Peptidylprolyl Isomerase Pin1 Directly Enhances the DNA Binding Functions of Estrogen Receptor α.

The transcriptional activity of estrogen receptor α (ERα), the key driver of breast cancer proliferation, is enhanced by multiple cellular interactions, including phosphorylation-dependent interaction with Pin1, a proline isomerase, which mediates cis-trans isomerization of the N-terminal Ser(P)(118)-Pro(119) in the intrinsically disordered AF1 (activation function 1) domain of ERα. Because both ERα and Pin1 have multiple cellular partners, it is unclear how Pin1 assists in the regulation of ERα transactivation mechanisms and whether the functional effects of Pin1 on ERα signaling are direct or indirect. Here, we tested the specific action of Pin1 on an essential step in ERα transactivation, binding to specific DNA sites. DNA binding analysis demonstrates that stable overexpression of Pin1 increases endogenous ERα DNA binding activity when activated by estrogen but not by tamoxifen or EGF. Increased DNA binding affinity is a direct effect of Pin1 on ERα because it is observed in solution-based assays with purified components. Further, our data indicate that isomerization is required for Pin1-modulation of ERα-DNA interactions. In an unbiased in vitro DNA binding microarray with hundreds of thousands of permutations of ERα-binding elements, Pin1 selectively enhances the binding affinity of ERα to consensus DNA elements. These studies reveal that Pin1 isomerization of phosphorylated ERα can directly regulate the function of the adjacent DNA binding domain, and this interaction is further modulated by ligand binding in the ligand-binding domain, providing evidence for Pin1-dependent allosteric regulation of ERα function.

Elevated GRHL2 Imparts Plasticity in ER-Positive Breast Cancer Cells.

Estrogen receptor (ER)-positive breast cancer is characterized by late recurrences following initial treatment. The epithelial cell fate transcription factor Grainyhead-like protein 2 (GRHL2) is overexpressed in ER-positive breast cancers and is linked to poorer prognosis as compared to ER-negative breast cancers. To understand how GRHL2 contributes to progression, GRHL2 was overexpressed in ER-positive cells. We demonstrated that elevated GRHL2 imparts plasticity with stem cell- and dormancy-associated traits. RNA sequencing and immunocytochemistry revealed that high GRHL2 not only strengthens the epithelial identity but supports a hybrid epithelial to mesenchymal transition (EMT). Proliferation and tumor studies exhibited a decrease in growth and an upregulation of dormancy markers, such as NR2F1 and CDKN1B. Mammosphere assays and flow cytometry revealed enrichment of stem cell markers CD44 and ALDH1, and increased self-renewal capacity. Cistrome analyses revealed a change in transcription factor motifs near GRHL2 sites from developmental factors to those associated with disease progression. Together, these data support the idea that the plasticity and properties induced by elevated GRHL2 may provide a selective advantage to explain the association between GRHL2 and breast cancer progression.

Analysis of ligand-dependent recruitment of coactivator peptides to RXRbeta in a time-resolved fluorescence resonance energy transfer assay.

Because RXR plays a significant role in nuclear receptor signaling as a common heterodimeric partner for TR, PPAR, RAR, VDR, LXR and others, the ability of RXRbeta ligand binding domain (LBD) to interact with coregulator peptides bearing LXXLL or other interaction motifs was investigated using time-resolved fluorescence resonance energy transfer (TR-FRET). The random phage display peptide D22 and peptides derived from PGC1alpha, SRC1-4, SRC2-3, PRIP/RAP250 and RIP140 yielded the highest TR-FRET signal with RXRbeta LBD in the presence of saturating 9-cis retinoic acid (9-cisRA). Several peptides including D22, PGC1alpha, SRC3-2, PRIP/RAP250 and SRC1-4 also formed a complex with RXRbeta LBD in the presence of all-trans retinoic acid (at-RA) and the fatty acids, phytanic acid (PA) and docosahexaenoic acid (DHA). Determination of the dose dependency (EC50) of these compounds to recruit D22 to RXRbeta LBD indicated that the rank order potency was 9-cisRA>PA>at-RA>DHA. The ligands 9-cisRA and at-RA yielded an overall higher fold-change in D22 recruitment to RXRbeta LBD suggesting that more RXRbeta LBD-D22 complex was formed in the presence of these ligands under the assay conditions tested. The statistical parameter Z' factor for 9-cisRA-induced recruitment of D22 to RXRbeta LBD was 0.6 after 2h incubation, indicating a robust methodology that could be applied to high throughput screening. These results demonstrate that RXRbeta occupied with the fatty acid ligands, DHA and PA, can recruit coactivator peptides in a ligand-dependent manner.

Analysis of ligand-dependent recruitment of coactivator peptides to estrogen receptor using fluorescence polarization.

