Identification of Response Elements on Promoters Using Site-Directed Mutagenesis and Chromatin Immunoprecipitation

  • Salah Boudjadi
  • Julie C. Carrier
  • Jean-François Beaulieu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1765)

Abstract

Proximal promoters are located upstream of the transcription start sites of genes, and they contain regulatory sequences on which bind different transcription factors for promoting colorectal cancer progression. Here we describe the comprehensive methodology used previously for the identification and functional characterization of MYC-responsive elements in the integrin α1 subunit (ITGA1) gene using a combination of in silico analysis, site-directed mutagenesis, and chromatin immunoprecipitation.

Key words

Promoters ChIP Response element Mutagenesis Transcription factor MYC ITGA1 

Notes

Acknowledgments

The original work was supported by the Canadian Institute of Health Research Grants MOP-97836 and MOP-123415. J-FB was the recipient of the Canadian Research Chair in Intestinal Physiopathology. JCC is a scholar of the Fonds de la Recherche du Québec-Santé (FRQS). SB is a recipient of the FRQS postdoctoral fellowship.

References

  1. 1.
    Zambelli F, Pesole G, Pavesi G (2013) Motif discovery and transcription factor binding sites before and after the next-generation sequencing era. Brief Bioinform 14(2):225–237.  https://doi.org/10.1093/bib/bbs016CrossRefPubMedGoogle Scholar
  2. 2.
    Osada M, Park HL, Nagakawa Y, Yamashita K, Fomenkov A, Kim MS, Wu G, Nomoto S, Trink B, Sidransky D (2005) Differential recognition of response elements determines target gene specificity for p53 and p63. Mol Cell Biol 25(14):6077–6089.  https://doi.org/10.1128/MCB.25.14.6077-6089.2005CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Boudjadi S, Bernatchez G, Beaulieu JF, Carrier JC (2013) Control of the human osteopontin promoter by ERRalpha in colorectal cancer. Am J Pathol 183(1):266–276.  https://doi.org/10.1016/j.ajpath.2013.03.021CrossRefPubMedGoogle Scholar
  4. 4.
    Boudjadi S, Beaulieu JF (2016) MYC and integrins interplay in colorectal cancer. Oncoscience 3(2):50–51.  https://doi.org/10.18632/oncoscience.293PubMedPubMedCentralGoogle Scholar
  5. 5.
    Boudjadi S, Carrier JC, Beaulieu JF (2013) Integrin alpha 1 subunit is up-regulated in colorectal cancer. Biomarker Res 1(16):1–7Google Scholar
  6. 6.
    Boudjadi S, Carrier JC, Groulx JF, Beaulieu JF (2016) Integrin alpha1beta1 expression is controlled by c-MYC in colorectal cancer cells. Oncogene 35(13):1671–1678.  https://doi.org/10.1038/onc.2015.231CrossRefPubMedGoogle Scholar
  7. 7.
    Thomas P, Smart TG (2005) HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods 51(3):187–200.  https://doi.org/10.1016/j.vascn.2004.08.014CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Salah Boudjadi
    • 1
  • Julie C. Carrier
    • 2
  • Jean-François Beaulieu
    • 3
  1. 1.Cancer Molecular Pathology Section, Laboratory of PathologyNational Cancer InstituteBethesdaUSA
  2. 2.Department of Medicine, Faculty of Medicine and Health ScienceUniversité de SherbrookeSherbrookeCanada
  3. 3.Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology, Faculty of Medicine and Health SciencesUniversité de SherbrookeSherbrookeCanada

Personalised recommendations