The Naturally Occurring Variant of Estrogen Receptor (ER) ERΔE7 Suppresses Estrogen-Dependent Transcriptional Activation by Both Wild-Type ERα and ERβ

Abstract We have isolated and functionally characterized the exon 7-skipped variant (ERΔE7) of estrogen receptor (ER)α, which has emerged as the predominant variant expressed in multiple normal and tumoral tissues. However, to date no function has been established for this variant in mammalian cells. ERΔE7 exhibits a negligible ability to bind ligands, insensitivity to allosteric modulation by estrogen and antiestrogens, and loss of estrogen-dependent interaction with p160 coactivators such as SRC-1 and AIB1. ERΔE7 is able to form heterodimers with both ERα and ERβ in a ligand-independent manner. Transient expression experiments in HeLa cells show that increasing amounts of ERΔE7 result in a progressive inhibition of the estrogen-dependent transcriptional activation by both wild-type ERα and ERβ on estrogen response element-driven promoters. The inhibitory effect of ERΔE7 is due to the inhibition of binding of wild-type receptors to their responsive elements. Surprisingly, the activation function (AF)-1-dependent transactivation triggered by epithelial growth factor and phorbol-12-myristate-13-acetate is also abolished in ERΔE7 despite AF1 integrity, suggesting a cross-talk between AF1 and AF2 regions of the receptor. These results indicate that the naturally occurring variant ERΔE7 is a dominant negative receptor that, when expressed at high levels relative to wild-type ERs, might have profound effects on several estrogen-dependent functions.

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A Naturally Occurring hPMS2 Mutation Can Confer a Dominant Negative Mutator Phenotype

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1635 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1635-1641 ◽

Cited By ~ 63

Author(s):

Nicholas C. Nicolaides ◽

Susan J. Littman ◽

Paul Modrich ◽

Kenneth W. Kinzler ◽

Bert Vogelstein

Keyword(s):

Mammalian Cells ◽

Germ Line ◽

Dominant Negative ◽

Wild Type ◽

Mutator Phenotype ◽

Mmr Genes ◽

Naturally Occurring ◽

Hereditary Nonpolyposis ◽

The Relationship ◽

Present Experimental Evidence

ABSTRACT Defects in mismatch repair (MMR) genes result in a mutator phenotype by inducing microsatellite instability (MI), a characteristic of hereditary nonpolyposis colorectal cancers (HNPCC) and a subset of sporadic colon tumors. Present models describing the mechanism by which germ line mutations in MMR genes predispose kindreds to HNPCC suggest a “two-hit” inactivation of both alleles of a particular MMR gene. Here we present experimental evidence that a nonsense mutation at codon 134 of the hPMS2 gene is sufficient to reduce MMR and induce MI in cells containing a wild-type hPMS2 allele. These results have significant implications for understanding the relationship between mutagenesis and carcinogenesis and the ability to generate mammalian cells with mutator phenotypes.

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Defining the Roles of β-Catenin and Plakoglobin in LEF/T-Cell Factor-Dependent Transcription Using β-Catenin/Plakoglobin-Null F9 Cells

Molecular and Cellular Biology ◽

10.1128/mcb.02375-06 ◽

2007 ◽

Vol 28 (2) ◽

pp. 825-835 ◽

Cited By ~ 25

Author(s):

Masayuki Shimizu ◽

Yoshitaka Fukunaga ◽

Junichi Ikenouchi ◽

Akira Nagafuchi

Keyword(s):

