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Chromosome Conformation Capture on Chip (4C): Data Processing

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Polycomb Group Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1480))

Abstract

4C methods are useful to investigate dependencies between regulatory mechanisms and chromatin structures by revealing the frequency of chromatin contacts between a locus of interest and remote sequences on the chromosome. In this chapter we describe a protocol for the data analysis of microarray-based 4C experiments, presenting updated versions of the methods we used in a previous study of the large-scale chromatin interaction profile of a Polycomb response element in Drosophila. The protocol covers data preparation, normalization, microarray probe selection, and the multi-resolution detection of regions with enriched chromatin contacts. A reanalysis of two independent mouse datasets illustrates the versatility of this protocol and the importance of data processing in 4C. Methods were implemented in the R package MRA.TA (Multi-Resolution Analyses on Tiling Array data), and they can be used to analyze ChIP-on-chip data on broadly distributed chromatin components such as histone marks.

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References

  1. Dekker J, Rippe K, Dekker M et al ((2002) Capturing chromosome conformation. Science 295:1306–1311

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  3. Sexton T, Cavalli G (2015) The role of chromosome domains in shaping the functional genome. Cell 160:1049–1059

    Article  CAS  PubMed  Google Scholar 

  4. Marti-Renom MA, Mirny LA (2011) Bridging the resolution gap in structural modeling of 3D genome organization. PLoS Comput Biol 7:e1002125

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Comet I, Savitskaya E, Schuettengruber B et al (2006) PRE-mediated bypass of two Su(Hw) insulators targets PcG proteins to a downstream promoter. Dev Cell 11:117–124

    Article  CAS  PubMed  Google Scholar 

  6. Lanzuolo C, Roure V, Dekker J et al (2007) Polycomb response elements mediate the formation of chromosome higher-order structures in the bithorax complex. Nat Cell Biol 9:1167–1174

    Article  CAS  PubMed  Google Scholar 

  7. Sexton T, Yaffe E, Kenigsberg E et al (2012) Three-dimensional folding and functional organization principles of the Drosophila genome. Cell 148(3):458–472

    Article  CAS  PubMed  Google Scholar 

  8. Rao SSP, Huntley MH, Durand NC et al (2014) A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159:1665–1680

    Article  CAS  PubMed  Google Scholar 

  9. Dixon JR, Selvaraj S, Yue F et al (2012) Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485:1–5

    Article  Google Scholar 

  10. Nora EP, Lajoie BR, Schulz EG et al (2012) Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485:381–385

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhao Z, Tavoosidana G, Sjölinder M et al (2006) Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat Genet 38:1341–1347

    Article  CAS  PubMed  Google Scholar 

  12. Göndör A, Rougier C, Ohlsson R (2008) High-resolution circular chromosome conformation capture assay. Nat Protoc 3:303–313

    Article  PubMed  Google Scholar 

  13. Simonis M, Klous P, Splinter E et al (2006) Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C). Nat Genet 38:1348–1354

    Article  CAS  PubMed  Google Scholar 

  14. Schoenfelder S, Sexton T, Chakalova L et al (2009) Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells. Nat Genet 42:53–61

    Article  PubMed  PubMed Central  Google Scholar 

  15. Bantignies F, Roure V, Comet I et al (2011) Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila. Cell 144:214–226

    Article  CAS  PubMed  Google Scholar 

  16. Lomvardas S, Barnea G, Pisapia DJ et al (2006) Interchromosomal interactions and olfactory receptor choice. Cell 126:403–413

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. Sexton T, Kurukuti S, Mitchell JA et al (2012) Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip. Nat Protoc 7:1335–1350

    Article  CAS  PubMed  Google Scholar 

  19. Tolhuis B, Blom M, van Lohuizen M (2012) Chromosome conformation capture on chip in single Drosophila melanogaster tissues. Methods 58:231–242

    Article  CAS  PubMed  Google Scholar 

  20. 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yang YH, Dudoit S, Luu P et al (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30:e15

    Article  PubMed  PubMed Central  Google Scholar 

  22. Cleveland WS (1979) Robust locally weighted regression and smoothing scatterplots. J Am Stat Assoc 74:829–836

    Article  Google Scholar 

  23. Palstra R-J, Simonis M, Klous P et al (2008) Maintenance of long-range DNA interactions after inhibition of ongoing RNA polymerase II transcription. PloS One 3:e1661

    Article  PubMed  PubMed Central  Google Scholar 

  24. Tolhuis B, Blom M, Kerkhoven RM et al (2011) Interactions among Polycomb domains are guided by chromosome architecture. PLoS Genet 7:e1001343

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Montavon T, Soshnikova N, Mascrez B et al (2011) A regulatory archipelago controls hox genes transcription in digits. Cell 147:1132–1145

    Article  CAS  PubMed  Google Scholar 

  26. Lonfat N, Montavon T, Jebb D et al (2013) Transgene- and locus-dependent imprinting reveals allele-specific chromosome conformations. Proc Natl Acad Sci U S A 110:11946–11951

