A novel estrogen receptor alpha-associated protein alters receptor-deoxyribonucleic acid interactions and represses receptor-mediated transcription.

Estrogen receptor alpha (ER alpha) serves as a ligand-activated transcription factor, turning on transcription of estrogen-responsive genes in target cells. Numerous regulatory proteins interact with the receptor to influence ER alpha-mediated transactivation. In this study, we have identified pp32, which interacts with the DNA binding domain of ER alpha when the receptor is free, but not when it is bound to an estrogen response element. Coimmunoprecipitation experiments demonstrate that endogenously expressed pp32 and ER alpha from MCF-7 breast cancer cells interact. Although pp32 substantially enhances the association of the receptor with estrogen response element-containing DNA, overexpression of pp32 in MCF-7 cells decreases transcription of an estrogen-responsive reporter plasmid. pp32 Represses p300-mediated acetylation of ER alpha and histones in vitro and inhibits acetylation of ER alpha in vivo. pp32 Also binds to other nuclear receptors and inhibits thyroid hormone receptor beta-mediated transcription. Taken together, our studies provide evidence that pp32 plays a role in regulating transcription of estrogen-responsive genes by modulating acetylation of histones and ER alpha and also influences transcription of other hormone-responsive genes as well.

A novel estrogen receptor alpha-associated protein, template-activating factor Ibeta, inhibits acetylation and transactivation.

Estrogen receptor-alpha (ERalpha) functions as a ligand-activated transcription factor that alters expression of estrogen-responsive genes in target cells. Numerous regulatory proteins interact with ERalpha to influence estrogen-mediated transactivation. We have identified a novel coregulatory protein, template-activating factor-Ibeta (TAF-Ibeta), which binds to ERalpha in vitro when the receptor is not complexed with an estrogen response element. The central region of TAF-Ibeta interacts with both the DNA-binding domain and the carboxy-terminal region of ERalpha. Coimmunoprecipitation experiments demonstrate that TAF-Ibeta is associated with the unoccupied, but not the estrogen-occupied, ERalpha in MCF-7 breast cancer cells. Overexpression of TAF-Ibeta inhibits ERalpha-mediated transcription in a dose- dependent manner. TAF-Ibeta represses p300-mediated acetylation of histones and ERalpha in vitro and decreases ERalpha acetylation in vivo. TAF-Ibeta also binds to other nuclear receptor superfamily members and represses thyroid hormone receptor beta- induced transcription in transient transfection assays. Taken together, these data provide evidence that TAF-Ibeta regulates transcription of estrogen- responsive genes by modulating acetylation of histones and ERalpha and that the effects of TAF-Ibeta extend to other nuclear receptor superfamily members as well.

Estrogen receptor alpha and activating protein-1 mediate estrogen responsiveness of the progesterone receptor gene in MCF-7 breast cancer cells.

The progesterone receptor (PR) gene is activated by estrogen in MCF-7 human breast cancer cells. Although the human PR gene does not contain an estrogen response element (ERE), we have identified a putative activating protein-1 (AP-1) site at +90 in the PR gene that was hypersensitive to deoxyribonuclease I cleavage in genomic Southern analysis, bound purified Fos and Jun, formed a complex with Fos/Jun heterodimers present in MCF-7 nuclear extracts in gel mobility shift assays, and functioned as an estrogen-responsive enhancer in transient cotransfection assays. When the +90 AP-1 site was mutated in the context of the PR gene, estrogen responsiveness was significantly decreased. Purified estrogen receptor (ER) enhanced binding of Fos and Jun to the +90 AP-1 site and bound to an adjacent imperfect ERE half-site. Mutating this ERE half-site diminished the binding of ER, Fos, and Jun and decreased transcription. Chromatin immunoprecipitation assays demonstrated that the ER, Fos, and Jun were present at the +90 AP-1 site in the endogenous PR gene only after treatment of MCF-7 cells with estrogen. These studies suggest that the cooperative interaction of the ER with Fos and Jun proteins helps confer estrogen responsiveness to the endogenous PR gene.

Differential modulation of DNA conformation by estrogen receptors alpha and beta.

The human estrogen receptor (ER) induces transcription of estrogen-responsive genes upon binding to estrogen and the estrogen response element (ERE). To determine whether receptor-induced changes in DNA structure are related to transactivation, we compared the abilities of ER alpha and ER beta to activate transcription and induce distortion and bending in DNA. ER alpha induced higher levels of transcription than ER beta in the presence of 17 beta-estradiol. In circular permutation experiments ER alpha induced greater distortion in DNA fragments containing the consensus ERE sequence than ER beta. Phasing analysis indicated that ER alpha induced a bend directed toward the major groove of the DNA helix but that ER beta failed to induce a directed DNA bend. Likewise, the ER alpha DNA binding domain (DBD) and hinge region induced a bend directed toward the major groove of the DNA helix, but the ER beta DBD and hinge region failed to bend ERE-containing DNA fragments. Using receptor chimeras we demonstrated that the ER alpha DBD C-terminal extension is required for directed DNA bending. Transient transfection assays revealed that appropriately oriented DNA bending enhances receptor-mediated transactivation. The different abilities of ER alpha and ER beta to induce change in DNA structure could foster or inhibit the interaction of regulatory proteins with the receptor and other transcription factors and help to explain how estrogen-responsive genes are differentially regulated by these two receptors.