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Coactivation of Estrogen Receptor α (ERα)/Sp1 By Vitamin D Receptor Interacting Protein 150 (DRIP150)
Abstract
Vitamin D receptor interacting protein (DRIP150) coactivates estrogen receptor alpha (ERalpha)-mediated transactivation in breast cancer cell lines transfected with a construct (pERE(3)) containing three estrogen responsive elements (EREs). In this study, we show that DRIP150 also coactivates ERalpha/Sp1-mediated transactivation in ZR-75, MCF-7, and MDA-MB-231 breast cancer cells transfected with a construct (pSp1(3)) containing three consensus GC-rich motifs. Studies on coactivation of wild-type and variant ERalpha/Sp1 by DRIP150 indicates that the DNA-binding domain and helix 12 in the ligand binding domain of ERalpha are required and the coactivation response is squelched by overexpressing an NR-box peptide that contains two LXXLL motifs from GRIP2. In contrast, coactivation of ERalpha/Sp1 by wild-type and mutant DRIP150 expression plasmids show that coactivation of ERalpha/Sp1 by DRIP150 is independent of the NR-boxes. Deletion analysis of DRIP150 demonstrates that coactivation requires an alpha-helical NIFSEVRVYN (amino acids 795-804) motif within 23 amino acid sequence (789-811) in the central region of DRIP150 and similar results were obtained for coactivation of ERalpha by DRIP150. Thus, although different domains of ERalpha are required for hormone-dependent activation of ERalpha and ERalpha/Sp1, coactivation of these transcription factors by DRIP150 requires the alpha-helical amino acids 795-804. This is the first report of a coactivator that enhances ERalpha/Sp1-mediated transactivation in breast cancer cells.
... Mdm2 which exhibits ubiquitin E3 ligase activity is involved in this cycling process, and previous studies show that Mdm2 overexpression coactivates ERα-dependent transactivation in cells transfected with ERE-promoter constructs (Saji et al. 2001). Research in this laboratory and others have demonstrated that E2 also activates genes in breast cancer cells through ERα/Sp interactions with GC-rich promoters (O'Malley 2007), and we have been investigating coactivation of ERα and ERα/Sp in breast cancer cells transfected with pERE 3 and pSp1 3 , respectively (Kim et al. 2003;Wu et al. 2004;Lee et al. 2005;Lee & Safe 2007). Vitamin D interacting protein 150 (DRIP150) is a mediator complex protein (Koh et al. 2002;Kouzmenko et al. 2004) that coactivates ERα and ERα/Sp1, and this involves an α-helical NIFSEVRVYN (amino acids 795-804) motif within a twenty-three amino acid sequence (789-811) in the central region of DRIP150 which does not contain an LXXLL box (Lee et al. 2005;Lee & Safe 2007). ...
... Research in this laboratory and others have demonstrated that E2 also activates genes in breast cancer cells through ERα/Sp interactions with GC-rich promoters (O'Malley 2007), and we have been investigating coactivation of ERα and ERα/Sp in breast cancer cells transfected with pERE 3 and pSp1 3 , respectively (Kim et al. 2003;Wu et al. 2004;Lee et al. 2005;Lee & Safe 2007). Vitamin D interacting protein 150 (DRIP150) is a mediator complex protein (Koh et al. 2002;Kouzmenko et al. 2004) that coactivates ERα and ERα/Sp1, and this involves an α-helical NIFSEVRVYN (amino acids 795-804) motif within a twenty-three amino acid sequence (789-811) in the central region of DRIP150 which does not contain an LXXLL box (Lee et al. 2005;Lee & Safe 2007). DRIP150 had minimal effects on Sp-dependent transactivation and coactivated ERα and ERα/Sp1 primarily through interactions with ERα (Lee & Safe 2007). ...
... Vitamin D interacting protein 150 (DRIP150) is a mediator complex protein (Koh et al. 2002;Kouzmenko et al. 2004) that coactivates ERα and ERα/Sp1, and this involves an α-helical NIFSEVRVYN (amino acids 795-804) motif within a twenty-three amino acid sequence (789-811) in the central region of DRIP150 which does not contain an LXXLL box (Lee et al. 2005;Lee & Safe 2007). DRIP150 had minimal effects on Sp-dependent transactivation and coactivated ERα and ERα/Sp1 primarily through interactions with ERα (Lee & Safe 2007). In this study, we demonstrate that E2-induced transactivation in breast cancer cells transfected with pSp1 3 or pERE 3 was significantly inhibited by knockdown of Mdm2 by RNA interference (siMdm2) (Figs. ...
... Mdm2 which exhibits ubiquitin E3 ligase activity is involved in this cycling process, and previous studies show that Mdm2 overexpression coactivates ERα-dependent transactivation in cells transfected with ERE-promoter constructs (Saji et al. 2001). Research in this laboratory and others have demonstrated that E2 also activates genes in breast cancer cells through ERα/Sp interactions with GC-rich promoters (O'Malley 2007), and we have been investigating coactivation of ERα and ERα/Sp in breast cancer cells transfected with pERE 3 and pSp1 3 , respectively (Kim et al. 2003;Wu et al. 2004;Lee et al. 2005;Lee & Safe 2007). Vitamin D interacting protein 150 (DRIP150) is a mediator complex protein (Koh et al. 2002;Kouzmenko et al. 2004) that coactivates ERα and ERα/Sp1, and this involves an α-helical NIFSEVRVYN (amino acids 795-804) motif within a twenty-three amino acid sequence (789-811) in the central region of DRIP150 which does not contain an LXXLL box (Lee et al. 2005;Lee & Safe 2007). ...
... Research in this laboratory and others have demonstrated that E2 also activates genes in breast cancer cells through ERα/Sp interactions with GC-rich promoters (O'Malley 2007), and we have been investigating coactivation of ERα and ERα/Sp in breast cancer cells transfected with pERE 3 and pSp1 3 , respectively (Kim et al. 2003;Wu et al. 2004;Lee et al. 2005;Lee & Safe 2007). Vitamin D interacting protein 150 (DRIP150) is a mediator complex protein (Koh et al. 2002;Kouzmenko et al. 2004) that coactivates ERα and ERα/Sp1, and this involves an α-helical NIFSEVRVYN (amino acids 795-804) motif within a twenty-three amino acid sequence (789-811) in the central region of DRIP150 which does not contain an LXXLL box (Lee et al. 2005;Lee & Safe 2007). DRIP150 had minimal effects on Sp-dependent transactivation and coactivated ERα and ERα/Sp1 primarily through interactions with ERα (Lee & Safe 2007). ...
... Vitamin D interacting protein 150 (DRIP150) is a mediator complex protein (Koh et al. 2002;Kouzmenko et al. 2004) that coactivates ERα and ERα/Sp1, and this involves an α-helical NIFSEVRVYN (amino acids 795-804) motif within a twenty-three amino acid sequence (789-811) in the central region of DRIP150 which does not contain an LXXLL box (Lee et al. 2005;Lee & Safe 2007). DRIP150 had minimal effects on Sp-dependent transactivation and coactivated ERα and ERα/Sp1 primarily through interactions with ERα (Lee & Safe 2007). In this study, we demonstrate that E2-induced transactivation in breast cancer cells transfected with pSp1 3 or pERE 3 was significantly inhibited by knockdown of Mdm2 by RNA interference (siMdm2) (Figs. ...
Murine double minute clone 2 (MDM2) is a multifunctional protein, which modulates nuclear receptor-mediated transactivation. In this study, we show that MDM2 significantly enhanced estrogen receptor α (ERα) and ERα/specificity protein-mediated transactivation in MCF-7 and ZR-75 breast cancer cells. This was demonstrated by both MDM2 overexpression and knockdown experiments by RNA interference. ERα interacted with wild-type MDM2 and deletion mutants of MDM2 containing amino acids 1-342 (C-terminal deletion) and 134-490 (N-terminal deletion), but not 134-342. In contrast, only wild-type but not mutant MDM2 enhanced ERα-mediated transactivation. Protein-protein interactions in vitro were 17β-estradiol (E(2)) independent, whereas fluorescent resonance energy transfer experiments in living cells showed that E(2) enhanced ERα-MDM2 interactions. Subsequent RNA interference and mammalian two-hybrid experiments suggested that MDM2 did not directly interact with endogenous coactivators such as the steroid receptor coactivators but played a role in enhancing ERα-mediating gene expression and estrogen responsiveness through interactions with ERα.
... The IFIT1 gene was chosen in spite of this modest increase because it codes for a protein that has been suggested to function in the pathogenesis of SLE [28]. Second, was the DRIP150 gene since this gene product functions as a cofactor for ER mediated transcription [29] and could be responsible for the increased sensitivity of SLE T cells to estradiol. ...
... In addition, coactivators can recruit other factors to the promoter of responsive genes that enhance the transcriptional response. DRIP150 is a member of a multi-protein complex that shares several subunits with the mammalian Mediator complex [29,54]. Mediator complexes function as a bridge between distal transcription activators and RNA polymerase II. ...
