplasmic hybrid cells harboring type 2 diabetes‐associated mitochondrial DNA haplogroups. PLoS One 6: e22116.

Jang JY, Choi Y, Jeon YK, Aung KC and Kim CW (2008) Over‐expression of adenine nucleotide translocase 1 (ANT1) induces apoptosis and tumor regression in vivo. BMC Cancer 8: 160.

Jemal A, Bray F, Center MM et al. (2011) Global cancer statistics. CA: A Cancer Journal for Clinicians 61: 69–90.

Laoudj‐Chenivesse D, Carnac G, Bisbal C et al. (2005) Increased levels of adenine nucleotide translocator 1 protein and response to oxidative stress are early events in facioscapulohumeral muscular dystrophy muscle. Journal of Molecular Medicine (Berlin) 83: 216–224.

Lemmers RJ, de Kievit P, Sandkuijl L et al. (2002) Facioscapulohumeral muscular dystrophy is uniquely associated with one of the two variants of the 4q subtelomere. Nature Genetics 32: 235–236.

Li G, Thomas AM, Hart SN et al. (2010) Farnesoid X receptor activation mediates head‐to‐tail chromatin looping in the Nr0b2 gene encoding small heterodimer partner. Molecular Endocrinology 24: 1404–1412.

Lieberman‐Aiden E, van Berkum NL, Williams L et al. (2009) Comprehensive mapping of long‐range interactions reveals folding principles of the human genome. Science 326: 289–293.

Ling JQ, Li T, Hu JF et al. (2006) CTCF mediates interchromosomal colocalization between Igf2/H19 and Wsb1/Nf1. Science 312: 269–272.

Liu J and Francke U (2006) Identification of cis‐regulatory elements for MECP2 expression. Human Molecular Genetics 15: 1769–1782.

Meyer KB, Maia AT, O'Reilly M et al. (2011) A functional variant at a prostate cancer predisposition locus at 8q24 is associated with PVT1 expression. PLoS Genetics 7: e1002165.

Neul JL, Fang P, Barrish J et al. (2008) Specific mutations in methyl‐CpG‐binding protein 2 confer different severity in Rett syndrome. Neurology 70: 1313–1321.

Osborne CS, Chakalova L, Brown KE et al. (2004) Active genes dynamically colocalize to shared sites of ongoing transcription. Nature Genetics 36: 1065–1071.

Percy AK, Lane JB, Childers J et al. (2007) Rett syndrome: North American database. Journal of Child Neurology 22: 1338–1341.

Pirozhkova I, Petrov A, Dmitriev P et al. (2008) A functional role for 4qA/B in the structural rearrangement of the 4q35 region and in the regulation of FRG1 and ANT1 in facioscapulohumeral dystrophy. PLoS One 3: e3389.

Pittman AM, Naranjo S, Jalava SE et al. (2010) Allelic variation at the 8q23.3 colorectal cancer risk locus functions as a cis‐acting regulator of EIF3H. PLoS Genetics 6: e1001126.

Rett A (1966) On a unusual brain atrophy syndrome in hyperammonemia in childhood. Wien Med Wochenschr 116: 723–726.

Robyr D, Friedli M, Gehrig C et al. (2011) Chromosome conformation capture uncovers potential genome‐wide interactions between human conserved non‐coding sequences. PLoS One 6: e17634.

Savinainen KJ, Helenius MA, Lehtonen HJ and Visakorpi T (2006) Overexpression of EIF3S3 promotes cancer cell growth. Prostate 66: 1144–1150.

Splinter E, de Wit E, Nora EP et al. (2011) The inactive X chromosome adopts a unique three‐dimensional conformation that is dependent on Xist RNA. Genes & Development 25: 1371–1383.

Statland JM and Tawil R (2011) Facioscapulohumeral muscular dystrophy: molecular pathological advances and future directions. Current Opinion in Neurology 24: 423–428.

Stierer M, Rosen H, Weber R et al. (1993) Immunohistochemical and biochemical measurement of estrogen and progesterone receptors in primary breast cancer. Correlation of histopathology and prognostic factors. Annals of Surgery 218: 13–21.

