The activator protein 2 (AP-2) transcription factors are essential proteins for oestrogenic repression of the ERBB2 proto-oncogene in breast cancer cells. In the present study, we have examined the possible oestrogenic regulation of AP-2 genes themselves in breast-tumour-derived lines. As early as 1 h after oestrogen treatment, AP-2γ mRNA was markedly increased, whereas AP-2α was down-regulated, but with slower kinetics, and AP-2β was not affected at all. Addition of anti-oestrogens ablated these effects. Modulation of the protein levels corresponded to changes in the transcript levels, thus suggesting that in oestrogen-treated cells, an inversion of the balance between AP-2α and AP-2γ isoforms occurs. The 5´-untranslated region (5´-UTR) of the human AP-2γ gene contains one consensus and one degenerate oestrogen-responsive element (ERE). Reporter constructs carrying the AP-2γ promoter and the 5´-UTR were up-regulated by oestrogens in transient transfection assays. Deletion of the most conserved (but not of the degenerate) ERE from reporter constructs abrogated the oestrogenic response, although both ERE-containing segments were footprinted in DNaseI protection assays. In vitro binding assays demonstrated the ability of oestrogen receptor α (ERα) to bind to this site, and chromatin immunoprecipitation analysis of the endogenous gene showed that ERα occupies this region in response to oestrogens. We conclude that AP-2γ is a primary oestrogen-responsive gene and suggest that AP-2 proteins may mediate some oestrogenic responses.
- activator protein-2 transcription factor
- breast cancer
- oestrogen receptor α
- oestrogen-regulated genes
- transcriptional regulation
Abbreviations used: AP-2γ, activator protein 2, isoform γ (TFAP2C); BCA, bicinchoninic acid; CAT, chloramphenicol acetyltransferase; ChIP, chromatin immunoprecipitation; CITED, CREB-binding protein/p300-interacting transactivator with ED (Glu/Asp)-rich tail); CM, complete medium; CREB, cAMP-response-element-binding protein; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility-shift assay; ERα, oestrogen receptor α; rERα, recombinant ERα; ERE, oestrogen-response element; FCS, foetal calf serum; FPA (etc.), footprint A (etc.); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Inr, initiator; LUC, luciferase; 4OH-TAM, 4-hydroxy-tamoxifen; RT, reverse transcriptase; SM, stripped medium; Sp1, specificity protein 1; TK, thymidine kinase; 5´-UTR, 5´-untranslated region; Vit, vitellogenin.
- The Biochemical Society, London ©2004