Mouse Mammary Tumor Virus Promoter Research Articles

Superantigen expression is driven by both mouse mammary tumor virus long terminal repeat-associated promoters in transgenic mice.

In addition to the usual retroviral promoter, the mouse mammary tumor virus (MMTV) long terminal repeat carries a second promoter located in the U3 region. Here we show that both of these promoters are independently able to give rise to superantigen activity in transgenic mice. The ability of multiple MMTV promoters to drive superantigen expression underscores its importance in the virus life cycle.

Hormone activation induces nucleosome positioning in vivo.

The mouse mammary tumor virus (MMTV) promoter is induced by glucocorticoid hormone. A robust hormone- and receptor-dependent activation could be reproduced in Xenopus laevis oocytes. The homogeneous response in this system allowed a detailed analysis of the transition in chromatin structure following hormone activation. This revealed two novel findings: hormone activation led to the establishment of specific translational positioning of nucleosomes despite the lack of significant positioning in the inactive state; and, in the active promoter, a subnucleosomal particle encompassing the glucocorticoid receptor (GR)-binding region was detected. The presence of only a single GR-binding site was sufficient for the structural transition to occur. Both basal promoter elements and ongoing transcription were dispensable. These data reveal a stepwise process in the transcriptional activation by glucocorticoid hormone.

Strength and specificity of different gene promoters in oral cancer cells.

Gene therapy of oral cancer will require expression of genes by promoters that are both powerful and relatively tumor specific. We compared the level of expression of a reporter gene from promoters of human cytomegalovirus (CMV), SV40 virus, mouse mammary tumor virus (MMTV), human papillomaviruses (HPV) types 16 and 18, and the human multi-drug-resistance gene (mdr1), in several lines of oral cancer cells. In the oral cancer cell line 686LN the rank order of expression levels was: CMV > SV40 > HPV > mdr1 > MMTV. Unlike in previous reports the mdr1 promoter was no more active in two cancer cell lines with mutations in the p53 gene than in two other lines with wild-type p53, and its expression level could not be increased by either doxorubicin or taxol. On the other hand, expression from the MMTV promoter was increased over 10-fold by the presence of 1 microM dexamethasone. Thus, by an appropriate choice of promoter and inducer a wide variety of expression levels, over a 3-log range, could be attained in 686LN cells. The oral cancer-specificity of each promoter was judged by comparing expression in the neuroblastoma line IMR32. The most specific promoters were those from papillomaviruses, which were up to 45 times more active in the oral cancer cells, and the least specific was the CMV promoter. In order to find if an HPV-derived promoter was sufficient to produce expression of a suicide phenotype the 686 promoter was cloned adjacent to the thymidine kinase gene of herpes simplex and the construct was expressed from an adenovirus vector. The vector reduced the growth of 686LN cells over a 5-day period by up to 32% when optimal concentrations of virus and ganciclovir were used. These data will be valuable in the design of new constructs for gene therapy of oral cancer.

Engineering CHO cells to overexpress a secreted reporter protein upon induction from mouse mammary tumor virus promoter

The mouse mammary tumor virus (MMTV) promoter is induced by the addition of a glucocorticoid hormone or analog such as dexamethasone. The hormone binds to its specific transcription factor, glucocorticoid receptor (GR), and the activated complex then binds to the glucocorticoid response elements (GREs) in the enhancer region of the MMTV promoter to induce the overexpression of downstream genes. We have constructed an expression vector for a reporter protein, secreted alkaline phosphatase (SEAP),controlled by the MMTV promoter and co-transfected this vector along with a GR expression cassette into Chinese hamster ovary (CHO) cells. High producers were cloned and grown in suspension cultures. A very high titer, over 0.4 mg/mL, of SEAP was obtained from this inducible overexpression system, about ten times that achievable with the same reporter protein using the strong constitutive SV40 promoter in CHO cells. A peak production rate of 187 pg SEAP per cell per day was observed within 3 days after induction, compared to the peak rate of 23 pg SEAP per cell per day expressed using the constitutive SV40 promoter. With the reduced or zero growth rate during the protein production phase, this novel MMTV overexpression system is highly suited for optimizing glycoprotein synthesis rates in high cell density fed-batch or perfusion bioreactors.

Engineering CHO cells to overexpress a secreted reporter protein upon induction from mouse mammary tumor virus promoter

Engineering CHO cells to overexpress a secreted reporter protein upon induction from mouse mammary tumor virus promoter

Synergistic Action of GA-Binding Protein and Glucocorticoid Receptor in Transcription from the Mouse Mammary Tumor Virus Promoter

B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position −139 to −146 from the cap site) and fp2 (at −157 to −164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.

