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In Cellulo DNA Analysis: LMPCR Footprinting

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DNA-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1334))

Abstract

The in cellulo analysis of protein-DNA interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene expression. The nuclease-hypersensitive sites and sequences bound by transcription factors often correspond to genetic regulatory elements. Using the ligation-mediated polymerase chain reaction (LMPCR) technology, it is possible to precisely analyze these DNA sequences to demonstrate the existence of DNA-protein interactions or unusual DNA structures directly in living cells. Indeed, the ideal chromatin substrate is, of course, found inside intact cells. LMPCR, a genomic sequencing technique that map DNA single-strand breaks at the sequence level of resolution, is the method of choice for in cellulo footprinting and DNA structure studies because it can be used to investigate complex animal genomes, including human. The detailed conventional and automated LMPCR protocols are presented in this chapter.

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References

  1. Pfeifer GP, Tanguay RL, Steigerwald SD, Riggs AD (1990) In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes Dev 4:1277–1287

    Article  CAS  PubMed  Google Scholar 

  2. Pfeifer GP, Drouin R, Riggs AD, Holmquist GP (1991) In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6-4) pyrimidone photoproducts by ligation-mediated polymerase chain reaction. Proc Natl Acad Sci U S A 88:1374–1378

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Chen CJ, Li LJ, Maruya A, Shively JE (1995) In vitro and in vivo footprint analysis of the promoter of carcinoembryonic antigen in colon carcinoma cells: effects of interferon gamma treatment. Cancer Res 55:3873–3882

    CAS  PubMed  Google Scholar 

  4. Tornaletti S, Pfeifer GP (1995) UV light as a footprinting agent: modulation of UV-induced DNA damage by transcription factors bound at the promoters of three human genes. J Mol Biol 249:714–728

    Article  CAS  PubMed  Google Scholar 

  5. Pfeifer GP, Riggs AD (1991) Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR. Genes Dev 5:1102–1113

    Article  CAS  PubMed  Google Scholar 

  6. Mueller PR, Wold B (1989) In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246:780–786

    Article  CAS  PubMed  Google Scholar 

  7. Pfeifer GP, Steigerwald SD, Mueller PR, Wold B, Riggs AD (1989) Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246:810–813

    Article  CAS  PubMed  Google Scholar 

  8. Pfeifer GP, Drouin R, Riggs AD, Holmquist GP (1992) Binding of transcription factors creates hot spots for UV photoproducts in vivo. Mol Cell Biol 12:1798–1804

    PubMed Central  CAS  PubMed  Google Scholar 

  9. Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci U S A 81:1991–1995

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Pfeifer GP (1992) Analysis of chromatin structure by ligation-mediated PCR. PCR Methods Appl 2:107–111

    Article  CAS  PubMed  Google Scholar 

  11. Pfeifer GP, Riggs AD (1993) Genomic footprinting by ligation mediated polymerase chain reaction. In: White B (ed) PCR protocols: Current Methods and Applications. Humana, Totowa, NJ, pp 169–181

    Google Scholar 

  12. Pfeifer GP, Riggs AD (1993) Genomic sequencing, Methods Mol. Biol 23:169–181

    CAS  Google Scholar 

  13. Pfeifer GP, Singer-Sam J, Riggs AD (1993) Analysis of methylation and chromatin structure. Methods Enzymol 225:567–583

    Article  CAS  PubMed  Google Scholar 

  14. Gao S, Drouin R, Holmquist GP (1994) DNA repair rates mapped along the human PGK1 gene at nucleotide resolution. Science 263:1438–1440

    Article  CAS  PubMed  Google Scholar 

  15. Tornaletti S, Pfeifer GP (1994) Slow repair of pyrimidine dimers at p53 mutation hotspots in skin cancer. Science 263:1436–1438

    Article  CAS  PubMed  Google Scholar 

  16. Rodriguez H, Drouin R, Holmquist GP, O’Connor TR, Boiteux S, Laval J, Doroshow JH, Akman SA (1995) Mapping of copper/hydrogen peroxide-induced DNA damage at nucleotide resolution in human genomic DNA by ligation-mediated polymerase chain reaction. J Biol Chem 270:17633–17640

    Article  CAS  PubMed  Google Scholar 

  17. Drouin R, Therrien JP (1997) UVB-induced cyclobutane pyrimidine dimer frequency correlates with skin cancer mutational hotspots in p53. Photochem Photobiol 66:719–726

    Article  CAS  PubMed  Google Scholar 

  18. Drouin R, Therrien JP, Angers M, Ouellet S (2001) In vivo DNA analysis. In: Moss T (ed) Methods in molecular Biology. Humana Press Inc, Totowa, NJ, pp 175–219

    Google Scholar 

  19. Dai SM, Chen HH, Chang C, Riggs AD, Flanagan SD (2000) Ligation-mediated PCR for quantitative in vivo footprinting. Nat Biotechnol 18:1108–1111