T Cell ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Dominant Negative ◽

Nuclear Accumulation ◽

Dominant Negative Effect ◽

Wild Type ◽

F9 Cells ◽

T Cell Factor ◽

Negative Effect

ABSTRACT β-Catenin functions as a transcriptional regulator in Wnt signaling. Its function is regulated by a specific destruction system. Plakoglobin is a close homologue of β-catenin in mammalian cells and is regulated in a similar fashion. When β-catenin or plakoglobin is exogenously expressed in cells, endogenous β-catenin is stabilized, which complicates estimation of the transcriptional activities of exogenously expressed proteins. To facilitate the design of experiments aimed at investigating the transcriptional activities of β-catenin and plakoglobin, we utilized F9 cells in which we knocked out endogenous β-catenin and/or plakoglobin by gene deletion and exogenously expressed wild-type and mutant β-catenin and/or plakoglobin. We show that C-terminally deleted β-catenin, but not plakoglobin, has a strong dominant-negative effect on transcription without altering the nuclear accumulation of β-catenin. Moreover, we show that Wnt-3a activation of LEF/T-cell factor (TCF)-dependent transcription depends on β-catenin but not on plakoglobin. Using chimeras of β-catenin and plakoglobin, we demonstrate that plakoglobin has the potential to function in transcriptional regulation but is not responsible for Wnt-3a signaling in F9 cells. Our data show that preferential nuclear accumulation of β-catenin is not necessarily linked to its transcriptional activity. We also clearly demonstrate that plakoglobin is insufficient for LEF/TCF-dependent transcriptional activation by Wnt-3a in F9 cells.

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pp90rsk1 Regulates Estrogen Receptor-Mediated Transcription through Phosphorylation of Ser-167

Molecular and Cellular Biology ◽

10.1128/mcb.18.4.1978 ◽

1998 ◽

Vol 18 (4) ◽

pp. 1978-1984 ◽

Cited By ~ 238

Author(s):

Peteranne B. Joel ◽

Jeffrey Smith ◽

Thomas W. Sturgill ◽

Tracey L. Fisher ◽

John Blenis ◽

...

Keyword(s):

Estrogen Receptor ◽

Transcriptional Activation ◽

Ectopic Expression ◽

Activation Function ◽

Estrogen Receptor Α ◽

Kinase Domain ◽

Stable Complex ◽

Wild Type

ABSTRACT The estrogen receptor α (ER), a member of the steroid receptor superfamily, contains an N-terminal hormone-independent transcriptional activation function (AF-1) and a C-terminal hormone-dependent transcriptional activation function (AF-2). Here, we used in-gel kinase assays to determine that pp90rsk1 activated by either epidermal growth factor (EGF) or phorbol myristate acetate specifically phosphorylates Ser-167 within AF-1. In vitro kinase assays demonstrated that pp90rsk1 phosphorylates the N terminus of the wild-type ER but not of a mutant ER in which Ser-167 was replaced by Ala. In vivo, EGF stimulated phosphorylation of Ser-167 as well as Ser-118. Ectopic expression of active pp90rsk1increased the level of phosphorylation of Ser-167 compared to that of either a mutant pp90rsk1, which is catalytically inactive in the N-terminal kinase domain, or to that of vector control. The ER formed a stable complex with the mutant pp90rsk1in vivo. Transfection of the mutant pp90rsk1 depressed ER-dependent transcription of both a wild-type ER and a mutant ER that had a defective AF-2 domain (ER TAF-1). Furthermore, replacing either Ser-118 or Ser-167 with Ala in ER TAF-1 showed similar decreases in transcription levels. A double mutant in which both Ser-118 and Ser-167 were replaced with Ala demonstrated a further decrease in transcription compared to either of the single mutations. Taken together, our results strongly suggest that pp90rsk1 phosphorylates Ser-167 of the human ER in vivo and that Ser-167 aids in regulating the transcriptional activity of AF-1 in the ER.

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Naturally occurring dominant negative variants of Stat5.

Molecular and Cellular Biology ◽

10.1128/mcb.16.11.6141 ◽

1996 ◽

Vol 16 (11) ◽

pp. 6141-6148 ◽

Cited By ~ 181

Author(s):

D Wang ◽

D Stravopodis ◽

S Teglund ◽

J Kitazawa ◽

J N Ihle

Keyword(s):

