Functional genomics identifies a mechanism for estrogen activation of the retinoic acid receptor alpha1 gene in breast cancer cells.
The identification of estrogen receptor (ERalpha) target genes is crucial to our understanding of its predominant role in breast cancer. In this study, we used a chromatin immunoprecipitation (ChIP)-cloning strategy to identify ERalpha-regulatory modules and associated target genes in the human breast cancer cell line MCF-7. We isolated 12 transcriptionally active genomic modules that recruit ERalpha and the coactivator steroid receptor coactivator (SRC)-3 to different intensities in vivo. One of the ERalpha-regulatory modules identified is located 3.7 kb downstream of the first transcriptional start site of the RARA locus, which encodes retinoic acid receptor alpha1 (RARalpha1). This module, which includes an estrogen response element (ERE), is conserved between the human and mouse genomes. Direct binding of ERalpha to the ERE was shown using EMSAs, and transient transfections in MCF-7 cells demonstrated that endogenous ERalpha can induce estrogen-dependent transcriptional activation from the module or the ERE linked to a heterologous promoter. Furthermore, ChIP assays showed that the coregulators SRC-1, SRC-3, and receptor-interacting protein 140 are recruited to this intronic module in an estrogen-dependent manner. As expected from previous studies, the transcription factor Sp1 can be detected at the RARA alpha1 promoter by ChIP. However, treatment with estradiol did not influence Sp1 recruitment nor help recruit ERalpha to the promoter. Finally, ablation of the intronic ERE was sufficient to abrogate the up-regulation of RARA alpha1 promoter activity by estradiol. Thus, this study uncovered a mechanism by which ERalpha significantly activates RARalpha1 expression in breast cancer cells and exemplifies the utility of functional genomics strategies in identifying long-distance regulatory modules for nuclear receptors.
Laganière
Josée
J
Molecular Oncology Group, McGill University Health Center, and Department of Biochemistry, McGill University, Room H5-21, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1.
Deblois
Geneviève
G
Giguère
Vincent
V
D000123:Acetyltransferases / Q000378:metabolism; D001483:Base Sequence; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004958:Estradiol / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism*; D016678:Genome; D051548:Histone Acetyltransferases; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D056921:Nuclear Receptor Coactivator 3; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1; D015513:Oncogene Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020794:Receptor Protein-Tyrosine Kinases; D057050:Receptor Tyrosine Kinase-like Orphan Receptors; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D020218:Response Elements; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D047628:Estrogen Receptor alpha; D004967:Estrogens; D057094:Nuclear Receptor Subfamily 1, Group F, Member 1; D015513:Oncogene Proteins; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D004958:Estradiol; D000123:Acetyltransferases; D051548:Histone Acetyltransferases; C538866:NCOA3 protein, human; C538867:Ncoa3 protein, mouse; D056921:Nuclear Receptor Coactivator 3; C078716:ROR1 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D057050:Receptor Tyrosine Kinase-like Orphan Receptors