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ong CT, Van Bortle K, Ramos E et al (2013) XPoly(ADP-ribosyl)ation regulates insulator function and intrachromosomal interactions in drosophila. Cell 155:148–159

    Article  CAS  PubMed  Google Scholar 

  28. de Wit E, Braunschweig U, Greil F et al (2008) Global chromatin domain organization of the Drosophila genome. PLoS Genet 4:e1000045

    Article  PubMed  PubMed Central  Google Scholar 

  29. Noordermeer D, de Wit E, Klous P et al (2011) Variegated gene expression caused by cell-specific long-range DNA interactions. Nat Cell Biol 13:1–10

    Article  Google Scholar 

  30. Yaffe E, Tanay A (2011) Probabilistic modeling of Hi-C contact maps eliminates systematic biases to characterize global chromosomal architecture. Nat Genet 43:1059–1065

    Article  CAS  PubMed  Google Scholar 

  31. van de Werken HJG, Landan G, Holwerda SJB et al (2012) Robust 4C-seq data analysis to screen for regulatory DNA interactions. Nat Methods 9:969–972

    Article  PubMed  Google Scholar 

  32. van de Werken HJG, de Vree PJP, Splinter E et al (2012) 4C technology: protocols and data analysis. Methods Enzymol 513:89–112

    Article  PubMed  Google Scholar 

  33. Splinter E, de Wit E, van de Werken HJG et al (2012) Determining long-range chromatin interactions for selected genomic sites using 4C-seq technology: from fixation to computation. Methods 58:221–230

    Article  CAS  PubMed  Google Scholar 

  34. Thongjuea S, Stadhouders R, Grosveld FG et al (2013) R3Cseq: an R/Bioconductor package for the discovery of long-range genomic interactions from chromosome conformation capture and next-generation sequencing data. Nucleic Acids Res 41:1–12

    Article  Google Scholar 

  35. Walter C, Schuetzmann D, Rosenbauer F et al (2014) Basic4Cseq: an R/Bioconductor package for analyzing 4C-seq data. Bioinformatics 30:3268–3269

    Article  CAS  PubMed  Google Scholar 

  36. Williams RL, Starmer J, Mugford JW et al (2014) FourSig: a method for determining chromosomal interactions in 4C-Seq data. Nucleic Acids Res 42:1–16

    Article  Google Scholar 

  37. 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 Dev 25:1371–1383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. R Core Team (2015) R: a language and environment for statistical computing. http://www.r-project.org/

  39. Gentleman RC, Carey VJ, Bates DM et al (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5:R80

    Article  PubMed  PubMed Central  Google Scholar 

  40. Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25

    Article  PubMed  PubMed Central  Google Scholar 

  41. Edgar R, Domrachev M, Lash AE (2002) Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30:207–210

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Fukunaga K, Hostetler L (1975) The estimation of the gradient of a density function, with applications in pattern recognition. IEEE Trans Inf Theory 21:32–40

    Article  Google Scholar 

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Acknowledgements

We thank Elzo de Wit and Bas Tolhuis for their help sharing source code and biological data, and Tom Sexton for feedback and discussions. B.L. was supported by the Ministère de l’Enseignement Supérieur et de la Recherche and the European Research Council (ERC-2008-AdG No 232947), I.C. was supported by the Ministère de l’Enseignement Supérieur et de la Recherche and by the Ligue contre le Cancer. F.B. and G.C. are supported by the CNRS. G.C. research was supported by grants from the European Research Council (ERC-2008-AdG No 232947), the CNRS, the European Network of Excellence EpiGeneSys, the Agence Nationale de la Recherche, and the Association pour la Recherche sur le Cancer.

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Authors and Affiliations

  1. Institut de Génétique Humaine, CNRS UPR 1142, 141, rue de la Cardonille, 34396, Montpellier, France

    Benjamin Leblanc, Itys Comet, Frédéric Bantignies & Giacomo Cavalli

  2. BRIC - University of Copenhagen, Ole Maaløes Vej 5, DK-2200, Copenhagen N, Denmark

    Benjamin Leblanc & Itys Comet

Authors
  1. Benjamin Leblanc
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  2. Itys Comet
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  3. Frédéric Bantignies
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  4. Giacomo Cavalli
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Corresponding author

Correspondence to Benjamin Leblanc .

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Editors and Affiliations

  1. IBCN-CNR, Institute of Cell Biology and Neurobiology and Istituto Nazionale di Genetica Molecolare “Romeo ed Enrica Invernizzi”, Rome, Italy

    Chiara Lanzuolo

  2. Istituto Nazionale di Genetica Molecolare “Romeo ed Enrica Invernizzi”, Milan, Italy

    Beatrice Bodega

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Leblanc, B., Comet, I., Bantignies, F., Cavalli, G. (2016). Chromosome Conformation Capture on Chip (4C): Data Processing. In: Lanzuolo, C., Bodega, B. (eds) Polycomb Group Proteins. Methods in Molecular Biology, vol 1480. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6380-5_21

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  • DOI: https://doi.org/10.1007/978-1-4939-6380-5_21

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6378-2

  • Online ISBN: 978-1-4939-6380-5

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