    Article  CAS  PubMed  Google Scholar 

  20. Dai SM, O’Connor TR, Holmquist GP, Riggs AD, Flanagan SD (2002) Ligation-mediated PCR: robotic liquid handling for DNA damage and repair. Biotechniques 33:1090–1097

    CAS  PubMed  Google Scholar 

  21. Cartwright IL, Kelly SE (1991) Probing the nature of chromosomal DNA-protein contacts by in vivo footprinting. Biotechniques 11(188–190):192–184, 196 passim

    Google Scholar 

  22. Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560

    Article  CAS  PubMed  Google Scholar 

  23. Chin PL, Momand J, Pfeifer GP (1997) In vivo evidence for binding of p53 to consensus binding sites in the p21 and GADD45 genes in response to ionizing radiation. Oncogene 15:87–99

    Article  CAS  PubMed  Google Scholar 

  24. Angers M, Drouin R, Bachvarova M, Paradis I, Marceau F, Bachvarov DR (2000) In vivo protein-DNA interactions at the kinin B(1) receptor gene promoter: no modification on interleukin-1 beta or lipopolysaccharide induction. J Cell Biochem 78:278–296

    Article  CAS  PubMed  Google Scholar 

  25. Becker MM, Wang JC (1984) Use of light for footprinting DNA in vivo. Nature 309:682–687

    Article  CAS  PubMed  Google Scholar 

  26. Pfeifer GP, Tornaletti S (1997) Footprinting with UV irradiation and LMPCR. Methods 11:189–196

    Article  CAS  PubMed  Google Scholar 

  27. Pfeifer GP, Chen HH, Komura J, Riggs AD (1999) Chromatin structure analysis by ligation-mediated and terminal transferase-mediated polymerase chain reaction. Methods Enzymol 304:548–571

    Article  CAS  PubMed  Google Scholar 

  28. Cadet J, Anselmino C, Douki T, Voituriez L (1992) Photochemistry of nucleic acids in cells. J Photochem Photobiol B 15:277–298

    Article  CAS  PubMed  Google Scholar 

  29. Mitchell DL, Nairn RS (1989) The biology of the (6-4) photoproduct. Photochem Photobiol 49:805–819

    Article  CAS  PubMed  Google Scholar 

  30. Holmquist GP, Gao S (1997) Somatic mutation theory, DNA repair rates, and the molecular epidemiology of p53 mutations. Mutat Res 386:69–101

    Article  CAS  PubMed  Google Scholar 

  31. Gale JM, Nissen KA, Smerdon MJ (1987) UV-induced formation of pyrimidine dimers in nucleosome core DNA is strongly modulated with a period of 10.3 bases. Proc Natl Acad Sci U S A 84:6644–6648

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Gale JM, Smerdon MJ (1990) UV induced (6-4) photoproducts are distributed differently than cyclobutane dimers in nucleosomes. Photochem Photobiol 51:411–417

    Article  CAS  PubMed  Google Scholar 

  33. Mitchell DL, Nguyen TD, Cleaver JE (1990) Nonrandom induction of pyrimidine-pyrimidone (6-4) photoproducts in ultraviolet-irradiated human chromatin. J Biol Chem 265:5353–5356

    CAS  PubMed  Google Scholar 

  34. Rigaud G, Roux J, Pictet R, Grange T (1991) In vivo footprinting of rat TAT gene: dynamic interplay between the glucocorticoid receptor and a liver-specific factor. Cell 67:977–986

    Article  CAS  PubMed  Google Scholar 

  35. Miller MR, Castellot JJ Jr, Pardee AB (1978) A permeable animal cell preparation for studying macromolecular synthesis. DNA synthesis and the role of deoxyribonucleotides in S phase initiation. Biochemistry 17:1073–1080

    Article  CAS  PubMed  Google Scholar 

  36. Contreras R, Fiers W (1981) Initiation of transcription by RNA polymerase II in permeable, SV40-infected or noninfected, CVI cells; evidence for multiple promoters of SV40 late transcription. Nucleic Acids Res 9:215–236

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Tanguay RL, Pfeifer GP, Riggs AD (1990) PCR-aided DNaseI footprinting of single copy gene sequences in permeabilized cells. Nucleic Acids Res 18:5902

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Tormanen VT, Swiderski PM, Kaplan BE, Pfeifer GP, Riggs AD (1992) Extension product capture improves genomic sequencing and DNase I footprinting by ligation-mediated PCR. Nucleic Acids Res 20:5487–5488

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Rozek D, Pfeifer GP (1993) In vivo protein-DNA interactions at the c-jun promoter: preformed complexes mediate the UV response. Mol Cell Biol 13:5490–5499

    PubMed Central  CAS  PubMed  Google Scholar 

  40. Tornaletti S, Bates S, Pfeifer GP (1996) A high-resolution analysis of chromatin structure along p53 sequences. Mol Carcinog 17:192–201

    Article  CAS  PubMed  Google Scholar 

  41. Szabo PE, Pfeifer GP, Mann JR (1998) Characterization of novel parent-specific epigenetic modifications upstream of the imprinted mouse H19 gene. Mol Cell Biol 18:6767–6776

