FIG. 5.

A 3′ estrogen-responsive enhancer in the progesterone receptor gene is regulated by cyclin D1. (A and B) Cloning of the 3′ ERE of the PR gene downstream of a basal promoter-luciferase construct activates transcription that responds to estrogen and cyclin D1. We cloned 1.3 kb of the PR 3′ UTR into luciferase reporter vectors. We performed reporter assays using T47D cells in the absence and presence of estradiol for 24 h. Knockdown efficiency for the siRNAs for these experiments is shown in Fig. 4A. Levels of firefly luciferase from the progesterone receptor plasmids are compared to those of an internal pCMV-renilla control (FF/RL ratio). Means ± standard errors of the results determined in three replicated experiments are shown. Reporter activity levels in cells transfected with a scrambled control siRNA (scramble) and an siCCND1 (siCCND1) in the presence of vehicle (A) or estradiol (B) were compared. Results for a pGL2-basic (pGL2N) vector with no promoter, a reporter containing a basal promoter (pGL2P), the basal vector containing the PR gene 3′ UTR (pGL2N-UTR), and the PR gene 3′ UTR region cloned in the promoter reporter (pGL2P-UTR) are shown. (C and D) Activity levels determined using the same reporters were further compared between cells transfected with an empty vector control (vector) or an expression plasmid for cyclin D1 (pCCND1) in the presence of vehicle (C) or estradiol (D). (E) The 3′ ERE of the PR gene is activated by cyclin D1 in a CDK4/CDK6-independent manner in the absence of estrogen and in a CDK4/CDK6-dependent manner in its presence. We evaluated the effect of wild-type cyclin D1 (D1) and the KE cyclin D1 mutant (D1 KE) on the 3′ UTR region by use of the PR gene 3′ UTR region cloned in the promoter reporter (pGL2P-UTR). Transfections were performed as described for panels C and D. We further compared cells transfected with vehicle-treated cells (left panel [Vehicle]) to cells treated with estrogen (right panel [Estradiol]). (F) Changes in cyclin D1 levels do not change levels of expression from a reporter plasmid containing a 3× isolated estrogen response element. Standard reporter vector containing a basal promoter activated by a 3× estrogen receptor binding element (14) was cotransfected with a scrambled control siRNA (scr) and an siCCND1 (siD1), and reporter activity in the presence of estradiol was determined (left panel). The same reporter was cotransfected with an empty vector control (Vec) or an expression plasmid for cyclin D1 (pc-D1 [right panel]).