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DNase I Digestion of Isolated Nulcei for Genome-Wide Mapping of DNase Hypersensitivity Sites in Chromatin

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 977))

Abstract

DNase I hypersensitivity (DHS) analysis is a powerful method to analyze chromatin structure and identify genomic regulatory elements. Integration of a high-throughput detection method into DHS analysis makes genome-wide mapping of DHS sites possible at a reasonable cost. Here we describe methods for DHS analysis carried out with mouse liver nuclei, involving DNase I digestion followed by isolation of DNase I-released DNA fragments suitable for high-throughput, next generation DNA sequencing (DNase-seq). A real-time PCR-based assay used to optimize DNase I digestion conditions is also described.

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References

  1. Cockerill PN (2011) Structure and function of active chromatin and DNase I hypersensitive sites. FEBS J 278(13):2182–2210, 3060

    Article  PubMed  CAS  Google Scholar 

  2. Lu Q, Richardson B (2004) DNaseI hypersensitivity analysis of chromatin structure. Methods Mol Biol 287:77–86

    PubMed  CAS  Google Scholar 

  3. West AG, Fraser P (2005) Remote control of gene transcription. Hum Mol Genet 14(Spec No 1):R101–R111

    Article  PubMed  CAS  Google Scholar 

  4. Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, Furey TS, Crawford GE (2008) High-resolution mapping and characterization of open chromatin across the genome. Cell 132:311–322

    Article  PubMed  CAS  Google Scholar 

  5. Crawford GE, Davis S, Scacheri PC, Renaud G, Halawi MJ, Erdos MR, Green R, Meltzer PS, Wolfsberg TG, Collins FS (2006) DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays. Nat Methods 3:503–509

    Article  PubMed  CAS  Google Scholar 

  6. Sabo PJ, Kuehn MS, Thurman R, Johnson BE, Johnson EM, Cao H, Yu M, Rosenzweig E, Goldy J, Haydock A, Weaver M, Shafer A, Lee K, Neri F, Humbert R, Singer MA, Richmond TA, Dorschner MO, McArthur M, Hawrylycz M, Green RD, Navas PA, Noble WS, Stamatoyannopoulos JA (2006) Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays. Nat Methods 3:511–518

    Article  PubMed  CAS  Google Scholar 

  7. Song L, Crawford GE (2010) DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells. Cold Spring Harb Protoc 2010:pdb.prot5384

    Article  PubMed  Google Scholar 

  8. ENCODE Project Consortium, Myers RM, Stamatoyannopoulos J, Snyder M, Dunham I, Hardison RC, Bernstein BE, Gingeras TR, Kent WJ, Birney E, Wold B, Crawford GE (2011) A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol 9(4):e1001046

    Article  CAS  Google Scholar 

  9. Ling G, Sugathan A, Mazor T, Fraenkel E, Waxman DJ (2010) Unbiased, genome-wide in vivo mapping of transcriptional regulatory elements reveals sex differences in chromatin structure associated with sex-specific liver gene expression. Mol Cell Biol 30(23):5531–5544

    Article  PubMed  CAS  Google Scholar 

  10. Weis JH, Quertermous T (2001) Size fractionation using sucrose gradients. Curr Protoc Mol Biol, Chapter 5:Unit 5.3

    Google Scholar 

  11. Siersbæk R, Nielsen R, John S, Sung MH, Baek S, Loft A, Hager GL, Mandrup S (2011) Extensive chromatin remodelling and establishment of transcription factor ‘hotspots’ during early adipogenesis. EMBO J 30(8):1459–1472

    Article  PubMed  Google Scholar 

  12. McArthur M, Gerum S, Stamatoyannopoulos G (2001) Quantification of DNaseI-sensitivity by real-time PCR: quantitative analysis of DNaseI-hypersensitivity of the mouse beta-globin LCR. J Mol Biol 313(1):27–34

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

Supported in part by NIH grant DK33765 (to DJW).

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Ling, G., Waxman, D.J. (2013). DNase I Digestion of Isolated Nulcei for Genome-Wide Mapping of DNase Hypersensitivity Sites in Chromatin. In: Bina, M. (eds) Gene Regulation. Methods in Molecular Biology, vol 977. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-284-1_3

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  • DOI: https://doi.org/10.1007/978-1-62703-284-1_3

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-283-4

  • Online ISBN: 978-1-62703-284-1

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