Functional analysis of CpG methylation in the BRCA1 promoter region

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.

[1]  W. Hwu,et al.  FMR1 enhancer is regulated by cAMP through a cAMP-responsive element. , 1997, DNA and cell biology.

[2]  J. Rice,et al.  Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells , 1998, Oncogene.

[3]  A. Razin,et al.  CpG methylation, chromatin structure and gene silencing—a three‐way connection , 1998, The EMBO journal.

[4]  E. Atlas,et al.  GA-binding protein α/β is a critical regulator of the BRCA1 promoter , 2000, Oncogene.

[5]  T. Meyer,et al.  Cyclic adenosine 3',5'-monophosphate response element binding protein (CREB) and related transcription-activating deoxyribonucleic acid-binding proteins. , 1993, Endocrine reviews.

[6]  Peter A. Jones,et al.  Cancer-epigenetics comes of age , 1999, Nature Genetics.

[7]  J. Herman,et al.  5′ CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers , 1995, Nature Medicine.

[8]  A. Ferguson,et al.  Demethylation of the progesterone receptor CpG island is not required for progesterone receptor gene expression , 1998, Oncogene.

[9]  Chun-Fang Xu,et al.  Methylation of the BRCA1 promoter region in sporadic breast and ovarian cancer: correlation with disease characteristics , 1999, Oncogene.

[10]  Shiva M. Singh,et al.  Site-specific DNA methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB and SP1 transcription factors , 1999, Oncogene.

[11]  I. Andrulis,et al.  Methylation of the BRCA1 promoter is associated with decreased BRCA1 mRNA levels in clinical breast cancer specimens. , 2000, Carcinogenesis.

[12]  F. O'Malley,et al.  CpG methylation within the 5′ regulatory region of the BRCA1 gene is tumor specific and includes a putative CREB binding site , 1998, Oncogene.

[13]  G. Lenoir,et al.  Regional methylation of the 5’ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells , 2000, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[14]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[15]  G. Hager,et al.  Sequence-specific DNA binding by glucocorticoid receptor "zinc finger peptides". , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[16]  J. Jung,et al.  Homonucleotide tracts, short repeats and CpG/CpNpG motifs are frequent sites for heterogeneous mutations in the neurofibromatosis type 1 (NF1) tumour-suppressor gene. , 1997, Mutation research.

[17]  M. Westerfield,et al.  Characterization of paired tumor and non‐tumor cell lines established from patients with breast cancer , 1998, International journal of cancer.

[18]  C. Croce,et al.  Positive Regulation of the BRCA1 Promoter* , 1999, The Journal of Biological Chemistry.

[19]  Yonghong Xiao,et al.  Association of BRCA1 with Rad51 in Mitotic and Meiotic Cells , 1997, Cell.

[20]  J. Rice,et al.  Transcriptional repression of BRCA1 by aberrant cytosine methylation, histone hypoacetylation and chromatin condensation of the BRCA1 promoter. , 2000, Nucleic acids research.

[21]  B. Koller,et al.  BRCA1 required for transcription-coupled repair of oxidative DNA damage. , 1998, Science.

[22]  K. Kurihara,et al.  Tissue-specific methylation occurs in the essential promoter element of the tyrosine hydroxylase gene. , 1997, Brain research. Molecular brain research.

[23]  S. Baylin,et al.  Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon , 1994, Nature Genetics.

[24]  M. Skolnick,et al.  A collaborative survey of 80 mutations in the BRCA1 breast and ovarian cancer susceptibility gene. Implications for presymptomatic testing and screening. , 1995, JAMA.

[25]  P. Ainsworth,et al.  Constitutively methylated CpG dinucleotides as mutation hot spots in the retinoblastoma gene (RB1). , 1997, American journal of human genetics.

[26]  Chun-Fang Xu,et al.  Complex Regulation of the BRCA1 Gene* , 1997, The Journal of Biological Chemistry.

[27]  P. Goss,et al.  Transcription of BRCA1 Is Dependent on the Formation of a Specific Protein-DNA Complex on the Minimal BRCA1 Bi-directional Promoter* , 1999, The Journal of Biological Chemistry.

[28]  Steven E. Bayer,et al.  A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. , 1994, Science.

[29]  J. Herman,et al.  Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. , 1994, Proceedings of the National Academy of Sciences of the United States of America.