The major risk factor for developing systemic lupus erythematosus (SLE) is being female. The present study utilized gene profiles of activated T cells from females with SLE and healthy controls to identify signaling pathways uniquely regulated by estradiol that could contribute to SLE pathogenesis. Selected downstream pathway genes (+/- estradiol) were measured by real time polymerase chain amplification. Estradiol uniquely upregulated six pathways in SLE T cells that control T cell function including interferon-alpha signaling. Measurement of interferon-alpha pathway target gene expression revealed significant differences (p= 0.043) in DRIP150 (+/- estradiol) in SLE T cell samples while IFIT1 expression was bimodal and correlated moderately (r= 0.55) with disease activity. The results indicate that estradiol alters signaling pathways in activated SLE T cells that control T cell function. Differential expression of transcriptional coactivators could influence estrogen-dependent gene regulation in T cell signaling and contribute to SLE onset and disease pathogenesis.
... Both carcinogenesis models used in these studies were estrogen responsive and the tumors were estrogen receptor positive (10,11). A few studies have suggested a cross talk between 1α,25(OH) 2 D 3 and estrogen signaling pathway (12,13). Treatment with 1α,25(OH) 2 D 3 has been shown to down-regulate ERα mRNA level in breast cancer cells, preventing estrogen-induced cellular proliferation. ...
... Similarly, ERα expression decreased following 1α(OH)D5 treatment and this decrease was abolished in presence of protein synthesis inhibitor CHX. In another study, 1α,25(OH) 2 D 3 treatment reduced ERα mRNA level and estrogenic activity in MCF-7 cells (13). This decrease in mRNA was not abolished in presence of CHX. ...
Vitamin D analog, 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha(OH)D5), is a less toxic VDR agonist that suppresses proliferation of breast cancer cells in vitro and in vivo. The present study assessed 1alpha(OH)D5-mediated regulation of VDR, and its potential anti-estrogenic activity in BT-474 cells.
The mRNA and protein expression of steroid receptors were determined using RT-PCR and Western blot analyses, respectively.
VDR mRNA was up-regulated (180% of control) by 1alpha(OH)D5 within seven hours, whereas the expression of VDR protein increased by two-fold in 24 hours. This increase was abolished in presence of either actinomycin D or cyclohexamide. Additionally, there was a four-fold decrease in ERalpha mRNA and 40% decrease in ERalpha protein after 28 and 48 hours following 1alpha(OH)D5 treatment, respectively. Down-regulation of some of the estrogen-inducible genes was observed.
Although no VDR stabilization by 1alpha(OH)D5 was observed, there was an increased expression of the VDR followed by partial anti-estrogenic activity in hormone-responsive BT-474 cells.
... [58][59][60] In the later pathway, ER-Sp1 can recruit co-activator or co-repressor to activate or repress the transcription of target genes. 61,62 Interestingly, previous reports revealed that the Sp1 site (GC box), which can mediate nonclassical ERβ action, exists in ERα Promoter A-specific region (−245 to −182 bp). [39][40][41] Also, a previous report by Bartella et al showed that ERβ recruits NCoR and displaces RNA polymerase II at the Sp1 site to repress the transcription of ERα gene in breast cancer cells. ...
Normal thyroid tissue displays a prevalent expression of ERβ than ERα, which drastically turns upside down in the initiation and progression of papillary thyroid cancer (PTC). The underlying molecular mechanism of this phenomenon remains unclear. Here, we demonstrated that ERα and ERβ were coexpressed in human thyroid tissues and cells. ERα mRNA (A‐1) and ERβ mRNA (0N‐1), transcribed from Promoter A of ERα gene and Promoter 0N of ERβ gene, respectively, were the major mRNA isoforms which mainly contributed to total ERα mRNA and total ERβ mRNA in human thyroid‐derived cell lines and tissues. The expression levels of ERα mRNA (A‐1) and total ERα mRNA were gradually increased, and those of ERβ mRNA (0N‐1) and total ERβ mRNA were decreased by degree in the initiation and progression of PTC. No aberrant DNA methylation of ERα 5′‐untranslated region was involved in its up‐regulation; however, aberrant DNA methylation in Promoter 0N and Exon 0N of ERβ gene was found to be involved in its down‐regulation in the initiation and progression of PTC. ERβ can repress ERα gene transcription via recruitment of NCoR and displacement of RNA polymerase II at the Sp1 site in ERα Promoter A‐specific region in thyroid‐derived cells. It is suggested that DNA methylation of CpG islands in Promoter 0N and Exon 0N of ERβ gene leads to a decreased ERβ gene expression, which attenuates its inhibitory effect on ERα gene transcription and results in an increased ERα gene expression, cell proliferation, initiation, and progression of PTC.
... The expression of the ERβ is found in kidney, brain, bone, prostate, heart, lungs, and endothelial cells [2]. The binding and the functional selectivity of the ER helix with 12 domains play a vital role in the determination of the binding interactions with co activators and co repressors along with the effect of the ligand on their respective agonists and antagonists [3]. Estrogens play key roles in development and maintenance of normal sexual and reproductive function. ...
Estrogen receptors (ER) are overexpressed in human breast cancers and associated with differentiated tumors with a more favorable prognosis. Paradoxically, these receptors mediate the mitogenic action of estrogens in human breast cancer cells and the efficacy of antiestrogens in adjuvant therapy of primary tumors. The mode of action of overexpression.Estrogen receptors (ER) protein plays a critical role for causing the breast cancer. The exact mechanism underlying the ER protection against cancer progression to metastasis remains to be investigated. Here in this study we show that Pharmacophores of Diethylstilbestrol and other four drugs molecules were designed and screened. The main objective of this study is to design a New scaffold to block the estrogen receptor through 'Molecular Docking approach' by using the In silico tools. Further, Chemical Diversity and ADME screening are also desired which increase our ability to predict and model the most relevant pharmacokinetic and metabolic endpoints, thereby accelerating the drug discovery process.
... MED14 has been shown to interact with the N-terminal AF-1 region of GR (44), leaving the C-terminal AF-2 region free to interact with other transcriptional regulators. In breast cancer cell lines, MED14 has also been shown to act as an ERα co-activator through helix-12 interaction (45), further implicating this component of the mediator complex as a scaffold for recruitment of additional factors. This is the first data to suggest a novel integrator role for mediator proteins to modulate recruitment to sites throughout the genome of different transcription factors under various hormonal stimuli. ...
Nuclear receptors (NRs) are central regulators of pathophysiological processes; however, how their responses intertwine is
still not fully understood. The aim of this study was to determine whether and how steroid NRs can influence each other’s
activity under co-agonist treatment. We used a unique system consisting of a multicopy integration of an estrogen receptor
responsive unit that allows direct visualization and quantification of estrogen receptor alpha (ERα) DNA binding, co-regulator
recruitment and transcriptional readout. We find that ERα DNA loading is required for other type I nuclear receptors to be
co-recruited after dual agonist treatment. We focused on ERα/glucocorticoid receptor interplay and demonstrated that it requires
steroid receptor coactivators (SRC-2, SRC-3) and the mediator component MED14. We then validated this cooperative interplay
on endogenous target genes in breast cancer cells. Taken together, this work highlights another layer of mechanistic complexity
through which NRs cross-talk with each other on chromatin under multiple hormonal stimuli.
... Receptor-interacting protein 140 (RIP140) repressed E2-induced activation of ERα/ AP-1 by reversing the effects of SRC2 (Teyssier et al. 2003). In contrast, studies with the vitamin D-interacting protein 150 (DRIP150) coactivator demonstrated that DRIP150 coactivation of ERα/Sp was LXXLL-box-independent and required a novel helical region in DRIP150 (Lee & Safe 2007). Cell context-dependent induction and repression of VEGFR2 in ZR-75 and MCF-7 cells, respectively, is associated with the same GC-rich promoter sequences but there were differences in recruitment of coactivators (induction) and corepressors such as NCoR and SMRT (Higgins et al. 2006; Higgins et al. 2008). ...
17beta-estradiol binds to the estrogen receptor (ER) to activate gene expression or repression and this involves both genomic (nuclear) and non-genomic (extranuclear) pathways. Genomic pathways include the classical interactions of ligand-bound ER dimers with estrogen-responsive elements in target gene promoters. ER-dependent activation of gene expression also involves DNA-bound ER that subsequently interacts with other DNA-bound transcriptions factors and direct ER-transcription factor (protein-protein) interactions where ER does not bind promoter DNA. Ligand-induced activation of ER/specificity protein (Sp) and ER/activating protein-1 [(AP-1); consisting of jun/fos] complexes are important pathways for modulating expression of a large number of genes. This review summarizes some of the characteristics of ER/Sp- and ER/AP-1-mediated transactivation, which are dependent on ligand structure, cell context, ER-subtype (ERalpha and ERbeta), and Sp protein (SP1, SP3, and SP4) and demonstrates that this non-classical genomic pathway is also functional in vivo.
... Similarly, ESR1 and SP1 coactivation is well documented (56 -59). For example, vitamin D receptor interacting protein (DRIP150) coactivates ESR1 and SP1 in breast cancer cell lines (58). It is reasonable to predict that inactivating one may suppress the expression of the other. ...
