Hormone-dependent transactivation by estrogen receptor chimeras that do not interact with hsp90. Evidence for transcriptional repressors.
The ligand-free estrogen receptor (ER), like other steroid receptors, interacts with the 90-kDa heat shock protein hsp90 in vitro. Analysis of the effect of potential ER-hsp90 interactions in vivo on receptor function is complicated by the fact that hsp90 binds to ER domains required for hormone binding and stable DNA binding. ER chimeras were therefore created by replacing the ER DNA binding domain with that of GAL4. In addition, the N-terminal AF-1 domain of the ER was replaced with the strong constitutive activation domain of VP16 to create VP16-GAL-ERs. These chimeras bind DNA in a ligand-independent manner, but, importantly, are ligand-dependent transactivators, unlike VP16-GAL, which displays strong constitutive activity under the same conditions. Hormone induces transactivation by VP16-GAL-ERs to levels similar to the constitutive activity of VP16-GAL. Glycerol gradient and coimmunoprecipitation experiments showed that, unlike the wild-type ER, VP16-GAL-ER chimeras do not interact with hsp90. Deletion analyses indicate that a region of the ER, primarily between amino acids 370 and 470, is responsible for repressed transcription. Our results suggest that interaction with hsp90 is not necessary for controlling hormone-dependent transcription by the ER and provide evidence for repressor factors that interact with the N-terminal portion of the receptor's ligand binding domain in the absence of hormone.
Lee
H S
HS
Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Aumais
J
J
White
J H
JH
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D004247:DNA / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D017353:Gene Deletion; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D005656:Fungal Proteins; C070633:Gal-VP16; D018841:HSP90 Heat-Shock Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D004247:DNA