Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells

1994 ◽  
Vol 14 (1) ◽  
pp. 360-372
Author(s):  
S Minucci ◽  
D J Zand ◽  
A Dey ◽  
M S Marks ◽  
T Nagata ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Retinoic Acid ◽  
Embryonal Carcinoma ◽  
Hormone Receptors ◽  
Binding Activity ◽  
Dominant Negative ◽  
P19 Cells ◽  
Mobility Shift ◽  
Ec Cells ◽  
Dominant Negative Activity

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.

scholarly journals Dominant negative retinoid X receptor beta inhibits retinoic acid-responsive gene regulation in embryonal carcinoma cells.

1994 ◽  
Vol 14 (1) ◽  
pp. 360-372 ◽  
Author(s):  
S Minucci ◽  
D J Zand ◽  
A Dey ◽  
M S Marks ◽  
T Nagata ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Retinoic Acid ◽  
Embryonal Carcinoma ◽  
Hormone Receptors ◽  
Binding Activity ◽  
Dominant Negative ◽  
P19 Cells ◽  
Mobility Shift ◽  
Ec Cells ◽  
Dominant Negative Activity

Retinoid X receptors (RXRs) heterodimerize with multiple nuclear hormone receptors and are thought to exert pleiotropic functions. To address the role of RXRs in retinoic acid- (RA) mediated gene regulation, we designed a dominant negative RXR beta. This mutated receptor, termed DBD-, lacked the DNA binding domain but retained the ability to dimerize with partner receptors, resulting in formation of nonfunctional dimers. DBD- was transfected into P19 murine embryonal carcinoma (EC) cells, in which reporters containing the RA-responsive elements (RAREs) were activated by RA through the activity of endogenous RXR-RA receptor (RAR) heterodimers. We found that DBD- had a dominant negative activity on the RARE reporter activity in these cells. P19 clones stably expressing DBD- were established; these clones also failed to activate RARE-driven reporters in response to RA. Further, these cells were defective in RA-induced mRNA expression of Hox-1.3 and RAR beta, as well as in RA-induced down-regulation of Oct3 mRNA. Gel mobility shift assays demonstrated that RA treatment of control P19 cells induces RARE-binding activity, of which RXR beta is a major component. However, the RA-induced binding activity was greatly reduced in cells expressing DBD-. By genomic footprinting, we show that RA treatment induces in vivo occupancy of the RARE in the endogenous RAR beta gene in control P19 cells but that this occupancy is not observed with the DBD- cells. These data provide evidence that the dominant negative activity of DBD- is caused by the lack of receptor binding to target DNA. Finally, we show that in F9 EC cells expression of DBD- leads to inhibition of the growth arrest that accompanies RA-induced differentiation. Taken together, these results demonstrate that RXR beta and partner receptors play a central role in RA-mediated gene regulation and in the control of growth and differentiation in EC cells.

Ligand-dependent occupancy of the retinoic acid receptor beta 2 promoter in vivo

1994 ◽  
Vol 14 (12) ◽  
pp. 8191-8201
Author(s):  
A Dey ◽  
S Minucci ◽  
K Ozato
Keyword(s):  
Retinoic Acid ◽  
Dimethyl Sulfate ◽  
Dominant Negative ◽  
In Vitro Assays ◽  
Cell Extracts ◽  
Ec Cells ◽  
Beta 2 ◽  
Responsive Element ◽  
Receptor Beta

Retinoic acid (RA) activates transcription of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (beta RARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the beta RARE prior to RA treatment, but a footprint was detected within the first hour of RA treatment. Concomitantly, other elements in the promoter, the cyclic AMP-responsive element and tetradecanoyl phorbol acetate-like-responsive element became footprinted. Footprints at these elements were induced by RA without requiring new protein synthesis and remained for the entire duration of RA treatment but rapidly reversed upon withdrawal of RA. A delayed protection observed at the initiator site was also reversed upon RA withdrawal. The RA-inducible footprint was not due to induction of factors that bind to these element, since in vitro assays showed that these factors are present in P19 cell extracts before RA treatment. Significantly, no RA-induced footprint was observed at any of these elements in P19 cells expressing a dominant negative RXR beta, in which RXR heterodimers are unable to bind to the beta RARE. Results indicate that binding of a liganded heterodimer receptor to the beta RARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the beta RARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RAR beta 2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.

scholarly journals Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells

1994 ◽  
Vol 14 (8) ◽  
pp. 5309-5317
Author(s):  
S P Murphy ◽  
J J Gorzowski ◽  
K D Sarge ◽  
B Phillips
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Embryonal Carcinoma ◽  
Embryonic Stem ◽  
Binding Activity ◽  
Hsp70 Promoter ◽  
Ec Cells