    PubMed Central  CAS  PubMed  Google Scholar 

  42. Garrity PA, Wold BJ (1992) Effects of different DNA polymerases in ligation-mediated PCR: enhanced genomic sequencing and in vivo footprinting. Proc Natl Acad Sci U S A 89:1021–1025

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  43. Hornstra IK, Yang TP (1994) High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5′ region on the active and inactive X chromosomes: correlation with binding sites for transcription factors. Mol Cell Biol 14:1419–1430

    PubMed Central  CAS  PubMed  Google Scholar 

  44. Angers M, Cloutier JF, Castonguay A, Drouin R (2001) Optimal conditions to use Pfu exo(-) DNA polymerase for highly efficient ligation-mediated polymerase chain reaction protocols. Nucleic Acids Res 29, E83

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Rouget R, Vigneault F, Codio C, Rochette C, Paradis I, Drouin R, Simard LR (2005) Characterization of the survival motor neuron (SMN) promoter provides evidence for complex combinatorial regulation in undifferentiated and differentiated P19 cells. Biochem J 385:433–443

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. Vigneault F, Drouin R (2005) Optimal conditions and specific characteristics of Vent exo- DNA polymerase in ligation-mediated polymerase chain reaction protocols. Biochem Cell Biol 83:147–165

    Article  CAS  PubMed  Google Scholar 

  47. Drouin R, Gao S, Holmquist GP (1996) Agarose gel electrophoresis for DNA damage analysis. In: Pfeifer GP (ed) Technologies for Detection of DNA Damage and Mutations. Plenum, New York, pp 37–43

    Chapter  Google Scholar 

  48. Drouin R, Rodriguez H, Holmquist GP, Akman SA (1996) Ligation-mediated PCR for analysis of oxidative damage. In: Pfeifer GP (ed) Technologies for Detection of DNA Damage and Mutations. Plenum, New York, pp 211–225

    Chapter  Google Scholar 

  49. Mueller PR, Wold B (1991) Ligation-mediated PCR: applications to genomic footprinting. Methods 20–31

    Google Scholar 

  50. Iverson BL, Dervan PB (1987) Adenine specific DNA chemical sequencing reaction. Nucleic Acids Res 15:7823–7830

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  51. Zhang L, Gralla JD (1989) In situ nucleoprotein structure at the SV40 major late promoter: melted and wrapped DNA flank the start site. Genes Dev 3:1814–1822

    Article  CAS  PubMed  Google Scholar 

  52. Fry M, Loeb LA (1994) The fragile X syndrome d(CGG)n nucleotide repeats form a stable tetrahelical structure. Proc Natl Acad Sci U S A 91:4950–4954

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. Rychlik W (1993) Selection of primers for polymerase chain reaction. In: White B (ed) PCR protocols: Current Methods and Applications. Humana, Totowa, NJ, pp 31–40

    Chapter  Google Scholar 

  54. Hornstra IK, Yang TP (1992) Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanthine phosphoribosyltransferase gene 5′ region: implications for X chromosome inactivation. Mol Cell Biol 12:5345–5354

    PubMed Central  CAS  PubMed  Google Scholar 

  55. Hornstra IK, Yang TP (1993) In vivo footprinting and genomic sequencing by ligation-mediated PCR. Anal Biochem 213:179–193

    Article  CAS  PubMed  Google Scholar 

  56. McLellan JA (2001) Osmium tetroxide modification and the study of DNA-protein interactions. Methods Mol Biol 148:121–34

    Google Scholar 

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Acknowledgments

This work was supported by the Canadian Genetic Diseases Network (MRC/NSERC NCE program) and the Canada Research Chair. R. Drouin holds the Canada Research Chair in “Genetics, Mutagenesis and Cancer.”

Author information

Authors and Affiliations

  1. Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada

    Régen Drouin M.D., Ph.D., Nathalie Bastien & Isabelle Paradis

  2. Division of Genetics, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12th Avenue North, Sherbrooke, QC, Canada, J1H 5N4

    Régen Drouin M.D., Ph.D.

  3. Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada

    Jean-François Millau

  4. Department of Genetics, Harvard Medical School, Boston, MA, USA

    François Vigneault

Authors
  1. Régen Drouin M.D., Ph.D.
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  2. Nathalie Bastien
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  3. Jean-François Millau
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  4. François Vigneault
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  5. Isabelle Paradis
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Corresponding author

Correspondence to Régen Drouin M.D., Ph.D. .

Editor information

Editors and Affiliations

  1. Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada

    Benoît P. Leblanc

  2. Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada

    Sébastien Rodrigue

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Drouin, R., Bastien, N., Millau, JF., Vigneault, F., Paradis, I. (2015). In Cellulo DNA Analysis: LMPCR Footprinting. In: Leblanc, B., Rodrigue, S. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 1334. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2877-4_4

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  • DOI: https://doi.org/10.1007/978-1-4939-2877-4_4

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2876-7

  • Online ISBN: 978-1-4939-2877-4

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