In Vivo Genomic Footprinting Using LM–PCR Methods (original) (raw)

Abstract

Epigenetic regulatory proteins such as transcription factors, chromatin components, and chromatin modification activities alter gene activity during development University of Leeds;. The means by which alterations in these factors influence gene expression is poorly understood, but information of this kind is essential if we want to reprogram the epigenotype of specific cell types in a directed fashion. To facilitate chromatin structure-function analysis, we have developed a relatively simple procedure that uses magnetic beads to perform ligation-mediated polymerase chain reaction in solid phase. In this chapter, we describe detailed procedures for the examination of chromatin fine-structure and nucleosome positioning as well as changes in transcription factor binding-site occupancy during cellular differentiation.

Similar content being viewed by others

References

  1. Kontaraki, J., Chen, H. H., Riggs, A., and Bonifer, C. (2000) Chromatin fine structure profiles for a developmentally regulated gene: reorganization of the lysozyme locus before trans-activator binding and gene expression. Genes Dev. 14, 2106–2122.
    CAS PubMed Google Scholar
  2. Tagoh, H., Himes, R., Clarke, D., Leenen, P. J., Riggs, A. D., Hume, D., et al. (2002) Transcription factor complex formation and chromatin fine structure alterations at the murine c-fms (CSF-1 receptor) locus during maturation of myeloid precursor cells. Genes Dev. 16, 1721–1737.
    Article CAS PubMed Google Scholar
  3. Mueller, P. R. and Wold, B. (1989) In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246, 780–786.
    Article CAS PubMed Google Scholar
  4. Pfeifer, G. P., Steigerwald, S. D., Mueller, P. R., Wold, B., and Riggs, A. D. (1989) Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246, 810–813.
    Article CAS PubMed Google Scholar
  5. Pfeifer, G. P., Tanguay, R. L., Steigerwald, S. D., and Riggs, A. D. (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
  6. Telford, D. J. and Stewart, B. W. (1989) Micrococcal nuclease: its specificity and use for chromatin analysis. Int. J. Biochem. 21, 127–137.
    Article CAS PubMed Google Scholar
  7. McPherson, C. E., Shim, E. Y., Friedman, D. S., and Zaret, K. (1993) An active tissue-specific enhancer and transcription factors exist in a precisely positioned nucleosomal array. Cell 75, 387–398
    Article CAS PubMed Google Scholar
  8. Fragoso, G. and Hager, G,L. (1997) Analysis of in vivo nucleosome positions by determination of nucleosome-linker boundaries in crosslinked chromatin. Methods 11, 246–252.
    Article CAS PubMed Google Scholar
  9. Cockerill, P. N. (2000) Identification of DNaseI hypersensitive sites within nuclei. Methods Mol. Biol. 130, 29–46.
    CAS PubMed Google Scholar
  10. Pfeifer, G. P. and Riggs, A. D. (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
  11. Pheiffer, B. H. and Zimmerman, S. B. (1983) Polymer-stimulated ligation: enhanced blunt-or cohesive-end ligation of DNA or deoxyribooligonucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res. 11, 7853–7871.
    Article CAS PubMed Google Scholar
  12. Varadaraj, K. and Skinner, D. M. (1994) Denaturants or cosolvents improve the specificity of PCR amplification of G + C rich DNA using genetically engineered DNA polymerases. Gene 140, 1–5.
    Article CAS PubMed Google Scholar
  13. Mytelka, D. S. and Chamberlin, M. J. (1996) Analysis of DNA polymerase pauses and suppression associated with a trinucleotide consensus. Nucleic Acids Res. 24, 2774–2781.
    Article CAS PubMed Google Scholar

Download references

Author information

Authors and Affiliations

  1. Molecular Medicine Unit, St. James’s University Hospital, University of Leeds, Leeds, UK
    Hiromi Tagoh
  2. Molecular Medicine Unit, University of Leeds, St. James’s University Hospital, Clinical Sciences Building, Leeds, UK
    Peter N. Cockerill
  3. Molecular Medicine Unit, University of Leeds; St. James’s University Hospital, Leeds, UK
    Constanze Bonifer

Authors

  1. Hiromi Tagoh
    You can also search for this author inPubMed Google Scholar
  2. Peter N. Cockerill
    You can also search for this author inPubMed Google Scholar
  3. Constanze Bonifer
    You can also search for this author inPubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Gene Function and Development, Roslin Institute, Roslin, Midlothian, UK
    Steve Pells

Rights and permissions

© 2006 Humana Press Inc.

About this protocol

Cite this protocol

Tagoh, H., Cockerill, P.N., Bonifer, C. (2006). In Vivo Genomic Footprinting Using LM–PCR Methods. In: Pells, S. (eds) Nuclear Reprogramming. Methods in Molecular Biology™, vol 325. Humana Press. https://doi.org/10.1385/1-59745-005-7:285

Download citation

Publish with us