Two distinct murine heat shock transcription factors, HSF1 and HSF2, have been identified. HSF1 mediates the transcriptional activation of heat shock genes in response to environmental stress, while the function of HSF2 is not understood. Both factors can bind to heat shock elements (HSEs) but are maintained in a non-DNA-binding state under normal growth conditions. Mouse embryonal carcinoma (EC) cells are the only mammalian cells known to exhibit HSE-binding activity, as determined by gel shift assays, even when maintained at normal physiological temperatures. We demonstrate here that the constitutive HSE-binding activity present in F9 and PCC4.aza.R1 EC cells, as well as a similar activity found to be present in mouse embryonic stem cells, is composed predominantly of HSF2. HSF2 in F9 EC cells is trimerized and is present at higher levels than in a variety of nonembryonal cell lines, suggesting a correlation of these properties with constitutive HSE-binding activity. Surprisingly, transcription run-on assays suggest that HSF2 in unstressed EC cells does not stimulate transcription of two putative target genes, hsp70 and hsp86. Genomic footprinting analysis indicates that HSF2 is not bound in vivo to the HSE of the hsp70 promoter in unstressed F9 EC cells, although HSF2 is present in the nucleus and the promoter is accessible to other transcription factors and to HSF1 following heat shock. Thus trimerization and nuclear localization of HSF2 do not appear to be sufficient for in vivo binding of HSF2 to the HSE of the hsp70 promoter in unstressed F9 EC cells.

Regulated expression of a transfected human cardiac actin gene during differentiation of multipotential murine embryonal carcinoma cells

1988 ◽  
Vol 8 (1) ◽  
pp. 406-417
Author(s):  
M A Rudnicki ◽  
M Ruben ◽  
M W McBurney
Keyword(s):  
Cardiac Muscle ◽  
Thymidine Kinase ◽  
Embryonal Carcinoma ◽  
Actin Gene ◽  
P19 Cells ◽  
Cardiac Muscle Cells ◽  
Cardiac Actin ◽  
Ec Cells ◽  
Actin Promoter ◽  
Actin Mrna

P19 embryonal carcinoma (EC) cells are multipotential stem cells which can be induced to differentiate in vitro into a variety of cell types, including cardiac muscle cells. A cloned human cardiac actin (CH-actin) gene was transfected into P19 cells, and stable transformants were isolated. Low levels of CH-actin mRNA were present in transformed EC cells, but a marked increase in the level of CH-actin mRNA was found as these cells differentiated into cardiac muscle. The accumulation of CH-actin mRNA paralleled that of the endogenous mouse cardiac actin mRNA. A chimeric gene, which consisted of the CH-actin promoter linked to the herpes simplex virus thymidine kinase coding region, was constructed and transfected into P19 cells. In these transformants, the thymidine kinase protein was located almost exclusively in cardiac muscle cells and was generally not detectable in EC or other nonmuscle cells. These results suggest that the transfected CH-actin promoter functions in the appropriate developmental and tissue-specific manner during the differentiation of multipotential EC cells in culture.

scholarly journals Molecular Determinants of Differential Ligand Sensitivities of Insect Ecdysteroid Receptors

2000 ◽  
Vol 20 (11) ◽  
pp. 3870-3879 ◽  
Author(s):  
Sheng-Fu Wang ◽  
Stephen Ayer ◽  
William A. Segraves ◽  
Daryl R. Williams ◽  
Alexander S. Raikhel
Keyword(s):  
Dna Binding ◽  
Nuclear Receptors ◽  
Hormone Receptors ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Receptor Complex ◽  
S2 Cells ◽  
Ligand Specificity ◽  
Mobility Shift ◽  
Dna Binding Activity

ABSTRACT The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than theDrosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series ofAedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in AedesEcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.

scholarly journals Identification of a retinoic acid response element upstream of the murine Hox-4.2 gene.

1993 ◽  
Vol 13 (1) ◽  
pp. 257-265 ◽  
Author(s):  
H Pöpperl ◽  
M S Featherstone
Keyword(s):  
Retinoic Acid ◽  
Hox Genes ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Response Element ◽  
Retinoic Acid Response Element ◽  
Ec Cells

Hox genes play an important role in the process of vertebrate pattern formation, and their expression is intricately regulated both temporally and spatially. All-trans-retinoic acid (RA), a physiologically active metabolite of vitamin A, affects the expression of a large number of Hox genes in vitro and in vivo. However, the regulatory mechanisms underlying the RA response of these genes have not been extensively studied, and no response element for RA receptors (RARs) has been characterized in a Hox regulatory region. The expression of murine Hox-4.2 and its human homolog, HOX4B, is increased in embryonal carcinoma (EC) cell lines upon RA treatment (M. S. Featherstone, A. Baron, S. J. Gaunt, M.-G. Mattei, and D. Duboule, Proc. Natl. Acad. Sci. USA 85:4760-4764, 1988; A. Simeone, D. Acampora, V. Nigro, A. Faiella, M. D'Esposito, A. Stornaiuolo, F. Mavilio, and E. Boncinelli, Mech. Dev. 33:215-228, 1991). Using transient expression assays, we showed that luciferase reporter gene constructs carrying genomic sequences located upstream of Hox-4.2 responded to RA in murine P19 EC cells. A 402-bp NcoI fragment was necessary for the RA responsiveness of reporter constructs. This fragment contained a regulatory element, 5'-AGGTGA(N)5AGGTCA-3', that closely resembles the consensus sequence for an RA response element. The Hox-4.2 RA response element was critical for the RA induction and specifically bound RARs. In addition, the response to RA could be inhibited by expressing a dominant negative form of RAR alpha in transfected P19 EC cells. These results suggested that Hox-4.2 is a target for RAR-mediated regulation by RA.

scholarly journals Evidence for a stem cell-specific repressor of Moloney murine leukemia virus expression in embryonal carcinoma cells.

