FIGURE 3.

Atox1 binds to and activates the cyclin D1 promoter in a copper-dependent manner. A, effect of copper on transactivation of the cyclin D1 gene promoter in WT and Atox1-KO MEFs. Cells were transiently transfected with cyclin D1 promoter luciferase reporter constructs (pGL3-cyclin D1 (-962/+134)) or empty reporter constructs (pGL3-Basic) along with either pcDNA/Atox1 or pcDNA. Cells were treated with either BCS (200 μm) or CuCl₂ at the dose indicated. Two days after transfection, the luciferase activity was assayed and normalized to the Renilla luciferase activity produced by the co-transfected control plasmid pRL-CMV. Results shown are means ± S.E. from at least three independent transfection experiments, each performed in quadruplicate (^*, p < 0.01; #, p < 0.001 versus BCS-treated WT cells, or BCS-treated Atox1^-/- cells transfected with pcDNA/Atox1). B, identification of copper/Atox1-responsive elements in a proximal 90-bp cyclin D1 promoter element. WT and Atox1-KO MEFs were transiently transfected with 5′ deletion constructs of cyclin D1 in the presence of either CuCl₂ (10 μm, +Cu) or BCS (200 μm, -Cu). Left and middle panels, relative luciferase activity in WT or Atox1-KO MEFs in the presence of either CuCl₂ (10 μm, +Cu) or the copper chelator BCS (200 μm, -Cu). Right panel, relative luciferase activity in wild-type and Atox1^-/- cells in the presence of CuCl₂ (10 μm). Results shown are means ± S.E. from at least three independent transfection experiments, each performed in quadruplicate (^*, p < 0.01 versus BCS-treated WT (left panel), or CuCl₂-treated Atox1^-/-cells (right panel)). C and D, EMSA, showing the binding of Atox1 to the region -535 to -530 in the cyclin D1 promoter in a copper-dependent manner. C, nuclear extracts from MEFs were incubated with the biotinylated cyclin D1 promoter fragment with indicated treatments. D, left panel shows purified GST and GST-Atox1. Right panel, purified GST or GST-Atox1 was incubated with the DNA probe with indicated treatments. E, ChIP assay showing association of Atox1 with the cyclin D1 promoter in a copper-dependent manner in vivo. Cells were treated with either indicated treatments (upper panel) or CuCl₂ (10 μm) (lower panel) and cross-linked with 1% formaldehyde. Nuclear lysates were immunoprecipitated (IP) with anti-Atox1 antibody or normal IgG, and the promoter region of either cyclin D1, cyclin B1, or cyclin A was amplified by PCR. A small aliquot of lysates before IP were used for PCR amplification as the input control (Input). Results are representative of three independent experiments. F, region -535 to -530 is required for copper-induced activation of the cyclin D1 promoter. MEFs were transfected with a cyclin D1 promoter luciferase reporter construct (pGL3-cyclin D1 (-962/+134)) with or without mutation of the copper/Atox1-responsive element (-535 to -530 region). ^*, p < 0.01 versus BCS-treated cells.