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

Brad Saville,Mark Wormke,Fan Wang,Thu Nguyen,Eva Enmark,George Kuiper,Jan-Åke Gustafsson,Stephen Safe

doi:10.1074/jbc.275.8.5379

Abstract

17beta-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(alpha)/Sp1 and ER(beta)/Sp1 action using an E2-responsive construct (pSp1) containing a GC-rich promoter. Both ER(alpha) and ER(beta) 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(alpha)/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(beta)/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(alpha/beta) (AF-1alpha/AF-2beta) and ER(beta/alpha) (AF-1beta/AF-2alpha) proteins that resembled wild-type ER (alpha or beta) in terms of physical association with Sp1 protein. Transcriptional activation studies with chimeric ER(beta/alpha) and ER(alpha/beta) showed that only ER(alpha/beta) can activate transcription from an Sp1 element, not ER(beta/alpha). This indicates that the AF-1 domain from ER(alpha) 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(alpha) and ER(alpha/beta) 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.

Highlights

17␤-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
In rats, ER␤ mRNA transcripts were highly expressed in the prostate and ovary; moderate expression was observed in testis, uterus, lung, and bladder; and low ER␤ expression was observed in the spinal cord, various brain sections, pituitary, epididymis, and thymus [10]
Sp1 protein plays an important role in regulation of mammalian and viral genes, and results of ongoing research have shown that E2 responsiveness of c-fos, cathepsin D, retinoic acid receptor 1␣, adenosine deaminase, E2F1, bcl-2, and insulin-like growth factor-binding protein 4 gene expression in breast cancer cells is linked to specific GC-rich promoter sequences that bind ER␣/Sp1 complex in which only the Sp1 protein binds DNA [35,36,37,38,39,40,41,42]

Summary

Introduction

17␤-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␣/Sp1 and ER␤/Sp1 action using an E2-responsive construct (pSp1) containing a GC-rich promoter. Both ER␣ and ER␤ proteins physically interact with Sp1 (coimmunoprecipitation) and preferentially bind to the C-terminal region of this protein in pull-down assays. Sp1 protein plays an important role in regulation of mammalian and viral genes, and results of ongoing research have shown that E2 responsiveness of c-fos, cathepsin D, retinoic acid receptor 1␣, adenosine deaminase, E2F1, bcl-2, and insulin-like growth factor-binding protein 4 gene expression in breast cancer cells is linked to specific GC-rich promoter sequences that bind ER␣/Sp1 complex in which only the Sp1 protein binds DNA [35,36,37,38,39,40,41,42].

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Conclusion