Involvement of a specificity proteins-binding element in regulation of basal and estrogen-induced transcription activity of the BRCA1 gene

INTRODUCTION: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.CONCLUSION: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.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]

Regulation of Cyclooxygenase-2 by Environmental and Dietary Factors

Each year over 260,000 new cases of breast cancer will be diagnosed in the U.S. and approximately 40,000 women will die of metastatic breast cancer. The etiology of breast cancer is poorly understood and only 5 -10% of cases can be attributed to genetic factors. This suggests that the development of breast cancer may involve environmental factors including diet, lifestyle, and exposure to chemicals. Several lines of experimental and epidemiological evidence have highlighted COX-2 as a potential target for breast cancer prevention. The central hypothesis of this proposal is that activation of COX-2 transcription by epigenetic effectors can be prevented by dietary agents that target the activator protein-1 (AP-1) transcription factor and the aromatic hydrocarbon receptor (AhR). The first specific aim was to determine the mechanism through which conjugated linoleic acid (CLA) and rosmarinic acid (RA) inhibit TPA-induced COX-2 trancription. These studies documented that CLA and RA repressed COX-2 transcription by antagonizing the AP-1 transcription factor. The second specific aim was to investigate whether or not the AhR plays a role in TCDD-induced COX-2 transcription and effects of chemopreventive agents. Results indicated that AhR agonists induced the binding of the AhR to COX-2 and was prevented by CLA and the AhR antagonist, resveratrol (RES) and 3-methoxy-4-nitroflavone (3M4NF). The third specific aim was to examine the effects of AhR agonists and dietary selective AhR modulators on chromatin modifications associated with the COX-2 promoter. Chromatin immunoprecipitation (ChIP) assays revealed that the AhR is recruited to the region of the COX-2 promoter containing a xenobiotic response element and accompanied by recruitment of p300 and acetylation of histone H4. Transcriptional regulation of COX-2 by AhR agonists and dietary antagonists may also involve other post-transcriptional modifications of histones, which along with chromatin remodeling factors modulate the structure of chromatin and recruitment of RNA polymerase II. Overall, the results demonstrated that COX-2 transcription can be targeted by a variety of dietary agents that act through different mechanisms. Therefore, inhibition of transcriptional regulation of COX-2 by selected dietary factors may be a breast cancer preventive strategy that bypasses the side effects of drugs that target COX-2

Rosmarinic Acid Antagonizes Activator Protein-1–Dependent Activation of Cyclooxygenase-2 Expression in Human Cancer and Nonmalignant Cell Lines12

One mechanism through which bioactive food components may exert anticancer effects is by reducing the expression of the proinflammatory gene cyclooxygenase-2 (COX-2), which has been regarded as a risk factor in tumor development. Rosmarinic acid (RA) is a phenolic derivative of caffeic acid present in rosemary (Rosmarinus officinalis). Previous research documented that RA may exert antiinflammatory effects. However, the mechanisms of action of RA on COX-2 expression have not been investigated. Here, we report that in colon cancer HT-29 cells, RA (5, 10, and 20 μmol/L) reduced the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity (P < 0.05) and protein levels (P < 0.05). In addition, the cotreatment with RA reduced (5 μmol/L, P < 0.05; 10 and 20 μmol/L, P < 0.01) TPA-induced transcription from a control activator protein-1 (AP-1) promoter-luciferase construct and repressed binding of the AP-1 factors c-Jun (10 μmol/L; P < 0.01) and c-Fos (10 μmol/L; P < 0.05) to COX-2 promoter oligonucleotides harboring a cAMP-response element (CRE). The anti-AP1 effects of RA were also examined in a nonmalignant breast epithelial cell line (MCF10A) in which RA antagonized the stimulatory effects of TPA on COX-2 protein expression (5 μmol/L, P < 0.05; 10 and 20 μmol/L, P < 0.01), the recruitment of c-Jun and c-Fos (10 μmol/L; P < 0.01) to the COX-2/CRE oligonucleotides, and activation of the extracellular signal-regulated protein kinase-1/2 (ERK1/2) (10 μmol/L; P < 0.01), a member of the mitogen-activated protein kinase pathway. Additionally, RA antagonized ERK1/2 activation in colon HT-29 and breast MCF-7 cancer cells (10 μmol/L; P < 0.01). Thus, we propose that RA may be an effective preventative agent against COX-2 activation by AP-1-inducing agents in both cancer and nonmalignant mammary epithelial cells