Ligand-, Cell-, and Estrogen Receptor Subtype (α/β)-dependent Activation at GC-rich (Sp1) Promoter Elements

doi:10.1074/jbc.275.8.5379

Ligand-, Cell-, and Estrogen Receptor Subtype (α/β)-dependent Activation at GC-rich (Sp1) Promoter Elements*

From the ^‡Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466 and the ^§Center for Biotechnology and Medical Nutrition, Karolinska Institute, Novum, S-14186 Huddinge, Sweden

Abstract

17β-Estradiol (E2) induces expression of several genes via estrogen receptor (ER)-Sp1 protein interactions with GC-rich promoter elements in which Sp1 but not ER binds DNA. This study reports the ligand- and cell context-dependent ER_α/Sp1 and ER_β/Sp1 action using an E2-responsive construct (pSp1) containing a GC-rich promoter. Both ER_α and ER_β proteins physically interact with Sp1 (coimmunoprecipitation) and preferentially bind to the C-terminal region of this protein in pull-down assays. E2- and antiestrogen-dependent transcriptional activation of ER_α/Sp1 was observed in MCF-7, MDA-MB-231, and LnCaP cells, but not in HeLa cells. E2 did not affect or significantly decrease ER_β/Sp1 action, and antiestrogens had minimal effects in the same 4 cell lines. Exchange of activation function-1 (AF-1) domains of ER subtypes gave chimeric ER_α/β(AF-1α/AF-2β) and ER_β/α (AF-1β/AF-2α) proteins that resembled wild-type ER (α or β) in terms of physical association with Sp1 protein. Transcriptional activation studies with chimeric ER_β/α and ER_α/β showed that only ER_α/β can activate transcription from an Sp1 element, not ER_β/α. This indicates that the AF-1 domain from ER_α is responsible for activation at an Sp1 element, independent of ER subtype context. In order to further characterize this observation, deletion constructs in the AF-1 domain of both ER_α and ER_α/β were made, and transactivation studies indicated that the region between amino acids 79 and 117 of this domain is important for activation at an Sp1 element.

Footnotes

↵* Supported by the Welch Foundation, National Institutes of Health Grants CA76636 and ES09106, the Swedish Cancer Fund, and the Texas Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ Sid Kyle Professor of Toxicology. To whom correspondence should be addressed: Dept. of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466. Tel.: 409-845-5988; Fax: 409-862-4929; E-mail: ssafe@cvm.tamu.edu.
↵2 B. Saville, M. Wormke, F. Wang, T. Nguyen, E. Enmark, G. Kuiper, J.-Å. Gustafsson, and S. Safe, unpublished results.
Abbreviations:

ER

estrogen receptor

AF

activation function

E2

17β-estradiol

ERE

estrogen-responsive element

FBS

fetal bovine serum

hER

human estrogen receptor

aa

amino acid(s)

bp

base pair(s)

CAT

chloramphenicol acetyltransferase

DME/F12

Dulbecco's modified Eagle's medium/Ham's F-12 medium

PCR

polymerase chain reaction

GST

glutathione S-transferase

CAT

chloramphenicol acetyltransferase
- Received October 14, 1999.
- Revision received December 9, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.