Taft RJ, Pheasant M and Mattick JS (2007) The relationship between non‐protein‐coding DNA and eukaryotic complexity. Bioessays 29: 288–299.

Tang WY, Morey LM, Cheung YY et al. (2012) Neonatal exposure to estradiol/bisphenol A alters promoter methylation and expression of Nsbp1 and Hpcal1 genes and transcriptional programs of Dnmt3a/b and Mbd2/4 in the rat prostate gland throughout life. Endocrinology 153: 42–55.

Van Esch H, Bauters M, Ignatius J et al. (2005) Duplication of the MECP2 region is a frequent cause of severe mental retardation and progressive neurological symptoms in males. American Journal of Human Genetics 77 442–453.

Vu TH, Nguyen AH and Hoffman AR (2010) Loss of IGF2 imprinting is associated with abrogation of long‐range intrachromosomal interactions in human cancer cells. Human Molecular Genetics 19: 901–919.

Watson PH, Chia SK, Wykoff CC et al. (2003) Carbonic anhydrase XII is a marker of good prognosis in invasive breast carcinoma. British Journal of Cancer 88: 1065–1070.

de Wit E and de Laat W (2012) A decade of 3C technologies: insights into nuclear organization. Genes & Development 26: 11–24.

Yasui DH, Scoles HA, Horike S et al. (2011) 15q11.2‐13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain. Human Molecular Genetics 20: 4311–4323.

Zhang Z, Ott CJ, Lewandowska MA, Leir SH and Harris A (2012) Molecular mechanisms controlling CFTR gene expression in the airway. Journal of Cellular and Molecular Medicine 16: 1321–1330.

Zhao B, Zou J, Wang H et al. (2011) Epstein‐Barr virus exploits intrinsic B‐lymphocyte transcription programs to achieve immortal cell growth. Proceedings of the National Academy of Sciences of the USA 108: 14902–14907.

Further Reading

Altshuler DL, Durbin RM, Abecasis GR et al. Genomes Project Consortium (2010) A map of human genome variation from population‐scale sequencing. Nature 467: 1061–1073.

Ariani F, Hayek G, Rondinella D et al. (2008) FOXG1 is responsible for the congenital variant of Rett syndrome. American Journal of Human Genetics 83: 89–93.

Dhar SS, Ongwijitwat S and Wong‐Riley MT (2008) Nuclear respiratory factor 1 regulates all ten nuclear‐encoded subunits of cytochrome c oxidase in neurons. Journal of Biological Chemistry 283: 3120–3129.

Gleyzer N, Vercauteren K and Scarpulla RC (2005) Control of mitochondrial transcription specificity factors (TFB1M and TFB2M) by nuclear respiratory factors (NRF‐1 and NRF‐2) and PGC‐1 family coactivators. Molecular and Cellular Biology 25: 1354–1366.

de Greef JC, Frants RR and van der Maarel SM (2008) Epigenetic mechanisms of facioscapulohumeral muscular dystrophy. Mutation Research 647: 94–102.

Lin C, Franco B and Rosner MR (2005) CDKL5/Stk9 kinase inactivation is associated with neuronal developmental disorders. Human Molecular Genetics 14: 3775–3786.

Miele A and Dekker J (2009) Mapping cis‐ and trans‐ chromatin interaction networks using chromosome conformation capture (3C). Methods in Molecular Biology 464: 105–121.

Ongwijitwat S and Wong‐Riley MT (2005) Is nuclear respiratory factor 2 a master transcriptional coordinator for all ten nuclear‐encoded cytochrome c oxidase subunits in neurons? Gene 360: 65–77.

Palstra RJ (2009) Close encounters of the 3C kind: long‐range chromatin interactions and transcriptional regulation. Briefings in Functional Genomics and Proteomics 8: 297–309.

Simonis M, Kooren J and de Laat W (2007) An evaluation of 3C‐based methods to capture DNA interactions. Nature Methods 4: 895–901.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Horan, Martin P, and Ballard, J William O(Sep 2012) Application of Chromosome Conformation Capture (3C) to the Study of Human Genetic Disease. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0024383]