Behavioral and Immune Changes in V-Ha-ras Transgenic Mice

Transgenic mice (Oncomice) with an activated v-Ha-ras oncogene under the control of the mouse mammary tumor virus promoter develop mammary tumors. We wondered if the expression of the v-Ha-ras oncogene product would induce changes in mice behavioral activity, that could be associated with alterations in their immune system. Behavior was evaluated in an open field study considering line crossings and rears. Oncomice consistently showed less activity than FVB mice. Lieber-DeCarli diet decreased both types of activity in both strains. Cocaine treatment increased line crossings in both strains. Oncomice spleen and thymus cell supernatants contained higher levels of IL-2. Oncomice serum had higher levels of IL-1α. Our results suggest a direct association between higher levels of IL-1α and lower open field activity. Therefore, we can infer that the increased level of IL-1α found in Oncomice, could have a key role in oncogene induced immune and behavioral changes, and could be a requirement to facilitate its transforming activity.

Structure and Dynamic Properties of a Glucocorticoid Receptor-Induced Chromatin Transition

Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633–3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a “hit-and-run” mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262–1264, 2000).

Conditionally adherent growth of serum-independent CHO cells for automated drug screening and biopharmaceutical production

SSF3 is a CHO cell line adapted for growth in protein-free medium. It grows in suspension unless serum-derived attachment factors such as vitronectin are added to the medium. Serum-independent cell lines, which adhere to the substrate after induction with dexamethasone or constitutively, were created by transfection with a human vitronectin gene under control of the mouse mammary tumor-virus promoter. Substrate attachment and SSF3VN-cell spreading could be prevented with an RGD peptide (arginine-glycine-aspartic acid) confirming that attachment is mediated by an intregrin receptor. Hormone-inducible attachment could be blocked by glucocorticoid antagonist promegestone. All steps in the isolation of stable transfected SSF3VN cell lines could be done in a chemically defined medium avoiding the risk of introduction of serum-derived infectious agents. SSF3VN cells could be grown in protein-free medium in solid-phase large-scale bioreactors. Application in microplates as used in high-throughput screening was demonstrated in an assay of Ca(2+) release from internal stores induced by agonist-binding to recombinant human metabotropic glutamate receptor hmGluR1b.

A ligand binding domain mutation in the mouse glucocorticoid receptor functionally links chromatin remodeling and transcription initiation.

We utilized the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) in vivo to understand how the interaction of the glucocorticoid receptor (GR) with a nucleosome-assembled promoter allows access of factors required for the transition from a repressed promoter to a derepressed, transcriptionally competent promoter. A mutation (C644G) in the ligand binding domain (LBD) of the mouse GR has provided information regarding the steps required in the derepression/activation process and in the functional significance of the two major transcriptional activation domains, AF1 and AF2. The mutant GR activates transcription from a transiently transfected promoter that has a disordered nucleosomal structure, though significantly less well than the wild-type GR. With an integrated, replicated promoter, which is assembled in an ordered nucleosomal array, the mutant GR does not activate transcription, and it fails to induce chromatin remodeling of the MMTV LTR promoter, as indicated by nuclease accessibility assays. Together, these findings support a two-step model for the transition of a nucleosome-assembled, repressed promoter to its transcriptionally active, derepressed form. In addition, we find that the C-terminal GR mutation is dominant over the transcription activation function of the N-terminal GR activation domain. These findings suggest that the primary activation function of the C-terminal activation domain is different from the function of the N-terminal activation domain and that it is required for derepression of the chromatin-repressed MMTV promoter.

Inhibition of Histone Deacetylation Augments Dihydrotestosterone Induction of Androgen Receptor Levels: An Explanation for Trichostatin A Effects on Androgen-Induced Chromatin Remodeling and Transcription of the Mouse Mammary Tumor Virus Promoter