A major challenge in cancer chemotherapy has been developing safe and clinically efficacious chemotherapeutic agents. With its low toxicity profile, curcumin (diferuloylmethane), a naturally occurring flavinoid derived from the rhizome of Curcuma longa, has great promise. In vitro and in vivo preclinical studies have shown its inhibitory anticancer, antioxidant, anti-inflammatory, antiproliferative, and proapoptotic activities. The multiple mechanisms of the antitumor effect of curcumin putatively include down-regulating the expression of gene products such as nuclear factor-kappaB, growth suppression, inducing apoptosis, and modulating various signal transduction pathways and the expression of many oncogenes. The mechanisms underlying the antitumor activity of curcumin have not, however, been completely delineated.
An oligonucleotide microarray chip was developed and used to profile microRNA (miRNA) expressions in pancreatic cells treated with curcumin. Transcripts with regulated expression patterns on the arrays were validated by real-time PCRs. Additionally, potential mRNA targets were analyzed bioinformatically and confirmed with flow cytometry experiments.
Curcumin alters miRNA expression in human pancreatic cells, up-regulating miRNA-22 and down-regulating miRNA-199a*, as confirmed by TaqMan real-time PCR. Upregulation of miRNA-22 expression by curcumin or by transfection with miRNA-22 mimetics in the PxBC-3 pancreatic cancer cell line suppressed expression of its target genes SP1 transcription factor (SP1) and estrogen receptor 1 (ESR1), while inhibiting miRNA-22 with antisense enhanced SP1 and ESR1 expression.
These observations suggest that modulation of miRNA expression may be an important mechanism underlying the biological effects of curcumin.
ABSTRACT
Comparative docking studies have been performed on 10 drug molecules, which play a vital role in the treatment of breast cancer. These 10 drug molecules have the same target called Estrogen Receptor which acts as a DNA-binding transcription factor that regulates gene expression. The glide scores of these 10 drugs were compared to each other using the module Glide which is a part of the Schrödinger software, which provided a better understanding of the binding interactions of the ten drug molecules. Among the 10 drugs taken, toremifene had the lowest glide score, i.e. -9.82, which shows that it had better binding interaction with the protein.
Despite recent advances in targeted treatments for multiple myeloma, optimal molecular therapeutic targets have yet to be identified. To functionally identify critical molecular targets, we conducted a genome-scale lethality study in multiple myeloma cells using siRNAs. We validated the top 160 lethal hits with four siRNAs per gene in three multiple myeloma cell lines and two non-myeloma cell lines, cataloging a total of 57 potent multiple myeloma survival genes. We identified the Bcl2 family member MCL1 and several 26S proteasome subunits among the most important and selective multiple myeloma survival genes. These results provided biologic validation of our screening strategy. Other essential targets included genes involved in RNA splicing, ubiquitination, transcription, translation, and mitosis. Several of the multiple myeloma survival genes, especially MCL1, TNK2, CDK11, and WBSCR22, exhibited differential expression in primary plasma cells compared with other human primary somatic tissues. Overall, the most striking differential functional vulnerabilities between multiple myeloma and non-multiple myeloma cells were found to occur within the 20S proteasome subunits, MCL1, RRM1, USP8, and CKAP5. We propose that these genes should be investigated further as potential therapeutic targets in multiple myeloma.
Once shed from their fostering Sertoli cells, spermatozoa leave the testis and are transported passively by seminiferous fluid through the rete testis. Then, these immature cells enter the complex efferent duct system that is joined to the unique and convoluted epididymal duct. This epididymal duct, lined by a continuous layer of epithelial cells joined by tight junctions, is a tube several meters long (up to 60 m in domestic mammals) and forms an organ that is classically subdivided into 3 major anatomical regions: the head/caput, the corpus/body, and the tail/cauda. Spermatozoa travel throughout the duct for several days to weeks, depending on the species, and may be stored for even longer periods in the cauda part of the epididymis and vas deferens. During their journey the proportion of potentially "mature" spermatozoa increases, but it is only when they reach the cauda epididymidis that almost all spermatozoa have acquired their natural fertilizing ability, which involves progressive motility, the ability to undergo the postejaculatory events (capacitation and hyperactivation), and the capacity to recognize and to bind to the oocyte investments and egg plasma membrane. Recent secretomic, proteomic, and transcriptomic studies have provided new information on the functions and the regionalization of the epididymis and revealed some insights into the complexity of epididymal fluid. Among genes and proteins highly expressed by this tissue, many have roles related to sperm protection (such as oxidation), but a large number of new compounds related to innate immunity have also been discovered. This review will focus on possible new control mechanisms that these studies have suggested for this tissue.
Kisspeptins are natural ligands of G protein-coupled receptor-54. Activation of KiSS1/G protein-coupled receptor-54 signaling pathways results in potent activation of the hypothalamus-pituitary-gonadal axis and initiates puberty. Recent data have shown that in female mice, KiSS1 is positively regulated by estradiol (E(2)) in the anteroventral periventricular nucleus, an important reproductive neuroendocrine brain region, but negatively regulated in the arcuate nucleus. However, little is known about the molecular mechanisms governing E(2)-modulated KiSS1 expression. Here, we demonstrate that the expression level of the KiSS1 gene was up-regulated with the administration of E(2) in estrogen receptor alpha (ERalpha)-positive hypothalamic GT1-7 cells. Using transient transfection of human KiSS1 gene promoter coupled to a luciferase reporter, E(2) increases promoter activity in the presence of ERalpha. Deletion analysis of KiSS1 promoter indicates that the E(2)-regulated increase in promoter activity depends on the Sp1 sites of the proximal promoter region. Using both EMSAs and chromatin immunoprecipitation analysis, we determined that both Sp1 and Sp3 proteins constitutively associate with the four putative Sp1 sites in vitro, whereas the association of ERalpha with the KiSS1 promoter is dependent on E(2) exposure. Sp1 and ERalpha form a complex in vivo to mediate the E(2)-induced activation of KiSS1 promoter. Interestingly, Sp1 transactivates KiSS1 promoter activity, whereas Sp3 functions as a transcriptional repressor. Together, these results demonstrate that E(2)-dependent transcriptional activation of KiSS1 gene is mediated by ERalpha through the interaction of Sp1/Sp3 proteins with the GC-rich motifs of KiSS1 promoter, providing a molecular mechanism of how steroid hormone feedback regulates KiSS1 expression.
Estrogens act as potent mitogens in a large number of breast cancers, and the use of estrogen receptor (ER) antagonists is, there- fore, considered the endocrine therapy of choice in the management of this disease. We describe the molecular properties of EM-652, the active metabolite of EM-800, a novel nonsteroidal antiestrogen com- pound, on the transcriptional functions of ERa and ERb. Using RT-PCR, we show that ERa and ERb are expressed in mouse mam- mary glands, suggesting that both receptors should be considered putative targets for antiestrogen action in the breast. In cotransfec- tion assays using a synthetic estrogen-responsive promoter, EM-652 shows no agonistic activity on ERa and ERb transcriptional function and blocks the estradiol (E2)-mediated activation of both ERa and ERb. EM-652 is also very effective in abrogating E2-stimulated ERa and ERb trans-activation of the pS2 promoter in HeLa cells. EM-652 does not alter binding of ERa and ERb to DNA. The Ras-mediated induction of ERa and ERb transcriptional activity in the presence of E2 is also completely abolished by EM-652. In addition, EM-652 blocks the E2-dependent activation of ERa and ERb by the steroid hormone receptor coactivator-1 as well as the in vitro interaction between SRC-1 and the ligand-binding domains of both ERs. These results demonstrate that the novel antiestrogen EM-800 fully impedes AF-1 and AF-2 activities of ERa and ERb and can, therefore, be considered a potent and pure antagonist of both ER subtypes. (Endocrinology 139: 111-118, 1998)
The estrogen receptor (ER) is expressed in two forms, ERα and ERβ. Here we show that ERα and ERβ, expressed both in vitro and in vivo, form heterodimers which bind to DNA with an affinity (K
d of approximately 2 nm) similar to that of ERα and greater than that of ERβ homodimers. Mutation analysis of the hormone binding domain of ERα suggests
that the dimerization interface required to form heterodimers with ERβ is very similar but not identical to that required
for homodimer formation. The heterodimer, like the homodimers, are capable of binding the steroid receptor coactivator-1 when
bound to DNA and stimulating transcription of a reporter gene in transfected cells. Given the relative expression of ERα and
ERβ in tissues and the difference in DNA binding activity between ERα/ERβ heterodimers and ERβ it seems likely that the heterodimer
is functionally active in a subset of target cells.
Regulation of the low density lipoprotein (LDL) receptor promoter by cholesterol requires a well defined sterol regulatory site and an adjacent binding site for the universal transcription factor Sp1. These elements are located in repeats 2 and 3 of the wild type promoter, respectively. The experiments reported here demonstrate that Sp1 participates in sterol regulation of the LDL receptor in an orientation-specific fashion. We present data which suggest that sterol regulatory element-binding protein (SREBP) increases the binding of Sp1 to the adjacent repeat 3 sequence. We also demonstrate that SREBP and Sp1 synergistically activate expression from the LDL receptor promoter inside the cell by cotransfecting expression vectors encoding each protein into Drosophila tissue culture cells that are devoid of endogenous Sp1. In addition, other transcription factor sites were unable to substitute for Sp1 in sterol regulation when placed next to the SREBP-binding site. These studies together with recent data from others provide the basis of a working model for sterol regulation of the LDL receptor promoter. The presence of Sp1 sites in several other regulated promoters suggests that this universal transcription factor has been recruited to participate in many regulatory responses possibly by a similar mechanism.
