Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein-DNA complexes.
The human estrogen receptor (hER) is a trans-acting regulatory protein composed of a series of discrete functional domains. We have microinjected an hER expression vector (HEO) into Xenopus oocyte nuclei and demonstrate, using Western blot assay, that the hER is synthesized. When nuclear extracts from oocytes were prepared and incubated in the presence of a 2.7 kb DNA fragment comprising the 5' end of the vitellogenin gene B2, formation of estrogen-dependent complexes could be visualized by electron microscopy over the estrogen responsive element (ERE). Of crucial importance is the observation that the complex formation is inhibited by the estrogen antagonist tamoxifen, is restored by the addition of the hormone and does not take place with extracts from control oocytes injected with the expression vector lacking the sequences encoding the receptor. The presence of the biologically active hER is confirmed in co-injection experiments, in which HEO is co-introduced with a CAT reporter gene under the control of a vitellogenin promoter containing or lacking the ERE. CAT assays and primer extensions analyses reveal that both the receptor and the ERE are essential for estrogen induced stimulation of transcription. The same approach was used to analyze selective hER mutants. We find that the DNA binding domain (region C) is essential for protein--DNA complex formation at the ERE but is not sufficient by itself to activate transcription from the reporter gene. In addition to region C, both the hormone binding (region E) and amino terminal (region A/B) domains are needed for an efficient transcription activation.(ABSTRACT TRUNCATED AT 250 WORDS)
Theulaz
I
I
Institut de Biologie animale, Université de Lausanne, Switzerland.
Hipskind
R
R
ten Heggeler-Bordier
B
B
Green
S
S
Kumar
V
V
Chambon
P
P
Wahli
W
W
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D009865:Oocytes / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding / Q000187:drug effects; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D013045:Species Specificity; D013629:Tamoxifen / Q000494:pharmacology; D014158:Transcription, Genetic; D014982:Xenopus laevis
D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013629:Tamoxifen; D004247:DNA