Site-specific DNA methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB and SP1 transcription factors (original) (raw)

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• Published: 30 July 1999

Oncogene volume 18, pages 4108–4119 (1999)Cite this article

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Abstract

Tumour suppressor genes and growth regulatory genes are frequent targets for methylation defects that can result in aberrant expression and mutagenesis. We have established a methylation map of the promoter region of the neurofibromatosis (NF1) gene and demonstrated functional sensitivity for methylation at specific sites for the SP1 and CRE binding (CREB) proteins in the NF1 regulatory region. We evaluated the methylation status of CpG dinucleotides within five promoter subregions in the human and mouse homologues of the neurofibromatosis (NF1) genes. Three 5′ subregions were found to be consistently methylated in all the tissues analysed. In contrast, DNA methylation was absent in the vicinity of the transcription start site bounded by SP1 recognition sequences. Gelshift assays showed that methylation specifically inhibits the CREB transcription factor from binding to its recognition site at the NF1 transcription start site. Furthermore, SP1 elements within the NF1 promoter are methylation sensitive, particularly when methylation is present on the antisense strand. We propose that for NF1 as with several other tumour suppressor genes, CpG methylation occurs in a complex, site-specific manner with the maintenance of a methylation-free promoter region bounded by SP1 binding sites that allow an accessible promoter to be retained. When these SP1 boundaries are breached, methylation can sweep in, rendering the promoter inaccessible for specific methylation-sensitive transcription factors and leading to a loss of functional integrity of the methylation-free CpG island.

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Acknowledgements

We acknowledge the support of our colleagues in the Molecular Medical Genetics Program, Children's Hospital of Western Ontario and Dr Yves Ouellette for the use of his tissue culture facilities. We also gratefully acknowledge Dr Andre Bernards for providing several neuroblastoma cell lines and Dr David Viskochil for providing an upstream segment of the 5′ NF1 DNA sequence. Debora Mancini is a recipient of graduate scholarship support from NSERC (Canada). Trevor Archer is a scientist of the National Cancer Institute of Canada (NCIC) and is supported by funds from the Canadian Cancer Society. This research was funded by awards to David Rodenhiser by the Child Health Research Institute and the London Health Sciences Center Research Development Fund.

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Authors and Affiliations

1. Molecular Medical Genetics Program, Child Health Research Institute, London Health Sciences Centre, 800 Commissioner's Road East, London, Ontario, Canada, N6C 2V5
   Debora N Mancini, Shiva M Singh & David I Rodenhiser
2. London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada
   Trevor K Archer
3. Department of Biochemistry, University of Western Ontario, London, Ontario, Canada
   Trevor K Archer & David I Rodenhiser
4. Department of Obstetrics and Gynecology, University of Western Ontario, London, Ontario, Canada
   Trevor K Archer
5. Department of Oncology, University of Western Ontario, London, Ontario, Canada
   Trevor K Archer
6. Department of Paediatrics, University of Western Ontario, London, Ontario, Canada
   Debora N Mancini, Shiva M Singh & David I Rodenhiser
7. Department of Zoology, University of Western Ontario, London, Ontario, Canada
   Debora N Mancini, Shiva M Singh & David I Rodenhiser

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1. Debora N Mancini
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2. Shiva M Singh
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3. Trevor K Archer
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Mancini, D., Singh, S., Archer, T. et al. Site-specific DNA methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB and SP1 transcription factors.Oncogene 18, 4108–4119 (1999). https://doi.org/10.1038/sj.onc.1202764

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• Received: 30 July 1998
• Revised: 15 January 1999
• Accepted: 16 February 1999
• Published: 30 July 1999
• Issue Date: 15 July 1999
• DOI: https://doi.org/10.1038/sj.onc.1202764

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