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Functional analysis of CpG methylation in the BRCA1 promoter region

Oncogene volume 20, pages 5331–5340 (2001)Cite this article

Abstract

Understanding the role for DNA methylation in tumorigenesis has evolved from defining the location and extent of methylation in a variety of cancer-related genes to clarifying the functional and site-specific effects of aberrant methylation on gene expression. Our objectives were to characterize the functional effects of DNA methylation in the BRCA1 promoter and to clarify the functional status of the BRCA1 CRE (cAMP response element) motif. Luciferase reporter assays confirm that an intact CRE is important for BRCA1 expression in transient transfections. Luciferase activities were decreased in constructs where the CRE recognition sequence was altered and when constructs were methylated in vitro. Gel mobility shift and competition assays identified a DNA-protein complex recognizing the CRE motif that we were able to supershift using CREB-specific antibody. Furthermore this CRE is methylation sensitive, and we localized this methylation effect to a CpG dinucleotide within the BRCA1 CRE motif. The consequences of aberrant DNA methylation at specific transcription factor motifs, along with the multiple mutational events that can occur in a variety of essential genes such as BRCA1, paint a complex picture where both genetic and epigenetic changes contribute to tumour formation.

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Acknowledgements

We gratefully acknowledge Drs Harriet Kinyamu and Christy Fryer for their expertise in transfections and luciferase assays and the support of the CHRI and the LHSC Molecular Diagnostic Laboratory. We would also like to acknowledge Drs Shiva Singh and Gerald Kidder for their helpful discussions. Debora Mancini DiNardo is a recipient of a post-graduate scholarship from the Natural Sciences and Engineering Research Council. This research was supported by an operating grant to David Rodenhiser from the Canadian Breast Cancer Research Initiative.

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Author notes

  1. Debora N Mancini DiNardo

    Present address: Lewis Thomas Lab, room 128, Dept of Molecular Biology, Princeton University, Princeton, NJ 08544, New Jersey, USA

Authors and Affiliations

  1. London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada

    Darci T Butcher, Dale P Robinson & David I Rodenhiser

  2. Child Health Research Institute, University of Western Ontario, London, Ontario, Canada

    Debora N Mancini DiNardo, Darci T Butcher, Dale P Robinson & David I Rodenhiser

  3. Department of Biochemistry, University of Western Ontario, London, Ontario, Canada

    Darci T Butcher & David I Rodenhiser

  4. Department of Paediatrics, University of Western Ontario, London, Ontario, Canada

    Debora N Mancini DiNardo, Darci T Butcher, Dale P Robinson & David I Rodenhiser

  5. Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, NIEHS, Research Triangle Park, North Carolina, USA

    Trevor K Archer

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Correspondence to David I Rodenhiser.

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DiNardo, D., Butcher, D., Robinson, D. et al. Functional analysis of CpG methylation in the BRCA1 promoter region. Oncogene 20, 5331–5340 (2001). https://doi.org/10.1038/sj.onc.1204697

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