We have used a series of human estrogen receptor (ER) mutants to evaluate the cell- and promoter-specific transcriptional activities of the TAF1 and TAF2 transactivation regions within the human ER. We show that the manifestation of TAF1 or TAF2 function depends strongly upon promoter context; on certain promoters, both the TAF1 and TAF2 activators are required for wild-type transcriptional activity, whereas on other promoters, the TAF1 and TAF2 activators function independently. Using these constructs, we show that the antagonist activity of the triphenylethylene-derived antiestrogens, e.g. tamoxifen, arises from their intrinsic inability to activate ER TAF2 function. However, on certain promoters, these antiestrogens efficiently activate gene transcription through ER. Consistent with this observation, the TAF2 function of the ER is not required on all promoters. In these TAF2-independent promoter contexts, TAF2 function may be provided by a separate transcription factor bound to the promoter. These data suggest that 1) TAF1 may be the major transcriptional activator of the ER; and 2) TAF2 functions as a transcriptional facilitator. On promoters where TAF2 function is provided independently of the ER, the TAF1 function of the ER can function independently of TAF2 activity, allowing triphenylethylene-derived antiestrogens to demonstrate partial agonist activity. These observations provide a possible molecular explanation for the tissue-specific partial agonist properties of tamoxifen and related triphenylethylene antiestrogens observed in vivo.
The transcription factor LFB1/HNF1 from rat liver nuclei is a 628 amino acid protein that functions as a dimer binding to the inverted palindrome GTTAATN-ATTAAC consensus site. We have crystallized a 99 residue protein containing the homeodomain portion of LFB1, and solved its structure using X-ray diffraction data to 2.8 A resolution. The topology and orientation of the helices is essentially the same as that found in the engrailed, MAT alpha 2 and Antennapedia homeodomains, even though the LFB1 homeodomain contains 21 more residues. The 21 residue insertion is found in an extension of helix 2 and consequent lengthening of the connecting loop between helix 2 and helix 3. Comparison with the engrailed homeodomain-DNA complex indicates that the mode of interaction with DNA is similar in both proteins, with a number of conserved contacts in the major groove. The extra 21 residues of the LFB1 homeodomain are not involved in DNA binding. Binding of the LFB1 dimer to a B-DNA palindromic consensus sequence requires either a conformational change of the DNA (presumably bending), or a rearrangement of the subunits relative to the DNA.
The cloning of a novel estrogen receptor β (denoted ERβ) has recently been described (Kuiper, G. G. J. M., Enmark, E., Pelto-Huikko, M., Nilsson, S., and Gustafsson, J-A. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 5925-5930 and Mosselman, S., Polman, J., and Dijkema, R. (1996) FEBS Lett. 392, 49- 53). ERβ is highly homologous to the 'classical' estrogen receptor α (here referred to as ERα), has been shown to bind estrogens with an affinity similar to that of ERα, and activates expression of reporter genes containing estrogen response elements in an estrogen-dependent manner. Here we describe functional studies comparing the DNA binding abilities of human ERa and β in gel shift assays. We show that DNA binding by ERα and β are similarly affected by elevated temperature in the absence of ligand or in the presence of 17β-estradiol and the partial estrogen agonist 4-hydroxy- tamoxifen. In the absence of ligand, DNA binding by ERα and β is rapidly lost at 37 °C, while in the presence of 17β-estradiol and 4-hydroxy- tamoxifen, the loss in DNA binding at elevated temperature is much more gradual. We show that the loss in DNA binding is not due to degradation of the receptor proteins. However, while the complete antagonist ICI 182,780 does not 'protect' human ERα (hERα) from loss of DNA binding at elevated temperature in vitro, it does appear to protect human ERβ (hERβ), suggestive of differences in the way ICI 182,780 acts on hERα and β. We further report that ERα and β can dimerize with each other, the DNA binding domain of hERα being sufficient for dimerization with hERβ. Cell and promoter-specific transcription activation by ERα has been shown to be dependent on the differential action of the N- and C-terminal transcription activation functions AF-1 and AF-2, respectively. The existence of a second estrogen receptor gene and the dimerization of ERα and β add greater levels of complexity to transcription activation in response to estrogens.
Nuclear receptors transduce hormonal signals by binding directly to DNA target sites in promoters and modulating the transcription of linked genes. Receptor-mediated transactivation appears to be potentiated in response to ligand by a number of coactivators that may provide key interactions with components of the transcription preinitiation complex and/or alter chromatin structure. Here, we use the vitamin D3 receptor ligand-binding domain (VDR LBD) as an affinity matrix to identify components of a transcriptionally active nuclear extract that interact with VDR in response to ligand. We describe the purification of a complex of at least 10 VDR interacting proteins (DRIPs) ranging from 65 to 250 kD that associate with the receptor in a strictly 1,25-dihydroxyvitamin D3-dependent manner. These proteins also appear to interact with other, but not all, nuclear receptors, such as the thyroid hormone receptor. The DRIPs are distinct from known nuclear receptor coactivators, although like these coactivators, their interaction also requires the AF-2 transactivation motif of VDR. In addition, the DRIP complex contains histone acetyltransferase activity, indicating that at least one or more of the DRIPs may function at the level of nucleosomal modification. However, we show that the DRIPs selectively enhance the transcriptional activity of VDR on a naked DNA template utilizing a cell-free, ligand-dependent transcription assay. Moreover, this activity can be specifically depleted from the extract by liganded, but not unliganded, VDR-LBD. Overexpression of DRIP100 in vivo resulted in a strong squelching of VDR transactivation, suggesting the sequestration of other limiting factors, including components of the DRIP complex. These results demonstrate the existence of a new complex of novel functional nuclear receptor coactivators.
A multiprotein complex isolated from murine cells is identified as a counterpart of the yeast Mediator of transcriptional regulation on the basis of the following: homologs of two subunits of yeast Mediator, Srb7 and Med7, copurify with the complex; peptide sequencing reveals, in addition, homologs of the yeast Mediator subunits Rgr1 and Med6; as with yeast Mediator, the mouse complex binds to the RNA polymerase II C-terminal domain (CTD) and stimulates phosphorylation of the CTD by TFIIH. Peptide sequencing also identifies a component of mouse Mediator as a relative of Ring-3 protein, a mitogen-activated nuclear protein kinase, raising the possibility of Mediator as an end point of signal transduction pathways.
Many growth factors rapidly induce transcription of the c-fos proto-oncogene. We have investigated the pathways for induction of the c-fos promoter by serum and epidermal growth factor (EGF) in HeLa cells. Induction of the serum response element (SRE) of the c-fos promoter could be split into two parts, one involving the serum response factor-associated ternary complex factor (TCF) factors and the second mediated by core SRE sequences. Serum induction was mediated primarily by the core SRE, whereas EGF used both the TCF and core SRE pathways. Using activated and inhibitory signaling proteins, we found that phosphatidyl inositol 3-kinase (PI3K) and rho family members could mediate activation by serum. Activation by PI3K was mediated by core SRE sequences and was dependent upon rac and rho, suggesting a PI3K-to-rac-to-rho pathway for core SRE activation. The PI3K target Akt was also capable of activating the SRE but functioned through the TCF pathway, suggesting that Akt does not mediate the primary PI3K pathway to the SRE and that Akt is capable of activating TCF family members. Serum and EGF induction of the core SRE was partially inhibited by rho and PI3K inhibitors. The use of these inhibitors demonstrates the complexity of signaling pathways to the SRE and suggests that serum activates rho by PI3K-dependent and -independent pathways.
The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 > zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 > genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.
Transcriptional activation by nuclear receptors (NRs) involves the concerted action of coactivators, chromatin components, and the basal transcription machinery. Crucial NR coactivators, which target primarily the conserved ligand-regulated activation (AF-2) domain, include p160 family members, such as TIF2, as well as p160-associated coactivators, such as CBP/p300. Because these coactivators possess intrinsic histone acetyltransferase activity, they are believed to function mainly by regulating chromatin-dependent transcriptional activation. Recent evidence suggests the existence of an additional NR coactivator complex, referred to as the thyroid hormone receptor-associated protein (TRAP) complex, which may function more directly as a bridging complex to the basal transcription machinery. TRAP220, the 220-kDa NR-binding subunit of the complex, has been identified in independent studies using both biochemical and genetic approaches. In light of the functional differences identified between p160 and TRAP coactivator complexes in NR activation, we have attempted to compare interaction and functional characteristics of TIF 2 and TRAP220. Our findings imply that competition between the NR-binding subunits of distinct coactivator complexes may act as a putative regulatory step in establishing either a sequential activation cascade or the formation of independent coactivator complexes.