The integrated mouse mammary tumor virus (MMTV) promoter has provided an excellent model system with which to study the impact of steroid hormones on transcriptional activation in the context of a defined chromatin structure. The hormone response element (HRE) of this promoter is positioned on a phased nucleosome which becomes remodeled in response to steroids. One possible mechanism of chromatin remodeling by steroid receptors could involve recruitment of coactivators which alter the histone acetylation status of the HRE nucleosome. To examine how the androgen receptor (AR) influences transcription and chromatin remodeling and to assess whether changes in histone acetylation are involved in these effects, we determined whether the specific histone deacetylase inhibitor trichostatin A (TSA) influenced basal- and androgen-mediated transcriptional activation of the integrated MMTV promoter in the mouse L-cell fibroblast cell line 29+. These cells harbor the MMTV promoter integrated in the genome and express only one steroid hormone receptor subtype, i.e., the AR. Surprisingly, we found that treatment of the cells with TSA alone had virtually no effect on transcription and chromatin remodeling of the MMTV promoter nor on AR levels. However, pretreatment with TSA augmented the DHT effects on all three parameters. These results suggest that histone acetylation changes at the MMTV B nucleosome per se are not alone sufficient to induce chromatin remodeling and subsequent induction of MMTV transcription. Rather, the histone deacetylase inhibitor TSA exerts a portion of its effect on MMTV chromatin remodeling and transcriptional activation indirectly through increases in AR levels.

Transcriptional State of the Mouse Mammary Tumor Virus Promoter Can Affect Topological Domain Size in Vivo

Unrestrained DNA supercoiling and the number of topological domains were measured within a 1.8 megabase pair chromosomal region consisting of about 200 tandem repeats of a mouse mammary tumor virus promoter-driven ha-v-ras gene. When uninduced, unrestrained negative supercoiling was organized into 32-kilobase pair (kb) topological domains. Upon induction, DNA supercoiling throughout the region was completely relaxed. Supercoiling was detected, however, when elongation was blocked before or following induction. The formation of transcription initiation complexes upon addition of dexamethasone decreased the domain size to 16 kb. During transcription the domain size was 9 kb, the length of one repeat. These results suggest that topological domain boundaries can be "functional" in nature, being established by the formation of activated and elongating transcription complexes.

The Dominant Negative Activity of the Human Glucocorticoid Receptor β Isoform: SPECIFICITY AND MECHANISMS OF ACTION

Alternative splicing of the human glucocorticoid receptor gene generates a nonhormone binding splice variant (hGRbeta) that differs from the wild-type receptor (hGRalpha) only at the carboxyl terminus. Previously we have shown that hGRbeta inhibits the transcriptional activity of hGRalpha, which is consistent with reports of elevated hGRbeta expression in patients with generalized and tissue-specific glucocorticoid resistance. The potential role of hGRbeta in the regulation of target cell sensitivity to glucocorticoids prompted us to further evaluate its dominant negative activity in other model systems and to investigate its mode of action. We demonstrate in multiple cell types that hGRbeta inhibits hGRalpha-mediated activation of the mouse mammary tumor virus promoter. In contrast, the ability of the progesterone and androgen receptors to activate this promoter is only weakly affected by hGRbeta. hGRbeta also inhibits hGRalpha-mediated repression of an NF-kappaB-responsive promoter but does not interfere with homologous down-regulation of hGRalpha. We show that hGRbeta can associate with the heat shock protein hsp90 although with lower affinity than hGRalpha. In addition, hGRbeta binds GRE-containing DNA with a greater capacity than hGRalpha in the absence of glucocorticoids. Glucocorticoid treatment enhances hGRalpha, but not hGRbeta, binding to DNA. Moreover, we demonstrate that hGRalpha and hGRbeta can physically associate with each other in a heterodimer. Finally, we show that the dominant negative activity of hGRbeta resides within its unique carboxyl-terminal 15 amino acids. Taken together, our results suggest that formation of transcriptionally impaired hGRalpha-hGRbeta heterodimers is an important component of the mechanism responsible for the dominant negative activity of hGRbeta.

Induction of mammary gland hyperplasia in transgenic mice over-expressing human Cdc25B.

Cdc25 A and B are dual-specificity phosphatases which have been implicated in neoplastic transformation. Although Cdc25A and Cdc25B have been found to be over-expressed in many cancer cell lines and primary tumors, the physiological roles of Cdc25A and B in vivo are largely undefined. To investigate the roles of these proteins in the oncogenic transformation of the mammary gland we used the mouse mammary tumor virus (MMTV) promoter to target over-expression of the Cdc25B transgene in the mammary glands of transgenic mouse lines. Here we report that the over-expression of Cdc25B enhances the proliferation of mammary epithelial cells resulting in the formation of precocious alveolar hyperplasia. At the molecular level, marked increases in cyclin D1 protein have been found in transgenic mammary epithelial cells. The accelerated growth rate of the mammary epithelial cells could also be attributed to the increased levels of cyclin E/cdk2 activity. In addition, a pronounced decrease in apoptosis was also observed during the involution of mammary gland. The reduction of apoptosis during involution correlated well with the reduced expression of c-myc and p53, both of which have been implicated in apoptosis. Taken together, our results clearly indicate that the deregulated expression of Cdc25B generates mammary gland hyperplasia.

