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. 2004 Aug;24(16):7260–7274. doi: 10.1128/MCB.24.16.7260-7274.2004

FIG. 1.

FIG. 1.

(A) Analysis of CCND1 promoter responsiveness to estrogen upon stable transfection of the indicated luciferase reporter genes in hormone-responsive hBC cells, represented as fold induction by 50 nM E2 for 18 to 24 h (grey bars) versus the activity assayed in hormone-starved cells, arbitrarily set to 1. The black bar represents the fold induction measured in cells incubated simultaneously with 50 nM E2 and 5 μM ICI 182,780. On the left are schematically represented constructs used in transfections. TRE, TPA-responsive element; TREμ, same site mutated as previously described (22). Numbers at the top of the box mark the positions of the 5′-most nucleotides with respect to the transcription start site. Constructs D1Δ-138, D1Δ-754, D1Δ-860, D1Δ-956, and D1Δ-956μ were previously described (22). Constructs D1Δ-18, D1Δ-4S46, D1Δ-4S48, and D1Δ-18/ERE are described in Materials and Methods. F.i., fold induction. Bars indicate standard deviations. (B) In vivo footprinting of the CCND1 gene promoter region encompassing the TRE. The G residues marked by black arrowheads are specifically protected in E2-stimulated cells, whereas the G identified by a grey arrowhead is protected in the same cells depleted by E2. i.v., naked DNA methylated in vitro. (C) Estrogen responsiveness of luciferase reporter genes measured in hBC cells stably transfected with the plasmids schematically represented on the left. Synthetic wild-type or mutant 24-mer oligonucleotides reproducing the CCND1 gene sequence between residues −948 and −925 were cloned immediately upstream of the minimal promoter from the same gene (spanning residues −18 and + 14; white square), the thymidine kinase promoter (grey square; T-AP/dE), or the mouse mammary tumor virus promoter (black square; M-AP/dE). Mutant nucleotides in D1-APμ/dE are indicated. MCF-7 or ZR-75.1 cells were hormone deprived and restimulated as described above, including, where indicated, treatment with ICI 182,780. As for panel A, the results represent the means of three to five independent experiments. Luciferase activity assayed in starved cells transfected with each reporter was arbitrarily set to 1. Unless otherwise indicated, data displayed are from representative experiments carried out with MCF-7 cells; identical results were obtained with ZR-75.1 cells.