Nuclear receptors modulate the transcription of genes in direct response to small lipophilic ligands. Binding to ligands induces conformational changes in the nuclear receptors that enable the receptors to interact with several types of cofactor that are critical for transcription activation (transactivation). We previously described a distinct set of ligand-dependent proteins called DRIPs, which interact with the vitamin D receptor (VDR); together, these proteins constitute a new cofactor complex. DRIPs bind to several nuclear receptors and mediate ligand-dependent enhancement of transcription by VDR and the thyroid-hormone receptor in cell-free transcription assays. Here we report the identities of thirteen DRIPs that constitute this complex, and show that the complex has a central function in hormone-dependent transactivation by VDR on chromatin templates. The DRIPs are almost indistinguishable from components of another new cofactor complex called ARC, which is recruited by other types of transcription activators to mediate transactivation on chromatin-assembled templates. Several DRIP/ARC subunits are also components of other potentially related cofactors, such as CRSP, NAT, SMCC and the mouse Mediator, indicating that unique classes of activators may share common sets or subsets of cofactors. The role of nuclear-receptor ligands may, in part, be to recruit such a cofactor complex to the receptor and, in doing so, to enhance transcription of target genes.
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in the stimulation of bone growth, mineralization, and intestinal calcium and phosphate absorption; it also acts as a general inhibitor of cellular proliferation. Several new, clinically relevant compounds dissociate antiproliferative and calcemic activities of 1,25(OH)2D3, but the molecular basis for this has not been clearly elucidated. Here, we tested whether the potency of one class of compounds, 20-epi analogues, to induce myeloid cell differentiation, is because of direct molecular effects on vitamin D receptor (VDR). We report that two 20-epi analogues, MC1627 and MC1288, induced differentiation and transcription of p21(Waf1,Cip1), a key VDR target gene involved in growth inhibition, at a concentration 100-fold lower than that of 1,25(OH)2D3. We compared this sensitivity to analogue effects on VDR interacting proteins: RXR, GRIP-1, and DRIP205, a subunit of the DRIP coactivator complex. Compared with the interaction of VDR with RXR or GRIP-1, the differentiation dose-response most closely correlated to the ligand-dependent recruitment of the DRIP coactivator complex to VDR and to the ability of the receptor to activate transcription in a cell-free system. These results provide compelling links between the efficiency of the 20-epi analogue in inducing VDR/DRIP interactions, transactivation in vitro, and its enhanced ability to induce cellular differentiation.
The hormone-activated glucocorticoid receptor (GR), through its N- and C-terminal transcriptional activation functions AF-1 and AF-2, controls the transcription of target genes presumably through interaction(s) with transcriptional regulatory factors. Utilizing a modified yeast two-hybrid approach, we have identified the tumor susceptibility gene 101 (TSG101) and the vitamin D receptor-interacting protein 150 (DRIP150) as proteins that interact specifically with a functional GR AF-1 surface. In yeast and mammalian cells, TSG101 represses whereas DRIP150 enhances GR AF-1-mediated transactivation. Thus, GR AF-1 is capable of recruiting both positive and negative regulatory factors that differentially regulate GR transcriptional enhancement. In addition, we show that another member of the DRIP complex, DRIP205, interacts with the GR ligand binding domain in a hormone-dependent manner and facilitates GR transactivation in concert with DRIP150. These results suggest that DRIP150 and DRIP205 functionally link GR AF-1 and AF-2, and represent important mediators of GR transcriptional enhancement.
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 ERα/Sp1 and ERβ/Sp1 action using an E2-responsive construct (pSp1) containing a GC-rich promoter. Both ERα and ERβ 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 ERα/Sp1 was observed in MCF-7, MDA-MB-231, and LnCaP cells, but not in HeLa cells. E2 did not affect or significantly decrease
ERβ/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 ERα/β(AF-1α/AF-2β) and ERβ/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 ERβ/α and ERα/β showed that only ERα/β can activate transcription from an Sp1 element, not ERβ/α. This indicates that the AF-1 domain from ERα 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 ERα and ERα/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.
Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D(3) receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.
Nuclear receptors regulate transcription in direct response to their cognate hormonal ligands. Ligand binding leads to the dissociation of corepressors and the recruitment of coactivators. Many of these factors, acting in large complexes, have emerged as potential chromatin remodelers through intrinsic histone modifying activities. In addition, other ligand-recruited complexes appear to act more directly on the transcriptional apparatus. The DRIP complex is a 15-subunit complex required for nuclear receptor transcriptional activation in vitro. It is recruited to the receptor in response to ligand through specific interactions of one subunit, DRIP205. We present evidence that DRIP205 interacts with another member of the steroid receptor subfamily, estrogen receptor (ER). This interaction occurs in an agonist-stimulated fashion which in turn is inhibited by several ER antagonists. In vivo, a fragment of DRIP205 containing only its receptor interacting region acts to selectively inhibit ER's ability to activate transcription in response to estradiol. These observations suggest a key role for the DRIP coactivator complex in estrogen-ER signaling.
Treatment of HEC1A endometrial cancer cells with 10 nm 17β-estradiol (E2) resulted in decreased vascular endothelial growth factor (VEGF) mRNA expression, and a similar response
was observed using a construct, pVEGF1, containing a VEGF gene promoter insert from −2018 to +50. In HEC1A cells transiently
transfected with pVEGF1 and a series of deletion plasmids, it was shown that E2-dependent down-regulation was dependent on
wild-type estrogen receptor α (ERα) and reversed by the anti-estrogen ICI 182,780, and this response was not affected by progestins.
Deletion analysis of the VEGF gene promoter identified an overlapping G/GC-rich site between −66 to −47 that was required
for decreased transactivation by E2. Protein-DNA binding studies using electrophoretic mobility shift and DNA footprinting
assays showed that both Sp1 and Sp3 proteins bound this region of the VEGF promoter. Coimmunoprecipitation and pull-down assays
demonstrated that Sp3 and ERα proteins physically interact, and the interacting domains of both proteins are different from
those previously observed for interactions between Sp1 and ERα proteins. Using a dominant negative form of Sp3 and transcriptional
activation assays in Schneider SL-2 insect cells, it was confirmed that ERα-Sp3 interactions define a pathway for E2-mediated
inhibition of gene expression, and this represents a new mechanism for decreased gene expression by E2.
We previously demonstrated differential interactions of the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane (HPTE) with estrogen receptor alpha (ERalpha), ERbeta, and the androgen receptor (AR). In this study, we characterize the ERalpha, ERbeta, and AR activity of structurally related methoxychlor metabolites. Human hepatoma cells (HepG2) were transiently transfected with human ERalpha, ERbeta, and AR plus an appropriate steroid-responsive luciferase reporter vector. After transfection, cells were treated with various concentrations of HPTE or structurally related compounds in the presence (for detecting antagonism) and absence (for detecting agonism) of 17beta-estradiol and dihydrotestosterone. The monohydroxy analog of methoxychlor, as well as monohydroxy and dihydroxy analogs of 2, 2-bis(p-hydroxyphenyl)-1,1-dichloroethylene, had ERalpha agonist activity and ERbeta and AR antagonist activity similar to HPTE. The trihydroxy metabolite of methoxychlor displayed only weak ERalpha agonist activity and did not alter ERbeta or AR activities. Replacement of the trichloroethane or dichloroethylene group with a methyl group resulted in a compound with ERalpha and ERbeta agonist activity that retained antiandrogenic activities. This study identifies some of the structural requirements for ERalpha and ERbeta activity and demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that simultaneously act as agonists or antagonists through one or more hormone receptors.
Estrogen receptors (ERs) associate with distinct transcriptional coactivators to mediate activation of target genes in response
to estrogens. Previous work has provided multiple evidence for a critical role of p160 coactivators and associated histone
acetyltransferases in estrogen signaling. In contrast, the involvement of the mammalian mediator complex remains to be established.
Further, although the two subtypes ERα and ERβ appear to be similar in regard to principles of LXXLL-mediated coactivator binding to the AF-2 activation domain, there are indications that the context-dependent transcriptional
activation profiles of the two ERs can be quite distinct. Potentially, this could be attributed to differences with regard
to coregulator recruitment. We have here studied the interactions of the nuclear receptor-binding subunit of the mammalian
mediator complex, referred to as TRAP220, with ERα and ERβ. In comparison to the p160 coactivator TIF2, we find that TRAP220
displays ERβ preference. Here, we show that this is a feature of the binding specificity of the TRAP220 LXXLL motifs and demonstrate that the ER subtype-specific F-domain influences TRAP220 interaction. Such differences with regard
to coactivator recruitment indicate that the relative importance of individual coregulators in estrogen signaling could depend
on the dominant ER subtype.