Cell Proliferation ◽

Transcriptional Regulation ◽

Transcriptional Activation ◽

Sh2 Domain ◽

Dominant Negative ◽

Oncostatin M ◽

Specific Inhibition ◽

Signal Transducer ◽

Wild Type ◽

Naturally Occurring

Stat5 was initially identified as a prolactin-induced member of the signal transducer and activator of transcription (Stat) family in sheep. However, Stat5 is also activated in the response to a variety of cytokines. In mice, and possibly in other species, there exist two Stat5 genes (Stat5a and Stat5b) that encode proteins of 92 and 94 kDa that are 95% identical. In the studies described here, we demonstrate that naturally occurring carboxyl-truncated, variant Stat5 proteins of 77 and 80 kDa exist and that these proteins are inducibly tyrosine phosphorylated in the response to several cytokines and form heterodimers with the full-length, wild-type proteins. Using expression constructs encoding truncated forms, we demonstrate that the truncated forms can be tyrosine phosphorylated and bind DNA. Surprisingly, the tyrosine phosphorylation of the carboxyl-truncated forms is considerably more stable than that of the wild-type proteins. Overexpression of a carboxyl-truncated Stat5a in cells resulted in the specific inhibition of the transcriptional activation by interleukin-3 of the genes for oncostatin M (Osm) and the cytokine-inducible, SH2 domain-containing gene (Cis), both of which have been shown to be normally regulated by Stat5. Although Stat5 dominantly suppressed the induction of these genes, no effects on cell proliferation were observed. Together, the results demonstrate the natural existence of potentially dominantly suppressive variants of Stat5 and implicate the carboxyl domain of Stats in transcriptional regulation and functions related to dephosphorylation.

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MDM2 Suppresses p73 Function without Promoting p73 Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.19.5.3257 ◽

1999 ◽

Vol 19 (5) ◽

pp. 3257-3266 ◽

Cited By ~ 211

Author(s):

Xiaoya Zeng ◽

Lihong Chen ◽

Christine A. Jost ◽

Ruth Maya ◽

David Keller ◽

...

Keyword(s):

Transcriptional Activation ◽

Feedback Loop ◽

Human Lung ◽

Activation Function ◽

Wild Type ◽

Mdm2 Protein ◽

Induced Apoptosis ◽

P53 Inactivation

ABSTRACT The newly identified p53 homolog p73 can mimic the transcriptional activation function of p53. We investigated whether p73, like p53, participates in an autoregulatory feedback loop with MDM2. p73 bound to MDM2 both in vivo and in vitro. Wild-type but not mutant MDM2, expressed in human p53 null osteosarcoma Saos-2 cells, inhibited p73- and p53-dependent transcription driven by the MDM2 promoter-derived p53RE motif as measured in transient-transfection and chloramphenicol acetyltransferase assays and also inhibited p73-induced apoptosis in p53-null human lung adenocarcinoma H1299 cells. MDM2 did not promote the degradation of p73 but instead disrupted the interaction of p73, but not of p53, with p300/CBP by competing with p73 for binding to the p300/CBP N terminus. Both p73α and p73β stimulated the expression of the endogenous MDM2 protein. Hence, MDM2 is transcriptionally activated by p73 and, in turn, negatively regulates the function of this activator through a mechanism distinct from that used for p53 inactivation.

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Identification and Characterization of ART-27, a Novel Coactivator for the Androgen Receptor N Terminus

Molecular Biology of the Cell ◽

10.1091/mbc.01-10-0513 ◽

2002 ◽

Vol 13 (2) ◽

pp. 670-682 ◽

Cited By ~ 52

Author(s):

Steven M. Markus ◽

Samir S. Taneja ◽

Susan K. Logan ◽

Wenhui Li ◽

Susan Ha ◽

...

Keyword(s):

Androgen Receptor ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Hybrid Approach ◽

Independent Manner ◽

Activation Domains ◽

Cell Library ◽

Nuclear Extracts ◽

N Terminus ◽

Transcriptional Activation Domains

The androgen receptor (AR) is a ligand-regulated transcription factor that stimulates cell growth and differentiation in androgen-responsive tissues. The AR N terminus contains two activation functions (AF-1a and AF-1b) that are necessary for maximal transcriptional enhancement by the receptor; however, the mechanisms and components regulating AR transcriptional activation are not fully understood. We sought to identify novel factors that interact with the AR N terminus from an androgen-stimulated human prostate cancer cell library using a yeast two-hybrid approach designed to identify proteins that interact with transcriptional activation domains. A 157-amino acid protein termed ART-27 was cloned and shown to interact predominantly with the AR153–336, containing AF-1a and a part of AF-1b, localize to the nucleus and increase the transcriptional activity of AR when overexpressed in cultured mammalian cells. ART-27 also enhanced the transcriptional activation by AR153–336 fused to the LexA DNA-binding domain but not other AR N-terminal subdomains, suggesting that ART-27 exerts its effect via an interaction with a defined region of the AR N terminus. ART-27 interacts with AR in nuclear extracts from LNCaP cells in a ligand-independent manner. Interestingly, velocity gradient sedimentation of HeLa nuclear extracts suggests that native ART-27 is part of a multiprotein complex. ART-27 is expressed in a variety of human tissues, including sites of androgen action such as prostate and skeletal muscle, and is conserved throughout evolution. Thus, ART-27 is a novel cofactor that interacts with the AR N terminus and plays a role in facilitating receptor-induced transcriptional activation.

