Identification of a functional intermediate in receptor activation in progesterone-dependent cell-free transcription

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Abstract
The steroid hormone, progesterone, enhances transcription in vivo from promoters containing progesterone response elements (PREs). We have recently shown that the progesterone receptor (PR) modulates transcription in a cell-free system by facilitating the formation of a stable preinitiation complex, apparently through interaction with RNA polymerase II and other basic transcription factors. The precise role of ligand in this activation process remains unclear, however. In order to dissect the role of steroid ligand in gene action, we sought to devise an in vitro transcription system that mimics the hormone-dependent transcriptional activation observed in vivo. We now report the successful reconstitution in vitro of progesterone-dependent RNA synthesis from a PRE-driven promoter in nuclear extracts of human breast carcinoma (T47D) cells. The transcriptional activation is triggered by the hormone-induced binding of endogenous PR to PREs and exhibits hormone-specificity. The receptor exists in a 4S form in our initial salt-treated extract and is apparently dissociated from the heat-shock protein hsp90. Nevertheless, hormone is still required for DNA binding and transcriptional activation. These results suggest that dissociation of hsp90 and conversion to an inactive 4S intermediate could occur before the final event in ligand-mediated transactivation of gene expression.