title: Involvement of a specificity proteins-binding element in regulation of basal and estrogen-induced transcription activity of the BRCA1 gene.
abstract: BACKGROUND
  Increased estrogen level has been regarded to be a risk factor for breast cancer. However, estrogen has also been shown to induce the expression of the tumor suppressor gene, BRCA1. Upregulation of BRCA1 is thought to be a feedback mechanism for controlling DNA repair in proliferating cells. Estrogens enhance transcription of target genes by stimulating the association of the estrogen receptor (ER) and related coactivators to estrogen response elements or to transcription complexes formed at activator protein (AP)-1 or specificity protein (Sp)-binding sites. Interestingly, the BRCA1 gene lacks a consensus estrogen response element. We previously reported that estrogen stimulated BRCA1 transcription through the recruitment of a p300/ER-alpha complex to an AP-1 site harbored in the proximal BRCA1 promoter. The purpose of the study was to analyze the contribution of cis-acting sites flanking the AP-1 element to basal and estrogen-dependent regulation of BRCA1 transcription.
  
  
  METHODS
  Using transfection studies with wild-type and mutated BRCA1 promoter constructs, electromobility binding and shift assays, and DNA-protein interaction and chromatin immunoprecipitation assays, we investigated the role of Sp-binding sites and cAMP response element (CRE)-binding sites harbored in the proximal BRCA1 promoter.
  
  
  RESULTS
  We report that in the BRCA1 promoter the AP-1 site is flanked upstream by an element (5'-GGGGCGGAA-3') that recruits Sp1, Sp3, and Sp4 factors, and downstream by a half CRE-binding motif (5'-CGTAA-3') that binds CRE-binding protein. In ER-alpha-positive MCF-7 cells and ER-alpha-negative Hela cells expressing exogenous ER-alpha, mutation of the Sp-binding site interfered with basal and estrogen-induced BRCA1 transcription. Conversely, mutation of the CRE-binding element reduced basal BRCA1 promoter activity but did not prevent estrogen activation. In combination with the AP-1/CRE sites, the Sp-binding domain enhanced the recruitment of nuclear proteins to the BRCA1 promoter. Finally, we report that the MEK1 (mitogen-activated protein kinase kinase-1) inhibitor PD98059 attenuated the recruitment of Sp1 and phosphorylated ER-alpha, respectively, to the Sp and AP-1 binding element.
  
  
  CONCLUSIONS
  These cumulative findings suggest that the proximal BRCA1 promoter segment comprises cis-acting elements that are targeted by Sp-binding and CRE-binding proteins that contribute to regulation of BRCA1 transcription.
authors: Cancer Biology Interdisciplinary Graduate Program, Department of Nutritional Sciences, The University of Arizona, E 4th Street, Tucson, Arizona 85721-0038, USA.
authors: Jennifer K
authors: JK
authors: Hockings
authors: Stephanie C
authors: SC
authors: Degner
authors: Sherif S
authors: SS
authors: Morgan
authors: Michael Q
authors: MQ
authors: Kemp
authors: Donato F
authors: DF
authors: Romagnolo
mesh_terms: D051698:Activating Transcription Factor 2 / Q000378:metabolism*; D019313:BRCA1 Protein / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics*; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D019398:Genes, BRCA1; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D051698:Activating Transcription Factor 2; D019313:BRCA1 Protein; D004967:Estrogens; D005419:Flavonoids; D011960:Receptors, Estrogen; D018808:Transcription Factor AP-1; D011494:Protein Kinases; C065999:Sp1 kinase; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one