Ligand-dependent recruitment of coactivators to estrogen receptor (ER) plays an important role in transcriptional activation of target genes. Agonist-bound ER has been shown to adopt a favorable conformation for interaction with the LXXLL motifs of the coactivator proteins. To further examine the affinity and ligand dependence of the ER-coactivator interaction, several fluorescently tagged short peptides bearing an LXXLL motif (LXXLL peptide) from either natural coactivator sequences or random phage display sequences were used with purified ERalpha or ERbeta in an in vitro high-throughput fluorescence polarization assay. In the presence of saturating amounts of ligand, several LXXLL peptides bound to ERalpha and ERbeta with affinity ranging from 20-500 nm. The random phage display LXXLL peptides exhibited a higher affinity for ER than the natural single-LXXLL coactivator sequences tested. These studies indicated that ER agonists, such as 17beta-estradiol or estrone, promoted the interaction of ER with the coactivator peptides, whereas antagonists such as 4-hydroxytamoxifen or ICI-182,780 did not. Different LXXLL peptides demonstrated different affinities for ER depending on which ligand was bound to the receptor, suggesting that the peptides were recognizing different receptor conformations. Using the information obtained from direct measurement of the affinity of the ER-LXXLL peptide interaction, the dose dependency (EC50) of various ligands to either promote or disrupt this interaction was also determined. Interaction of ER with the LXXLL peptide was observed with ligands such as 17beta-estradiol, estriol, estrone, and genistein but not with ICI-182,780, 4-hydroxytamoxifen, clomiphene, or tamoxifen, resulting in distinct EC50 values for each ligand and correlating well with the ligand biological function as an agonist or antagonist. Ligand-dependent recruitment of the LXXLL peptide to ERbeta was observed in the presence of the ERbeta-selective agonist diarylpropionitrile, but not the ERalpha-selective ligand propyl pyrazole triol. This assay could be used to classify unknown ligands as agonists, antagonists, or partial modulators, based on either the receptor-coactivator peptide affinities or the dose dependency of this interaction in comparison with known compounds.

The androgen receptor T877A mutant recruits LXXLL and FXXLF peptides differently than wild-type androgen receptor in a time-resolved fluorescence resonance energy transfer assay.

The interactions of the ligand binding domain (LBD) of androgen receptor (AR) and the AR T877A mutant, found in prostate cancer, with peptides from coactivator and corepressor proteins or random phage display peptides were investigated using in vitro time-resolved fluorescence resonance energy transfer (TR-FRET). Interaction of wild-type AR LBD with the random phage display peptide D11FxxLF was observed with dihydrotestosterone (DHT), testosterone, R1881, estradiol, spironolactone, progesterone, and cortisol resulting in distinct dose dependency (EC50) values for each ligand and correlating well with the reported rank order potency of these agonists. Increasing concentrations of cyproterone acetate and mifepristone resulted in more complete disruption of the DHT-mediated AR-D11FxxLF peptide interaction, while flutamide, hydroxyflutamide, and bicalutamide caused only partial disruption of the complex. The mutant AR T877A LBD exhibited increased binding affinities for all ligands tested except for bicalutamide, mifepristone, DHT, and R1881 in a competitive binding assay as compared to wild-type AR LBD. This mutation was also characterized by increased ligand potency for agonist-induced peptide recruitment. Although usually an antagonist, hydroxyflutamide was more potent in the recruitment of D11FxxLF or an SRC3-1 LXXLL motif to AR T877A LBD than AR LBD. The antagonist cyproterone acetate behaved as a full antagonist of D11FxxLF recruitment to AR LBD and AR T877A LBD but as a more potent agonist in the recruitment of SRC3-1 to AR T877A LBD. These results suggest that the AR T877A mutation affects both ligand affinity and ligand dose dependency for peptide recruitment and may explain in part the altered responses of antagonists and increased transcriptional activation reported in androgen-independent prostate cancers.

Development of a coactivator displacement assay for the orphan receptor estrogen-related receptor-gamma using time-resolved fluorescence resonance energy transfer.

The estrogen-related receptor-gamma (ERRgamma) is a constitutively active orphan receptor that belongs to the nuclear receptor superfamily and is most closely related to the estrogen receptors. Although its physiological ligand is unknown, ERRgamma has been shown to interact with synthetic estrogenic compounds such as 4-hydroxytamoxifen (4-OHT), tamoxifen, and diethylstilbestrol (DES). To assess how coregulator proteins interact with ERRgamma in response to ligand, an in vitro interaction methodology using time-resolved fluorescence resonance energy transfer (TR-FRET) was developed using glutathione S-transferase (GST)-tagged ERRgamma ligand-binding domain (LBD), a terbium-labeled anti-GST antibody, a fluorescein-labeled peptide containing sequences derived from coregulator proteins, and various ligands. An initial screen of these coregulator peptides bearing the coactivator LXXLL motif, the corepressor LXXI/HIXXXI/L motif, or other interaction motifs from natural coactivator sequences or random phage display peptides indicated that the peptides PGC1alpha, D22, and SRC1-4, known as class III coregulators, interacted most strongly with ERRgamma in the absence of ligand. Given its assay window and biological relevance in energy metabolism and obesity, further studies were conducted with PGC1alpha. Fluorescein-labeled PGC1alpha peptide was displaced from the ERRgamma LBD in the presence of increasing concentrations of 4-OHT and tamoxifen, but DES was less effective in PGC1alpha displacement. The statistical parameter Z' factor that measures the robustness of the assay was greater than 0.8 for displacement of PGC1alpha from ERRgamma LBD in the presence of saturating 4-OHT over an assay incubation time of 1-6 h, indicating an excellent assay. These findings also suggest that binding of 4-OHT, tamoxifen, or DES to ERRgamma results in differential affinity of coregulators for ERRgamma due to unique ligand-induced conformations.