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.