Comparison of Chromatin Remodeling and Transcriptional Activation of the Mouse Mammary Tumor Virus Promoter by the Androgen and Glucocorticoid Receptor

We examined the interaction between the androgen (AR) and glucocorticoid receptor (GR) at the transcriptional level using mouse fibroblast cell lines harboring an integrated mouse mammary tumor virus (MMTV) promoter. We found that the AR, after induction with dihydrotestosterone (DHT), caused a progressive increase in MMTV-CAT reporter activity over 72 h which was correlated to an increase in chromatin remodeling of the MMTV promoter in the vicinity of the hormone response element (HRE). In contrast, stimulation of the GR by the synthetic glucocorticoid dexamethasone (Dex) caused a transient increase in MMTV transcriptional activity which returned to basal levels after 72 h. These changes were correlated to a transient increase in chromatin remodeling in the region of the HRE. Neither cotreatment nor pretreatment with Dex affected the DHT response. In fact, there was a more than additive effect of the two hormones on transcription at early time points. This suggests that the inability of GR to remodel chromatin, after 24 h of hormone treatment, is most likely related to changes in the GR itself and not the chromatin remodeling process. Consistent with this, nuclear GR levels dropped by greater than 50% after Dex treatment whereas the AR was induced fourfold after 24 h of DHT treatment. We conclude that a promoter with an ordered chromatin structure can still respond to androgens even after its glucocorticoid responsiveness is lost. This may be one mechanism cells utilize to establish target gene specificity for nuclear receptors that recognize identical DNA sequences.

Overproduction of histone H1 variants in vivo increases basal and induced activity of the mouse mammary tumor virus promoter.

BALB/c 3T3 cell lines containing integrated copies of the MMTV promoter driving a reporter gene were constructed. Expression vectors in which either of two H1 variants, H10 or H1c, were under control of an inducible promoter were introduced into these lines. Surprisingly, overproduction of either variant resulted in a dramatic increase in basal and hormone-induced expression from the MMTV promoter. H1 overproduction also slowed the loss of MMTV promoter activity associated with prolonged hormone treatment. Transiently transfected MMTV reporter genes, which do not adopt a phased nucleosomal arrangement, do not display increased activity upon H1 overproduction. Thus the effects observed for stable constructs most likely represents a direct effect of H1 on a chromatin-mediated process specific to the nucleosomal structure of the integrated constructs. Induction of increased levels of acetylated core histones by treatment with trichostatin A also potentiated MMTV activity and this effect was additive to that caused by H1 overproduction. However, the effects of TSA treatment, in control or H1-overproducing cells, were eliminated by inhibiting protein synthesis. TSA treatment does not necessarily potentiate MMTV promoter activity by increasing core histone acetylation within the MMTV promoter but perhaps by altering the synthesis of an unlinked transcriptional regulator.

Homeoproteins CDP and SATB1 interact: potential for tissue-specific regulation.

Homeoproteins are known to participate in development and cell type specification. The homeoproteins CCAAT displacement protein (CDP) and special AT-rich sequence binding protein 1 (SATB1) have been shown to bind to nuclear matrix-associated regions and to act as repressors of many cellular genes. Moreover, binding of SATB1 to the mouse mammary tumor virus (MMTV) promoter region dramatically affects the tissue-specific transcription of this retrovirus. Because protein-protein interactions are a common means of regulating homeoprotein function, we tested whether SATB1 and CDP interact in vivo and in vitro. SATB1 interacted with CDP through its DNA-binding domain, as demonstrated by glutathione S-transferase (GST) pull-down assays. GST pull-down assays also showed that CDP associated with SATB1 through three of its four DNA-binding domains (CR1, CR2, and the homeodomain). SATB1-specific antisera, but not preimmune sera, precipitated CDP from nuclear extracts, and CDP-specific antisera precipitated SATB1 from the same extracts. Far-Western blotting detected interaction of SATB1 and CDP in several different tissue extracts. Association of purified SATB1 and CDP in vitro resulted in the inability of each protein to bind to DNA in gel retardation assays. CDP overexpression in cultured T cells led to a loss of detectable SATB1 binding to the MMTV promoter region, as measured by gel shift experiments. CDP overexpression also elevated MMTV long terminal repeat reporter gene activity in transient-transfection assays, a result consistent with neutralization of the SATB1 repressor function in T cells. SATB1 is very abundant in certain tissues, particularly thymus, whereas CDP is relatively ubiquitous, except in certain terminally differentiated cell types. Because of the tissue and cell type distribution of SATB1 and CDP, we propose that the SATB1-to-CDP ratio in different tissues is a novel mechanism for homeoproteins to control gene expression and differentiation in mammals.

