Figure 3.

Functional reporter and in vivo chromatin immunoprecipitation assays with hTERT promoter in PCa cells. Transient transfections with (a) p1009 or p1009Mut reporters containing wild-type or mutated proximal hTERT-ERE; (b) p1009 reporter alone (–ER), or in combination with expression vectors for ER-α (+ER-α) or ER-β (+ER-β); (c) p1009 or VIT reporters with expression vectors for ER-β (+ER-β) were performed. Values are expressed as fold of hormone induction, calculated as the ratio of normalized luciferase activity (light units per β-galactosidase units per minute per μg of protein) in the presence or absence of E₂ (+/– E₂). Results represent the average (± SEM) of six independent experiments, each performed in duplicate. Similar results were obtained with a longer hTERT located at –949/–935 (proximal) and at –2757/–2738 (distal) relative to the ATG (data not shown). Formaldehyde cross-linked chromatin from LNCaP and DU145 cells untreated (0’), or treated with E₂ (10^–7 M) for 45 and 90 minutes (45’ and 90’), or with triiodothyronine (T₃ ,10^–7 M) for 45 minutes (+) was immunoprecipitated with Ab’s to ER-α (α–ER-α), ER-β (α–ER-β), NF-Y (α NF-Y), or in the absence of Ab’s (no Ab) and analyzed by PCR with primers specific for the hTERT (d) and CycB1 (e) promoters. Input corresponds to nonimmunoprecipitated cross-linked chromatin. Primer position (arrows) on schematic promoters relative to the start site of transcription (+1) and primer migration (*) are indicated.