The last two decades have witnessed a tremendous expansion in our knowledge of the mechanisms employed by eukaryotic cells to control gene activity. A critical insight to transcriptional control mechanisms was provided by the discovery of coactivators, a diverse array of cellular factors that connect sequence-specific DNA binding activators to the general transcriptional machinery, or that help activators and the transcriptional apparatus to navigate through the constraints of chromatin. A number of coactivators have been isolated as large multifunctional complexes, and biochemical, genetic, molecular, and cellular strategies have all contributed to uncovering many of their components, activities, and modes of action. Coactivator functions can be broadly divide into two classes: (a) adaptors that direct activator recruitment of the transcriptional apparatus, (b) chromatin-remodeling or -modifying enzymes. Strikingly, several distinct coactivator complexes nonetheless share many subunits and appear to be assembled in a modular fashion. Such structural and functional modularity could provide the cell with building blocks from which to construct a versatile array of coactivator complexes according to its needs. The extent of functional interplay between these different activities in gene-specific transcriptional regulation is only now becoming apparent, and will remain an active area of research for years to come.
Recent studies indicate that the expression of ERβ in breast cancer is lower than in the normal breast, suggesting that ERβ could play an important role in carcinogenesis. To investigate this hypothesis, we engineered ER-negative MDA-MB-231 (human breast cancer cells) to reintroduce either ERα or ERβ protein with an adenoviral vector. In these cells, ERβ (as ERα) expression was monitored using RT-PCR and Western blot. ERβ protein was localized in the nucleus (immunocytochemistry) and able to transactivate estrogen-responsive reporter constructs in the presence of E2. ERβ and ERα induced the expression of several endogenous genes such as pS2, TGFα, or the cyclin kinase inhibitor p21 but, in contrast to ERα, ERβ was unable to regulate c-myc proto-oncogene expression. The pure antiestrogen ICI 164, 384 completely blocked ERα and ERβ estrogen-induced activities. ERβ inhibited MDA-MB-231 cell proliferation in a ligand-independent manner, whereas ERα inhibition of proliferation is hormone dependent. Moreover, ERβ and ERα decreased cell motility and invasion. Our data bring the first evidence that ERβ is an important modulator of proliferation and invasion of breast cancer cells and support the hypothesis that the loss of ERβ expression could be one of the events leading to the development of breast cancer.
Background: Histamine plays important biological roles in cell-to-cell communication; it is a mediator in allergic responses, a regulator of gastric acid secretion, a messenger in bronchial asthma, and a neurotransmitter in the central nervous system. Histamine acts by binding to histamine receptors, and its local action is terminated primarily by methylation. Human histamine N-methyltransferase (HNMT) has a common polymorphism at residue 105 that correlates with the high- (Thr) and low- (Ile) activity phenotypes.Results: Two ternary structures of human HNMT have been determined: the Thr105 variant complexed with its substrate histamine and reaction product AdoHcy and the Ile105 variant complexed with an inhibitor (quinacrine) and AdoHcy. Our steady-state kinetic data indicate that the recombinant Ile105 variant shows 1.8- and 1.3-fold increases in the apparent KM for AdoMet and histamine, respectively, and slightly (16%) but consistently lower specific activity as compared to that of the Thr105 variant. These differences hold over a temperature range of 25°C–45°C in vitro. Only at a temperature of 50°C or higher is the Ile105 variant more thermolabile than the Thr105 enzyme.Conclusions: HNMT has a 2 domain structure including a consensus AdoMet binding domain, where the residue 105 is located on the surface, consistent with the kinetic data that the polymorphism does not affect overall protein stability at physiological temperatures but lowers KM values for AdoMet and histamine. The interactions between HNMT and quinacrine provide the first structural insights into a large group of pharmacologic HNMT inhibitors and their mechanisms of inhibition.
The recent discovery that an additional estrogen receptor subtype is present in various rat tissues has advanced our understanding of the mecha- nisms underlying estrogen signaling. Here we re- port on the cloning of the cDNA encoding the mouse homolog of estrogen receptor-b (ERb) and the functional characterization of mouse ERb pro- tein. ERb is shown to have overlapping DNA-bind- ing specificity with that of the estrogen receptor-a (ERa) and activates transcription of reporter gene constructs containing estrogen-response ele- ments in transient transfections in response to es- tradiol. Using a mammalian two-hybrid system, the formation of heterodimers of the ERb and ERa subtypes was demonstrated. Furthermore, ERb and ERa form heterodimeric complexes with re- tained DNA-binding ability and specificity in vitro. In addition, DNA binding by the ERb/ERa het- erodimer appears to be dependent on both sub- type proteins. Taken together these results sug- gest the existence of two previously unrecognized pathways of estrogen signaling; I, via ERb in cells exclusively expressing this subtype, and II, via the formation of heterodimers in cells expressing both receptor subtypes. (Molecular Endocrinology 11:
To investigate the functional differences between estrogen receptor (ER) α and β subtypes, we studied the expression and the transcription stimulating activities of these receptors. RT-PCR has demonstrated that ERα is expressed at a high level in MCF-7 cells derived from human breast cancer. Both ERα and ERβ were expressed at a lower level in HOS-TE85 and Saos2 cells derived from human osteosarcoma. Chloramphenicol acetyltransferase reporter assay detected the transcriptional activation by the endogenous receptor only in MCF-7 cells. Agonistic effect of tamoxifen was observed as strong as that of 17β-estradiol on ERE activation in MCF-7 cells at the concentration of 10−7M when ERE-containing reporter is constructed with β-globin promoter. The effect of tamoxifen was not apparent when the reporter was constructed with thymidine kinase promoter, suggesting that the differential gene activation between tamoxifen and estrogen may take place depending upon ERE-promoter context. Agonistic activity of tamoxifen was also detected in COS-7 and Saos-2 cells, but not in HEC-1 cells derived from human endometrial carcinoma via exogenously expressed ER. Interestingly, this effect was ERα specific. Thus, we demonstrate that agonistic effect of tamoxifen depends on the cell type, ERE-promoter context, and ER subtype. These parameters would explain at least a part of the tissue specific effects of antiestrogensin vivo.
To develop compounds that are antagonists on ER, but not ER, we have added basic side-chains typically found in non- steroidal antiestrogens to pyrazole compounds that bind with much higher affinity to ER than to ER. In this way we have developed basic side-chain pyrazoles (BSC-pyrazoles) that are high affinity, potent, selective antagonists on ER. These BSC- pyrazoles are themselves inactive on ER and ER, and they antagonize E2 stimulation by ER only. We investigated seven basic side-chain substituents on various alkyl-triaryl-substi- tuted pyrazoles, and the most ER-selective compound was methyl-piperidino-pyrazole (MPP). ER-selective antagonism was observed on diverse reporter-promoter gene constructs containing estrogen response elements that are consensus, nonconsensus (pS2), or comprised of multiple half-estrogen response elements (NHERF/EBP50) and on genes in which ER works indirectly by tethering to other DNA-bound proteins (TGF3). In contrast to these BSC-pyrazoles, the antiestro- gens trans-hydroxytamoxifen, raloxifene, and ICI 182,780 sup- press E2 activity via both ER and ER. The most effective BSC-pyrazole, MPP, fully antagonized E2 stimulation of pS2 mRNA in MCF-7 breast cancer cells, consistent with the fact that these cells contain almost exclusively ER. These com- pounds should be useful in studying the biological functions of ER and ER and in selectively blocking responses that are mediated through ER .( Endocrinology 143: 941-947, 2002)
Recent studies indicate that the expression of ER beta in breast cancer is lower than in the normal breast, suggesting that ER beta could play an important role in carcinogenesis. To investigate this hypothesis, we engineered ER-negative MDA-MB-231 (human breast cancer cells) to reintroduce either ER alpha or ER beta protein with an adenoviral vector. In these cells, ER beta (as ER alpha) expression was monitored using RT-PCR and Western blot. ER beta protein was localized in the nucleus (immunocytochemistry) and able to transactivate estrogen-responsive reporter constructs in the presence of E2. ER beta and ER alpha induced the expression of several endogenous genes such as pS2, TGF alpha, or the cyclin kinase inhibitor p21 but, in contrast to ER alpha, ER beta was unable to regulate c-myc proto-oncogene expression. The pure antiestrogen ICI 164, 384 completely blocked ER alpha and ER beta estrogen-induced activities. ER beta inhibited MDA-MB-231 cell proliferation in a ligand-independent manner, whereas ER alpha inhibition of proliferation is hormone dependent. Moreover, ER beta and ER alpha decreased cell motility and invasion. Our data bring the first evidence that ER beta is an important modulator of proliferation and invasion of breast cancer cells and support the hypothesis that the loss of ER beta expression could be one of the events leading to the development of breast cancer.
A yeast two-hybrid system was used to identify a protein that interacts with and enhances the human progesterone receptor (hPR) transcriptional activity without altering the basal activity of the promoter. Because the protein stimulated transactivation of all the steroid receptors tested, it has been termed steroid receptor coactivator-1 (SRC-1). Coexpression of SRC-1 reversed the ability of the estrogen receptor to squelch activation by hPR. Also, the amino terminal truncated form of SRC-1 acted as a dominant-negative repressor. Together, these results indicate that SRC-1 encodes a coactivator that is required for full transcriptional activity of the steroid receptor superfamily.