Conditional modulation of glucocorticoid receptor activities by CREB-binding protein (CBP) and p300

Coactivators of nuclear receptors are integral components of the signal transduction pathways of steroid hormones. Here, we show that one of the major coactivators of the glucocorticoid receptor (GR), CREB-binding protein (CBP), can also function conditionally as a negative regulator of its activities. Indeed, CBP suppressed the responsiveness of the mouse mammary tumor virus (MMTV) promoter to dexamethasone in a dose-dependent fashion in HeLa and A204 cells. Similarly, this protein suppressed the responsiveness of several glucocorticoid-responsive element (GRE)-containing synthetic promoters. Using deletion mutants of CBP, we localized the repressor effect of this protein to its N-terminal domain and showed that it was independent of the histone acetyltransferase and coactivator-binding domains but dependent upon its GR-binding domain. We also demonstrated functional differentiation between CBP and other coactivators, including SRC-1 and the CBP-related protein p300, both of which influenced GR signaling in a positive fashion. In fact, p300 completely antagonized the suppressive effects of CBP in a dose-dependent fashion, probably by competing with this protein at the level of the transcription complex. These findings suggest that CBP and p300 may function additively or antagonistically to each other depending on their relative concentrations and type of target tissue, to influence the sensitivity of tissues to glucocorticoids.

Synergism between mild hyperthermia and interferon-β gene expression

In the present study, the synergistic effect of mild hyperthermia in combination with gene expression of interferon-β (IFN-β) was examined in vitro in the human glioma cell line U87MG. The cells transiently expressed the IFN-β gene under the control of the mouse mammary tumor virus promoter and were then subjected to temperature elevation (41°C for 1 h). In terms of the cell killing effect, the optimum scheme was obtained by transfection for 4 days before hyperthermia, i.e. rate in the time course of IFN-β gene expression. The relative specific growth rate decreased to 32% compared with the control while it was only 40% under the hyperthermic conditions. These observations suggest that IFNβ expression was able to enhance the sensitivity of transfected glioma cells to mild hyperthermia.

Nuclear Factor I-mediated Repression of the Mouse Mammary Tumor Virus Promoter Is Abrogated by the Coactivators p300/CBP and SRC-1

To better understand the function of nuclear factor I (NFI) proteins in transcription, we have used transient transfection assays to assess transcriptional modulation by NFI proteins on the NFI-dependent mouse mammary tumor virus (MMTV) promoter. Expression of NFI-C or NFI-X, but not NFI-A or NFI-B proteins, represses glucocorticoid induction of the MMTV promoter in HeLa cells. Repression is DNA binding-independent as a deletion construct expressing the NH2-terminal 160 residues of NFI-C represses but does not bind DNA. Repression by NFI-C is cell type-dependent and occurs in HeLa and COS-1 cells but not 293 or JEG-3 cells. NFI-C does not repress progesterone induction of the MMTV promoter in HeLa cells, suggesting that progesterone induction of the promoter differs mechanistically from glucocorticoid induction. NFI-C-mediated repression is alleviated by overexpression of glucocorticoid receptor (GR), suggesting that NFI-C represses the MMTV promoter by preventing GR function. However, repression by NFI-C occurs with only a subset of glucocorticoid-responsive promoters, as the chimeric NFIGREbeta-gal promoter that is activated by GR is not repressed by NFI-C. Since the coactivator proteins p300/CBP, SRC-1A, and RAC3 had previously been shown to function at steroid hormone-responsive promoters, we asked whether they could influence NFI-C-mediated repression of MMTV expression. Expression of p300/CBP or SRC-1A alleviates repression by NFI-C, whereas RAC3 has no effect. This abrogation of NFI-C-mediated repression by p300/CBP and SRC-1A suggests that repression by NFI-C may occur by interference with coactivator function at the MMTV promoter.