1990 ◽  
Vol 10 (8) ◽  
pp. 4045-4057 ◽  
Author(s):  
T P Loh ◽  
L L Sievert ◽  
R W Scott
Keyword(s):  
Stem Cell ◽  
Base Pair ◽  
Murine Leukemia Virus ◽  
Embryonal Carcinoma ◽  
Leukemia Virus ◽  
Binding Activity ◽  
Moloney Murine Leukemia Virus ◽  
Nuclear Extracts ◽  
Ec Cells ◽  
Murine Leukemia

A negative regulatory element (NRE) spanning the tRNA primer-binding site (PBS) of Moloney murine leukemia virus (M-MuLV) mediates repression of M-MuLV expression specifically in embryonal carcinoma (EC) cells. We precisely defined the element by base-pair mutagenesis to an 18-base-pair segment of the tRNA PBS and showed that the element also restricted expression when moved upstream of the long terminal repeat. A DNA-binding activity specific for the M-MuLV NRE was detected in vitro by using crude EC nuclear extracts in exonuclease III protection assays. Binding was strongly correlated with repression in EC cells. Mutations within the NRE that relieved repression disrupted binding activity. Also, nuclear extracts prepared from permissive, differentiated EC cell cultures showed reduced binding activity for the NRE. These results indicate the presence of a stem cell-specific repressor that extinguishes M-MuLV expression via the NRE at the tRNA PBS.

scholarly journals Retinoid X Receptor (RXR) Agonist-Induced Activation of Dominant-Negative RXR-Retinoic Acid Receptor α403 Heterodimers Is Developmentally Regulated during Myeloid Differentiation

1999 ◽  
Vol 19 (5) ◽  
pp. 3372-3382 ◽  
Author(s):  
Barton S. Johnson ◽  
Roshantha A. S. Chandraratna ◽  
Richard A. Heyman ◽  
Elizabeth A. Allegretto ◽  
LeMoyne Mueller ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Trichostatin A ◽  
Receptor Activation ◽  
Nuclear Hormone Receptor ◽  
Retinoid X Receptor ◽  
Dominant Negative ◽  
Mouse Bone Marrow ◽  
Granulocytic Differentiation ◽  
Response Elements ◽  
Dominant Negative Activity

ABSTRACT The multiple biologic activities of retinoic acid (RA) are mediated through RAR and retinoid X receptor (RXR) nuclear receptors that interact with specific DNA target sequences as heterodimers (RXR-RAR) or homodimers (RXR-RXR). RA receptor activation appears critical to regulating important aspects of hematopoiesis, since transducing a COOH-terminally truncated RARα exhibiting dominant-negative activity (RARα403) into normal mouse bone marrow generates hematopoietic growth factor-dependent cell lines frozen at the multipotent progenitor (EML) or committed promyelocyte (MPRO) stages. Nevertheless, relatively high, pharmacological concentrations of RA (1 to 10 μM) overcome these differentiation blocks and induce terminal granulocytic differentiation of the MPRO promyelocytes while potentiating interleukin-3 (IL-3)-induced commitment of EML cells to the granulocyte/monocyte lineage. In the present study, we utilized RXR- and RAR-specific agonists and antagonists to determine how RA overcomes the dominant-negative activity of the truncated RARα in these different myeloid developmental stages. Unexpectedly, we observed that an RXR-specific, rather than an RAR-specific, agonist induces terminal granulocytic differentiation of MPRO promyelocytes, and this differentiation is associated with activation of DNA response elements corresponding to RAR-RXR heterodimers rather than RXR-RXR homodimers. This RXR agonist activity is blocked by RAR-specific antagonists, suggesting extensive cross-talk between the partners of the RXR-RARα403 heterodimer. In contrast, in the more immature, multipotent EML cells we observed that this RXR-specific agonist is inactive either in potentiating IL-3-mediated commitment of EML cells to the granulocyte lineage or in transactivating RAR-RXR response elements. RA-triggered GALdbd-RARα hybrid activity in these cells indicates that the multipotent EML cells harbor substantial nuclear hormone receptor coactivator activity. However, the histone deacetylase (HDAC) inhibitor trichostatin A readily activates an RXR-RAR reporter construct in the multipotent EML cells but not in the committed MPRO promyelocytes, indicating that differences in HDAC-containing repressor complexes in these two closely related but distinct hematopoietic lineages might account for the differential activation of the RXR-RARα403 heterodimers that we observed at these different stages of myeloid development.

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