We find that tamoxifen is a potent activator of estrogen receptor (ER)- mediated induction of promoters regulated by AP-1 sites including the human collagenase gene promoter and constructs in which an AP-1 site is fused to the herpes thymidine kinase promoter. This contrasts with the inability of tamoxifen to activate otherwise identical promoters bearing classical estrogen response elements. Tamoxifen agonism at AP-1 sites is cell type specific, occurring in cell lines of uterine, but not of breast, origin. It thus parallels tamoxifen agonism in vivo. AP-1 proteins such as Jun or Jun/Fos are needed for tamoxifen stimulation, and tamoxifen increases the transcriptional efficiency of these proteins even when they are provided at optimal amounts. The DNA binding domain (DBD) of ER is required for tamoxifen activation at AP-1 sites. In contrast, estrogen activation is partially independent of this domain. This suggests the existence of two pathways of ER action at AP-1: an alpha (DBD-dependent) pathway activated by tamoxifen, and a beta (DBD-independent) pathway activated by estrogen. Fusing VP16 transcriptional activation functions to ER potentiates the beta, but not the alpha, pathway. We discuss models for the two pathways and the possibility that the AP-1 pathway is a major route by which ER affects target tissue growth and differentiation in vivo.
The estrogen receptor is a transcription factor which, when bound to estradiol, binds DNA and regulates expression of estrogen-responsive
genes. A 160-kilodalton estrogen receptor-associated protein, ERAP160, was identified that exhibits estradiol-dependent binding
to the receptor. Mutational analysis of the receptor shows that its ability to activate transcription parallels its ability
to bind ERAP160. Antiestrogens are unable to promote ERAP160 binding and can block the estrogen-dependent interaction of the
receptor and ERAP160 in a dose-dependent manner. This evidence suggests that ERAP160 may mediate estradiol-dependent transcriptional
activation by the estrogen receptor. Furthermore, the ability of antiestrogens to block estrogen receptor-ERAP160 complex
formation could account for their therapeutic effects in breast cancer.
The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.
Both cyclin D1 and estrogens have an essential role in regulating proliferation of breast epithelial cells. We show here a novel role for cyclin D1 in growth regulation of estrogen-responsive tissues by potentiating transcription of estrogen receptor-regulated genes. Cyclin D1 mediates this activation independent of complex formation to a CDK partner. Cyclin D1 activates estrogen receptor-mediated transcription in the absence of estrogen and enhances transcription in its presence. The activation of estrogen receptor by cyclin D1 is not inhibited by anti-estrogens. A direct physical binding of cyclin D1 to the hormone binding domain of the estrogen receptor results in an increased binding of the receptor to estrogen response element sequences, and upregulates estrogen receptor-mediated transcription. These results highlight a novel role for cyclin D1 as a CDK-independent activator of the estrogen receptor.
The estrogen receptor (ER) is expressed in two forms, ERalpha and ERbeta. Here we show that ERalpha and ERbeta, expressed both in vitro and in vivo, form heterodimers which bind to DNA with an affinity (Kd of approximately 2 nM) similar to that of ERalpha and greater than that of ERbeta homodimers. Mutation analysis of the hormone binding domain of ERalpha suggests that the dimerization interface required to form heterodimers with ERbeta is very similar but not identical to that required for homodimer formation. The heterodimer, like the homodimers, are capable of binding the steroid receptor coactivator-1 when bound to DNA and stimulating transcription of a reporter gene in transfected cells. Given the relative expression of ERalpha and ERbeta in tissues and the difference in DNA binding activity between ERalpha/ERbeta heterodimers and ERbeta it seems likely that the heterodimer is functionally active in a subset of target cells.
The transactivation properties of the two estrogen receptors, ERα and ERβ, were examined with different ligands in the context
of an estrogen response element and an AP1 element. ERα and ERβ were shown to signal in opposite ways when complexed with
the natural hormone estradiol from an AP1 site: with ERα, 17β-estradiol activated transcription, whereas with ERβ, 17β-estradiol
inhibited transcription. Moreover, the antiestrogens tamoxifen, raloxifene, and Imperial Chemical Industries 164384 were potent
transcriptional activators with ERβ at an AP1 site. Thus, the two ERs signal in different ways depending on ligand and response
element. This suggests that ERα and ERβ may play different roles in gene regulation.
The recent discovery that an additional estrogen receptor subtype is present in various rat tissues has advanced our understanding of the mechanisms underlying estrogen signaling. Here we report on the cloning of the cDNA encoding the mouse homolog of estrogen receptor-beta (ER beta) and the functional characterization of mouse ER beta protein. ER beta is shown to have overlapping DNA-binding specificity with that of the estrogen receptor-alpha (ER alpha) and activates transcription of reporter gene constructs containing estrogen-response elements in transient transfections in response to estradiol. Using a mammalian two-hybrid system, the formation of heterodimers of the ER beta and ER alpha subtypes was demonstrated. Furthermore, ER beta and ER alpha form heterodimeric complexes with retained DNA-binding ability and specificity in vitro. In addition, DNA binding by the ER beta/ER alpha heterodimer appears to be dependent on both subtype proteins. Taken together these results suggest the existence of two previously unrecognized pathways of estrogen signaling; I, via ER beta in cells exclusively expressing this subtype, and II, via the formation of heterodimers in cells expressing both receptor subtypes.
A GC-rich oligonucleotide containing an estrogen responsive element (ERE) half-site from the heat shock protein 27 (Hsp 27) gene promoter (-105 to -84) [ie. GGGCGGG(N)10GGTCA; Sp1(N)10ERE] forms a complex with the Sp1 and estrogen receptor (ER) proteins. Moreover, promoter-reporter constructs containing this sequence (-108 to -84 or -108 to +23) are also estrogen-responsive. Mutation of the ERE half-site in the Hsp 27-derived oligonucleotides did not result in loss of estrogen responsiveness in transient transfection studies, suggesting that estrogen inducibility was mediated through the Sp1-DNA motif. Gel mobility shift assays using 32P-labeled wild type and ERE mutant Sp1(N)10ERE and consensus Sp1 oligonucleotides showed that Sp1 protein formed a DNA-protein complex with all three nucleotides, and the intensities of retarded bands were enhanced by coincubation with wild type ER and 11C-ER, which does not contain the DNA-binding domain. ER mutants in which N-terminal (19C-ER) and C-terminal (15C-ER) regions were deleted did not enhance Sp1-DNA binding or hormone-induced transactivation of GC-rich promoter-reporter constructs in ER-negative MDA-MB-231 cells, whereas both wild type and 11C-ER restored inducibility. Immunoprecipitation studies also confirmed that the Sp1 and ER proteins physically interact. The interaction of the Sp1 and ER proteins and the resulting enhanced Sp1-DNA binding is observed in the presence or absence of estrogen (hormone-independent), whereas transactivation of promoter-reporter constructs is estrogen-dependent. Thus, the results illustrate a new estrogen-dependent transactivation pathway that involves ER-protein interactions and is ERE-independent.
Estrogens act as potent mitogens in a large number of breast cancers, and the use of estrogen receptor (ER) antagonists is, therefore, considered the endocrine therapy of choice in the management of this disease. We describe the molecular properties of EM-652, the active metabolite of EM-800, a novel nonsteroidal antiestrogen compound, on the transcriptional functions of ER alpha and ER beta. Using RT-PCR, we show that ER alpha and ER beta are expressed in mouse mammary glands, suggesting that both receptors should be considered putative targets for antiestrogen action in the breast. In cotransfection assays using a synthetic estrogen-responsive promoter, EM-652 shows no agonistic activity on ER alpha and ER beta transcriptional function and blocks the estradiol (E2)-mediated activation of both ER alpha and ER beta. EM-652 is also very effective in abrogating E2-stimulated ER alpha and ER beta trans-activation of the pS2 promoter in HeLa cells. EM-652 does not alter binding of ER alpha and ER beta to DNA. The Ras-mediated induction of ER alpha and ER beta transcriptional activity in the presence of E2 is also completely abolished by EM-652. In addition, EM-652 blocks the E2-dependent activation of ER alpha and ER beta by the steroid hormone receptor coactivator-1 as well as the in vitro interaction between SRC-1 and the ligand-binding domains of both ERs. These results demonstrate that the novel antiestrogen EM-800 fully impedes AF-1 and AF-2 activities of ER alpha and ER beta and can, therefore, be considered a potent and pure antagonist of both ER subtypes.
Human estrogen receptor beta (hER beta) cDNA that encodes the full-length amino acid sequence has been isolated from testis poly(A)+ RNA with the combination of cDNA screening and reverse transcription-PCR. It is composed of a 1590-bp open reading frame and a segment of the 5'- and 3'-untranslated region (UTR) and encodes an additional 53 amino acids in the N-terminal region compared with the previously reported one. Protein interaction between ER alpha and ER beta was demonstrated in vitro by GST pull-down assay and in vivo by immunoprecipitation. Thus, this study indicates that ER alpha and ER beta can interact in vivo, cross-signaling each other.