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Transcriptional activation of the estrogen receptor

Clinical Chemistry ◽

10.1093/clinchem/39.2.341 ◽

1993 ◽

Vol 39 (2) ◽

pp. 341-345 ◽

Cited By ~ 3

Author(s):

L L Wei

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Estrogen Receptors ◽

Transcriptional Activation ◽

Breast Cancer Cells ◽

Wild Type ◽

Resistant Phenotype ◽

Estrogen Receptor Positive ◽

Almost All

Abstract Almost all breast cancer tumors progress to a hormone-resistant state. Evidence is presented that the existence of mutant estrogen receptors may explain some hormone-resistant phenotypes. Breast tumor cells bearing a mutant receptor that is constitutively active and does not bind hormone would have unregulated cell growth and thus appear to be hormone-independent. Alternatively, breast cancer cells may contain estrogen receptors that are transcriptionally inactive but when co-expressed with wild-type receptors render normal estrogen receptors inactive. These cells would be considered estrogen receptor-positive but would be hormone-resistant. The hormone-resistant phenotype could be further complicated by the finding that other nonreceptor proteins may also modulate the transcriptional activity of estrogen receptors. These findings, if substantiated in vivo, could add to the complexity of the hormone-resistant phenotype. Different strategies of treatment will need to be developed to effectively treat the various subtypes of hormone-resistant breast tumors.

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RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.758 ◽

1992 ◽

Vol 12 (2) ◽

pp. 758-766 ◽

Cited By ~ 60

Author(s):

R Ruggieri ◽

A Bender ◽

Y Matsui ◽

S Powers ◽

Y Takai ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Polarity ◽

Copy Number ◽

Mammalian Cells ◽

Direct Interaction ◽

Dominant Negative ◽

Yeast Cells ◽

Wild Type ◽

Ras Gene ◽

Ras Pathway

The Saccharomyces cerevisiae ras-like gene RSR1 is particularly closely related to the mammalian gene Krev-1 (also known as smg21A and rap1A). RSR1 was originally isolated as a multicopy suppressor of a cdc24 mutation, which causes an inability to bud or establish cell polarity. Deletion of RSR1 itself does not affect growth but causes a randomization of bud position. We have now constructed mutant alleles of RSR1 encoding proteins with substitutions of Val for Gly at position 12 (analogous to constitutively activated Ras proteins) or Asn for Lys at position 16 (analogous to a dominant-negative Ras protein). rsr1Val-12 could not restore a normal budding pattern to an rsr1 deletion strain but could suppress a cdc24 mutation when overexpressed. rsr1Asn-16 could randomize the budding pattern of a wild-type strain even in low copy number but was not lethal even in high copy number. These and other results suggest that Rsr1p functions only in bud site selection and not in subsequent events of polarity establishment and bud formation, that this function involves a cycling between GTP-bound and GDP-bound forms of the protein, and that the suppression of cdc24 involves direct interaction between Rsr1p[GTP] and Cdc24p. Functional homology between Rsr1p and Krev-1 p21 was suggested by the observations that expression of the latter protein in yeast cells could both suppress a cdc24 mutation and randomize the budding pattern of wild-type cells. As Krev-1 overexpression can suppress ras-induced transformation of mammalian cells, we looked for effects of RSR1 on the S. cerevisiae Ras pathway. Although no suppression of the activated RAS2Val-19 allele was observed, overexpression of rsr1Val-12 suppressed the lethality of strains lacking RAS gene function, apparently through a direct activation of adenyl cyclase. This interaction of Rsr1p with the effector of Ras in S. cerevisiae suggests that Krev-1 may revert ras-induced transformation of mammalian cells by affecting the interaction of ras p21 with its effector.

