Steroid hormone-dependent interaction of human progesterone receptor with its target enhancer element.
We investigated the requirement of steroid hormone for the specific binding of progesterone receptor to its cognate progesterone responsive element (PRE) in cell-free experiments. We prepared unfractionated nuclear extracts from human breast cancer (T47D) cells which are rich in progesterone receptors and used a gel retardation assay to monitor receptor-DNA complex formation. Exposure of receptor to either progesterone, R5020, or the antiprogestin RU38 486 in vivo or in vitro led to the formation of two protein-DNA complexes (1 and 2) which were not detected in nuclear extracts unexposed to hormone. Similar treatment with cortisol or estradiol failed to induce the formation of these complexes. The complexes were specific for PRE, since they could be competed efficiently in binding competition experiments by oligonucleotides containing PRE. A monoclonal antibody which recognizes both A and B forms of human progesterone receptor, interacted with both complexes 1 and 2 and shifted them to slower migrating forms. Another antibody which only recognizes the B form interacted with only complex 1 but not with complex 2, establishing that the complexes 1 and 2 were indeed formed by progesterone receptor forms B and A, respectively. We conclude from the above studies that in vivo or in vitro treatment of nuclear progesterone receptor with either progesterone or R5020 or RU38 486 alone can lead to detection of high affinity complexes formed between the PRE and the receptor present in unpurified nuclear extracts.
Bagchi
M K
MK
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Elliston
J F
JF
Tsai
S Y
SY
Edwards
D P
DP
Tsai
M J
MJ
O'Malley
B W
BW
D001943:Breast Neoplasms / Q000378:metabolism; D002460:Cell Line; D004742:Enhancer Elements, Genetic; D004958:Estradiol / Q000494:pharmacology*; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology*; D015735:Mifepristone; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology; D011397:Promegestone / Q000494:pharmacology; D011980:Receptors, Progesterone / Q000378:metabolism* / Q000648:ultrastructure; D014407:Tumor Cells, Cultured / Q000378:metabolism
D004963:Estrenes; D011980:Receptors, Progesterone; D015735:Mifepristone; D011374:Progesterone; D004958:Estradiol; D011397:Promegestone; D006854:Hydrocortisone