Cognate cDNAs are described for 2 of the 10 thyroid hormone receptor-associated proteins (TRAPs) that are immunopurified with thyroid hormone receptor alpha (TRalpha) from ligand-treated HeLa (alpha-2) cells. Both TRAP220 and TRAP100 contain LXXLL domains found in other nuclear receptor-interacting proteins and both appear to reside in a single complex with other TRAPs (in the absence of TR). However, only TRAP220 shows a direct ligand-dependent interaction with TRalpha, and these interactions are mediated through the C terminus of TRalpha and (at least in part) the LXXLL domains of TRAP220. TRAP220 also interacts with other nuclear receptors [vitamin D receptor, retinoic acid receptor alpha, retinoid X receptor alpha, peroxisome proliferation-activated receptor (PPAR) alpha, PPARgamma and, to a lesser extent, estrogen receptor] in a ligand-dependent manner, whereas TRAP100 shows only marginal interactions with estrogen receptor, retinoid X receptor alpha, PPARalpha, and PPARgamma. Consistent with these results, TRAP220 moderately stimulates human TRalpha-mediated transcription in transfected cells, whereas a fragment containing the LXXLL motifs acts as a dominant negative inhibitor of nuclear receptor-mediated transcription both in transfected cells (TRalpha) and in cell free transcription systems (TRalpha and vitamin D receptor). These studies indicate that TRAP220 plays a major role in anchoring other TRAPs to TRalpha during the function of the TRalpha-TRAP complex and, further, that TRAP220 (possibly along with other TRAPs) may be a global coactivator for the nuclear receptor superfamily.
A complex that represses activated transcription and contains the human homologs of the yeast Srb7, Srb10, Srb11, Rgr1, and Med6 proteins was isolated. The complex is devoid of the Srb polypeptides previously shown to be components of the yeast Mediator complex that functions in transcriptional activation. The complex phosphorylates the CTD of RNA polymerase II (RNAPII) at residues other than those phosphorylated by the kinase of TFIIH. Moreover, the complex specifically interacts with RNAPII. The interaction is not mediated by the CTD of RNAPII, but is precluded by phosphorylation of the CTD. Our results indicate that the complex is a subcomplex of the human RNAPII holoenzyme. We suggest that the RNAPII holoenzyme is a transcriptional control panel, integrating and responding to specific signals to activate or repress transcription.
Steroid hormones exert their major biological effects on differentiation, growth, homeostasis, and behavior by binding to nuclear receptors, which, in turn, regulate patterns of gene expression in appropriate target cells. Members of the steroid receptor superfamily include not only receptors for steroid hormones but also receptors for various other lipophilic ligands such as thyroid hormone, retinoids, and vitamin D3. Nuclear receptors are phosphoproteins, and most of the phosphorylation sites, which have been identified, are serine-threonine residues located in the amino terminus. Nuclear accessory proteins that facilitate the sequence-specific DNA-binding activity of the steroid class of receptors are described in this chapter. These proteins do not heterodimerize with steroid receptors per se, they form a ternary complex with receptor homodimers on target DNA. The solution structure of the high-mobility group box has revealed a novel DNA-binding motif consisting of three helices folded into an L shape. It is found that helices 1 and 2 form the short arm of the L, which is held at an approximately 80° angle from helix 3 and an extended amino terminal segment that together form the long arm.
The human thyroid hormone receptor-associated protein (TRAP) complex, an earlier described coactivator for nuclear receptors, and an SRB- and MED-containing cofactor complex (SMCC) that mediates activation by Gal4-p53 are shown to be virtually the same with respect to specific polypeptide subunits, coactivator functions, and mechanisms of action (activator interactions). In parallel with ligand-dependent interactions of nuclear receptors with the TRAP220 subunit, p53 and VP16 activation domains interact directly with a newly cloned TRAP80 subunit. These results indicate novel pathways for the function of nuclear receptors and other activators (p53 and VP16) through a common coactivator complex that is likely to target RNA polymerase II. Identification of the TRAP230 subunit as a previously predicted gene product also suggests a coactivator-related transcription defect in certain disease states.
Gene activation in eukaryotes is regulated by complex mechanisms in which the recruitment and assembly of the transcriptional machinery is directed by gene- and cell-type-specific DNA-binding proteins. When DNA is packaged into chromatin, the regulation of gene activation requires new classes of chromatin-targeting activity. In humans, a multisubunit cofactor functions in a chromatin-selective manner to potentiate synergistic gene activation by the transcriptional activators SREBP-1a and Sp1. Here we show that this activator-recruited cofactor (ARC) interacts directly with several different activators, including SREBP-1a, VP16 and the p65 subunit of NF-kappaB, and strongly enhances transcription directed by these activators in vitro with chromatin-assembled DNA templates. The ARC complex consists of 16 or more subunits; some of these are novel gene products, whereas others are present in other multisubunit cofactors, such as CRSP, NAT and mammalian Mediator. Detailed analysis indicates that the ARC complex is probably identical to the nuclear hormone-receptor cofactor DRIP. Thus, ARC/DRIP is a large composite co-activator that belongs to a family of related cofactors and is targeted by different classes of activator to mediate transcriptional stimulation.
Nuclear receptors regulate transcription in direct response to their cognate hormonal ligands. Ligand binding leads to the dissociation of corepressors and the recruitment of coactivators. Many of these factors, acting in large complexes, have emerged as chromatin remodelers through intrinsic histone modifying activities or through other novel functions. In addition, other ligand-recruited complexes appear to act more directly on the transcriptional apparatus, suggesting that transcriptional regulation by nuclear receptors may involve a process of both chromatin alterations and direct recruitment of key initiation components at regulated promoters.
Estrogen receptors (ERs alpha and beta) enhance transcription in response to estrogens by binding to estrogen response elements (EREs) within target genes and utilizing transactivation functions (AF-1 and AF-2) to recruit p160 coactivator proteins. The ERs also enhance transcription in response to estrogens and antiestrogens by modulating the activity of the AP-1 protein complex. Here, we examine the role of AF-1 and AF-2 in ER action at AP-1 sites. Estrogen responses at AP-1 sites require the integrity of the ERalpha AF-1 and AF-2 activation surfaces and the complementary surfaces on the p160 coactivator GRIP1 (glucocorticoid receptor interacting protein 1), the NID/AF-1 region, and NR boxes. Thus, estrogen-liganded ERalpha utilizes the same protein-protein contacts to transactivate at EREs and AP-1 sites. In contrast, antiestrogen responses are strongly inhibited by ERalpha AF-1 and weakly inhibited by AF-2. Indeed, ERalpha truncations that lack AF-1 enhance AP-1 activity in the presence of antiestrogens, but not estrogens. This phenotype resembles ERbeta, which naturally lacks constitutive AF-1 activity. We conclude that the ERs enhance AP-1 responsive transcription by distinct mechanisms with different requirements for ER transactivation functions. We suggest that estrogen-liganded ER enhances AP-1 activity via interactions with p160s and speculate that antiestrogen-liganded ER enhances AP-1 activity via interactions with corepressors.
The TRAP coactivator complex is a large, multisubunit complex of nuclear proteins which associates with nuclear hormone receptors (NRs) in the presence of cognate ligand and stimulates NR-mediated transcription. A single subunit, TRAP220, is thought to target the entire complex to a liganded receptor through a domain containing two of the signature LXXLL motifs shown previously in other types of coactivator proteins to be essential for mediating NR binding. In this work, we demonstrate that each of the two LXXLL-containing regions, termed receptor binding domains 1 and 2 (RBD-1 and RBD-2), is differentially preferred by specific NRs. The retinoid X receptor (RXR) displays a weak yet specific activation function 2 (AF2)-dependent preference for RBD-1, while the thyroid hormone receptor (TR), vitamin D(3) receptor (VDR), and peroxisome proliferator-activated receptor all exhibit a strong AF2-dependent preference for RBD-2. Using site-directed mutagenesis, we show that preference for RBD-2 is due to the presence of basic-polar residues on the amino-terminal end of the core LXXLL motif. Furthermore, we show that the presence and proper spacing of both RBD-1 and RBD-2 are required for an optimal association of TRAP220 with RXR-TR or RXR-VDR heterodimers bound to DNA and for TRAP220 coactivator function. On the basis of these results, we suggest that a single molecule of TRAP220 can interact with both subunits of a DNA-bound NR heterodimer.
The already known X-ray structures of lipases provide little evidence about initial, discrete structural steps occurring in the first phases of their activation in the presence of lipids (process referred to as interfacial activation). To address this problem, five new Thermomyces (formerly Humicola) lanuginosa lipase (TlL) crystal structures have been solved and compared with four previously reported structures of this enzyme. The bias coming from different crystallization media has been minimized by the growth of all crystals under the same crystallization conditions, in the presence of detergent/lipid analogues, with low or high ionic strength as the only main variable. Resulting structures and their characteristic features allowed the identification of three structurally distinct species of this enzyme: low activity form (LA), activated form (A), and fully Active (FA) form. The isomerization of the Cys268-Cys22 disulfide, synchronized with the formation of a new, short alpha(0) helix and flipping of the Arg84 (Arginine switch) located in the lid's proximal hinge, have been postulated as the key, structural factors of the initial transitions between LA and A forms. The experimental results were supplemented by theoretical calculations. The magnitude of the activation barrier between LA (ground state) and A (end state) forms of TlL (10.6 kcal/mol) is comparable to the enthalpic barriers typical for ring flips and disulfide isomerizations at ambient temperatures. This suggests that the sequence of the structural changes, as exemplified in various TlL crystal structures, mirror those that may occur during interfacial activation.