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Binding of Estrogen Receptor β to Estrogen Response Element in Situ Is Independent of Estradiol and Impaired by Its Amino Terminus

Molecular Endocrinology ◽

10.1210/me.2005-0120 ◽

2005 ◽

Vol 19 (11) ◽

pp. 2696-2712 ◽

Cited By ~ 27

Author(s):

Jing Huang ◽

Xiaodong Li ◽

Casey A. Maguire ◽

Russell Hilf ◽

Robert A. Bambara ◽

...

Keyword(s):

Estrogen Receptor ◽

Activation Function ◽

Amino Terminus ◽

Estrogen Response Element ◽

Response Element ◽

Estrogen Response ◽

17Β Estradiol ◽

Additional Explanation

Abstract The functions of 17β-estradiol (E2) are mediated by estrogen receptor (ER) α and β. ERs display similar DNA- and ligand-binding properties in vitro. However, ERβ shows lower transcriptional activity than ERα from the estrogen response element (ERE)-dependent signaling. We predicted that distinct amino termini contribute to differences in transcription efficacies of ERs by affecting in situ ER-ERE interactions. We used chromatin immunoprecipitation and a novel in situ ERE competition assay, which is based on the ability of ER to compete for ERE binding with a designer activator that constitutively induces transcription from an ERE-driven reporter construct. Interference of activator-mediated transcription by unliganded or liganded ERs was taken as an indication of ER-ERE interaction. Results revealed that ERs interacted with ERE similarly in the absence of E2. However, E2 enhanced the ERE binding of ERα but not that of ERβ. The removal of the amino terminus increased the ERβ-ERE interaction independent of E2. The ERβ amino terminus also prevented E2-mediated enhancement of the chimeric ERα-ERE interaction. Thus, the amino terminus of ERβ impairs the binding of ERβ to ERE. The abrogation of ligand-dependent activation function 2 of the amino-terminally truncated ERβ resulted in the manifestation of E2 effect on ERβ-ERE interaction. This implies that E2-mediated enhancement of ERβ-ERE interaction is masked by the activation function 2, whereas the intact amino terminus is a dominant region that decreases the binding of ERβ to ERE. Thus, ERβ-ERE interaction is independent of E2 and is impaired by its amino terminus. These findings provide an additional explanation for differences between ERα and ERβ functions that could differentially affect the physiology and pathophysiology of E2 signaling.

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Functional Interaction between the Estrogen Receptor and CTF1: Analysis of the Vitellogenin Gene B1 Promoter in Yeast

Molecular Endocrinology ◽

10.1210/mend.12.10.0182 ◽

1998 ◽

Vol 12 (10) ◽

pp. 1525-1541 ◽

Cited By ~ 3

Author(s):

Monika Tsai-Pflugfelder ◽

Susan M. Gasser ◽

Walter Wahli

Keyword(s):

Estrogen Receptor ◽

Chromatin Structure ◽

Transcriptional Activation ◽

Transactivation Domain ◽

Functional Interaction ◽

Estrogen Response ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene ◽

Human Estrogen Receptor ◽

Vitellogenin Gene

Abstract Eukaryotic gene expression depends on a complex interplay between the transcriptional apparatus and chromatin structure. We report here a yeast model system for investigating the functional interaction between the human estrogen receptor (hER) and CTF1, a member of the CTF/NFI transcription factor family. We show that a CTF1-fusion protein and the hER transactivate a synthetic promoter in yeast in a synergistic manner. This interaction requires the proline-rich transactivation domain of CTF1. When the natural estrogen-dependent vitellogenin B1 promoter is tested in yeast, CTF1 and CTF1-fusion proteins are unable to activate transcription, and no synergy is observed between hER, which activates the B1 promoter, and these factors. Chromatin structure analysis on this promoter reveals positioned nucleosomes at −430 to −270 (±20 bp) and at −270 to− 100 (±20 bp) relative to the start site of transcription. The positions of the nucleosomes remain unchanged upon hormone-dependent transcriptional activation of the promoter, and the more proximal nucleosome appears to mask the CTF/NFI site located at −101 to −114. We conclude that a functional interaction of hER with the estrogen response element located upstream of a basal promoter occurs in yeast despite the nucleosomal organization of this promoter, whereas the interaction of CTF1 with its target site is apparently precluded by a nucleosome.

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