Estrogen regulation of HSPC111 expression is dependent upon direct transcriptional activation by Myc. (a) Left panel: MCF-7 cells were arrested with ICI 182780 for 48 hours and then treated with cycloheximide (black bars) or control (white bars) before addition of estrogen or vehicle for 3 and 6 hours; levels of HSPC111 mRNA were determined by quantitative real-time PCR. Middle panel: MCF-7 cells were transfected with an HSPC111-luciferase reporter construct in the presence of increasing amounts of the c-Myc expression construct pCDNA3.1-cMyc. Right panel: MCF-7/MycWT (black bars) and empty vector controls (white bars) were transfected with the HSPC111-luciferase reporter construct and stimulated with increasing concentrations of zinc. Values are expressed as means ± standatrd deviation of triplicate samples from three independent experiments. (b) Left panel: MCF-7 cells were transfected with Myc-specific small interfering (si)RNA (siMyc17), RISC-free (RF), or nontargeting (NT) siRNA controls, or mock transfected with no siRNA. Transfected cells were arrested with ICI 182780 for 48 hours. Levels of MYC and HSPC111 mRNA were determined by quantitative real-time PCR after 24 hours of treatment with estradiol (black bars) or vehicle control (white bars). (c) Schematic showing the structure of the HSPC111 proximal promoter with the location of putative Myc-binding sites (E-boxes). Electrophoretic mobility shift assays demonstrate specific binding of c-Myc to E-boxes within the HSPC111 promoter. Radiolabeled oligonucleotides, as indicated above each gel, were incubated with nuclear extract from MCF-7 cells. Lane NC indicates no competitor oligonucleotides were added. The nonlabeled competitor oligonucleotides are indicated below each lane. Lane NS indictates nonspecific competitor oligonucleotide. (d) Chromatin was obtained from MCF-7/MycWT cells after 6 hours of treatment with zinc, and immunoprecipitated with c-Myc-specific or nonspecific (NS) antibodies as indicated. Left panel: Chromatin immunoprecipitation (ChIP) assay demonstrating the binding of c-Myc to the endogenous HSPC111 promoter. Lane I contains input chromatin that was not immunoprecipitated. Specific regions were then amplified by PCR using primers specific for site 1 or site 3, as indicated. Right panel: ChIP assay demonstrating the recruitment of c-Myc to the endogenous HSPC111 promoter in response to treatment with estradiol (E2) at 3 and 6 hours. Chromatin was immunoprecipitated with either a c-Myc-specific (C33; black bars) or a nonspecific (white bars) antibody and analyzed by quantitative real-time PCR using primers specific for site 1.