Advertisement

Chromatin Immunoprecipitation Assay for Analyzing Transcription Factor Activity at the Level of Peripheral Myelin Gene Promoters

  • Joelle Makoukji
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2011)

Abstract

Disruption of epigenetic regulators of transcription is a central mechanism of oncogenesis. Differential gene expression is facilitated by transcriptional regulatory mechanisms and chromatin modifications through DNA-protein interactions. One of the widely used assays to study this is chromatin immunoprecipitation (ChIP) assay, which enables the analysis of association between regulatory molecules, specific promoters, and histone modifications within the context of the cell. This is of immense value as ChIP assays can provide a glimpse of the regulatory mechanisms involved in gene expression in vivo. It is also a powerful technique for analyzing histone modifications as well as binding sites for proteins that bind either directly or indirectly to DNA. The basic steps in this protocol are fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. Although ChIP is a versatile tool, this procedure requires the optimization of the various reaction conditions. Here, we present a detailed application of the ChIP assay to study the differential recruitment of transcriptional factors at the level of peripheral myelin gene promoters.

Key words

Chromatin immunoprecipitation ChIP assay Quantitative real-time PCR TCF/LEF Transcription factor activity Regulation of gene expression Peripheral myelin genes MPZ PMP22 DNA-protein interactions Transcription factor recruitment β-Catenin 

Notes

Acknowledgments

I would like to thank Ms. Nadine J. Makhoul and Dr. Jamal Al Ali for the critical reading of the manuscript.

References

  1. 1.
    Kuo MH, Allis CD (1999) In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment. Methods 19:425–433CrossRefGoogle Scholar
  2. 2.
    Orlando V (2000) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem Sci 25:99–104CrossRefGoogle Scholar
  3. 3.
    Agalioti T, Lomvardas S, Parekh B, Yie J, Maniatis T, Thanos D (2000) Ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter. Cell 103:667–678CrossRefGoogle Scholar
  4. 4.
    Lee TI, Jenner RG, Boyer LA, Guenther MG, Levine SS, Kumar RM, Chevalier B, Johnstone SE, Cole MF, Isono K, Koseki H, Fuchikami T, Abe K, Murray HL, Zucker JP, Yuan B, Bell GW, Herbolsheimer E, Hannett NM, Sun K, Odom DT, Otte AP, Volkert TL, Bartel DP, Melton DA, Gifford DK, Jaenisch R, Young RA (2006) Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125:301–313CrossRefGoogle Scholar
  5. 5.
    Kurdistani SK, Grunstein M (2003) In vivo protein-protein and protein-DNA crosslinking for genomewide binding microarray. Methods 31:90–95CrossRefGoogle Scholar
  6. 6.
    Breiling A, Turner BM, Bianchi ME, Orlando V (2001) General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412:651–655CrossRefGoogle Scholar
  7. 7.
    Dedon PC, Soults JA, Allis CD, Gorovsky MA (1991) A simplified formaldehyde fixation and immunoprecipitation technique for studying protein-DNA interactions. Anal Biochem 197:83–90CrossRefGoogle Scholar
  8. 8.
    Mukhopadhyay A, Deplancke B, Walhout AJ, Tissenbaum HA (2008) Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nat Protoc 3:698–709CrossRefGoogle Scholar
  9. 9.
    Botquin V, Hess H, Fuhrmann G, Anastassiadis C, Gross MK, Vriend G, Scholer HR (1998) New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2. Genes Dev 12:2073–2090CrossRefGoogle Scholar
  10. 10.
    Hassan MQ, Javed A, Morasso MI, Karlin J, Montecino M, van Wijnen AJ, Stein GS, Stein JL, Lian JB (2004) Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin gene. Mol Cell Biol 24:9248–9261CrossRefGoogle Scholar
  11. 11.
    Dahl JA, Collas P (2007) Q2ChIP, a quick and quantitative chromatin immunoprecipitation assay, unravels epigenetic dynamics of developmentally regulated genes in human carcinoma cells. Stem Cells 25:1037–1046CrossRefGoogle Scholar
  12. 12.
    Boutry JM, Hauw JJ, Gansmuller A, Di-Bert N, Pouchelet M, Baron-Van Evercooren A (1992) Establishment and characterization of a mouse Schwann cell line which produces myelin in vivo. J Neurosci Res 32:15–26CrossRefGoogle Scholar
  13. 13.
    O’Neill LP, Turner BM (2003) Immunoprecipitation of native chromatin: NChIP. Methods 31:76–82CrossRefGoogle Scholar
  14. 14.
    Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K (2007) High-resolution profiling of histone methylations in the human genome. Cell 129:823–837CrossRefGoogle Scholar
  15. 15.
    Makoukji J, Shackleford G, Meffre D, Grenier J, Liere P, Lobaccaro JM, Schumacher M, Massaad C (2011) Interplay between LXR and Wnt/beta-catenin signaling in the negative regulation of peripheral myelin genes by oxysterols. J Neurosci 31:9620–9629CrossRefGoogle Scholar
  16. 16.
    Tawk M, Makoukji J, Belle M, Fonte C, Trousson A, Hawkins T, Li H, Ghandour S, Schumacher M, Massaad C (2011) Wnt/beta-catenin signaling is an essential and direct driver of myelin gene expression and myelinogenesis. J Neurosci 31:3729–3742CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Joelle Makoukji
    • 1
    • 2
  1. 1.Neurogenetics Program, Division of Pediatric Neurology, Department of Pediatrics and Adolescent Medicine, AUBMC Special Kids ClinicAmerican University of Beirut Medical CenterBeirutLebanon
  2. 2.Department of Biochemistry, Faculty of MedicineAmerican University of BeirutBeirutLebanon

Personalised recommendations