Distinct sequence elements involved in the glucocorticoid regulation of the mouse mammary tumor virus promoter identified by linker scanning mutagenesis

Buetti, Elena; Kühnel, Blanka

doi:10.1016/0022-2836(86)90009-4

Cited by 163 publications

(130 citation statements)

References 38 publications

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“…We therefore performed transient-expression assays in A20 cells with luciferase reporter plasmids under the control of an LTR with the distal GRE deleted (with a HindIII linker replacing the sequence between Ϫ193 and Ϫ162) or an LTR carrying an 8-base substitution in the GRE (mutant LS Ϫ175/Ϫ166). Both plasmids show a 10-to 20-fold reduction in glucocorticoid response in L cells (12). As shown in Fig.…”

Section: Identification Of Binding Sites For B-cell Nuclear Proteins mentioning

confidence: 55%

“…The wild-type MMTV LTR (GR strain; from the PstI site at position 11 in the LTR to the PvuII site at its 3Ј end: pLSwt [36]) and the mutants LS Ϫ175/Ϫ166 and ⌬Ϫ193/Ϫ162 (carrying an octameric HindIII linker inserted between the indicated positions from the RNA start site [12]) were subcloned as SalI (filled-in)-BamHI fragments into the firefly luciferase pGL3-Basic vector (Promega) at the SmaI and BglII sites. The pGL3 constructs with LTRs truncated at position Ϫ303 were made in the same vector by inserting the fragment from Ϫ303 (filled-in StyI site) to the artificial BamHI site next to the 3Ј end of the LTR.…”

Section: Methodsmentioning

confidence: 99%

“…DNase I footprinting assays were performed by the following modifications (41,47) of the original method (24). An asymmetrically radiolabeled DNA probe was prepared from a plasmid with a truncation at position Ϫ303 of pLSwt (12), obtained by religating an EcoRI-StyI-cut pLSwt after filling in with Klenow polymerase. The resulting pLSwt (Ϫ303) plasmid was digested at the (reconstituted) EcoRI site, end labeled with [␥-32 P]ATP and polynucleotide kinase, and digested with BamHI.…”

Section: Methodsmentioning

confidence: 99%

“…The proximity of fp1 and fp2 to the distal GRE, together with the observed suppression of the glucocorticoid response in fp1 and fp2 mutants, suggested that the in vitro complex I may interact with the glucocorticoid receptor bound in the adjacent GRE (positions Ϫ171 to Ϫ182). While this sequence element is essential for the glucocorticoid response in L cells (12), its function had to be tested in B cells. We therefore performed transient-expression assays in A20 cells with luciferase reporter plasmids under the control of an LTR with the distal GRE deleted (with a HindIII linker replacing the sequence between Ϫ193 and Ϫ162) or an LTR carrying an 8-base substitution in the GRE (mutant LS Ϫ175/Ϫ166).…”

Section: Identification Of Binding Sites For B-cell Nuclear Proteins mentioning

confidence: 99%

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

Aurrekoetxea-Hernández

Buetti

2000

J Virol

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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.Mouse mammary tumor virus (MMTV) is a type B retrovirus which causes carcinomas of the mammary gland in females of susceptible mouse strains (8, 13). The carcinogenic transformation results from transcriptional activation of a particular class of cellular oncogenes (int genes; reviewed in reference 59). The mammary gland specificity of the oncogenic property of MMTV depends on the high viral replication rate and consequent high reinfection rate in the mammary epithelial cells, which are stimulated by pregnancy-related hormones. This hormonal stimulation has made the MMTV promoter the beststudied model for investigating the regulation of gene expression by steroid hormones (for a review, see references 7 and 51). Glucocorticoids, progesterone, and androgens (but not estrogens) strongly stimulate the rate of MMTV transcription through the binding of hormone-receptor complexes in the hormone regulatory element (HRE) (see Fig. 1) region of the proviral DNA, located upstream of the promoter in the long terminal repeat (LTR). Binding sites for additional transcription factors have been characterized, in particular for CTF (also called nuclear factor 1 [NF-1]) and Oct-1 in the HRE, and for mammary gland-specific factors further upstream in the LTR (reviewed in reference 31).During primary infection, MMTV is transmitted in the milk from the mother to the newborn and is taken up in the intestine, where it infects local lymphocytes (rev...

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Section: Identification Of Binding Sites For B-cell Nuclear Proteins mentioning

confidence: 55%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Identification Of Binding Sites For B-cell Nuclear Proteins mentioning

confidence: 99%

See 2 more Smart Citations

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

Aurrekoetxea-Hernández

Buetti

2000

J Virol

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“…The MMTV LTR consists of multiple regulatory elements including an upstream enhancer with tissue-specific activity (36,43,45,63), repressor regions (28,34,44,46), and the proximal promoter with the hormone response elements, NF1 (15) and OTF1 (13,41) sites, and the TATA box and initiator element (51). We used deletion analysis to determine which region of the LTR is responsible for the synergistic effect.…”

Section: Experimental Designmentioning

confidence: 99%

Nucleoprotein Structure Influences the Response of the Mouse Mammary Tumor Virus Promoter to Activation of the Cyclic AMP Signalling Pathway

Pennie

Hager

Smith

1995

Molecular and Cellular Biology

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Recent studies have provided evidence of crosstalk between steroid receptors and cyclic AMP (cAMP) signalling pathways in the regulation of gene expression. A synergism between intracellular phosphorylation inducers and either glucocorticoids or progestins has been shown to occur during activation of the mouse mammary tumor virus (MMTV) promoter. We have investigated the effect of 8-Br-cAMP and okadaic acid, modulators of cellular kinases and phosphatases, on the hormone-induced activation of the MMTV promoter in two forms: a transiently transfected template with a disorganized, accessible nucleoprotein structure and a stably replicating template with an ordered, inaccessible nucleoprotein structure. Both okadaic acid and 8-Br-cAMP synergize significantly with either glucocorticoids or progestins in activating the transiently transfected MMTV template. In contrast, 8-Br-cAMP, but not okadaic acid, is antagonistic to hormoneinduced activation of the stably replicating MMTV template. Nuclear run-on experiments demonstrate that this inhibition is a transcriptional effect on both hormone-induced transcription and basal transcription. Surprisingly, 8-Br-cAMP does not inhibit glucocorticoid-induced changes in restriction enzyme access and nuclear factor 1 binding. However, association of a complex with the TATA box region is inhibited in the presence of 8-Br-cAMP. Thus, cAMP treatment interferes with the initiation process but does not inhibit interaction of the receptor with the template. Since the replicated, ordered MMTV templates and the transfected, disorganized templates show opposite responses to 8-Br-cAMP treatment, we conclude that chromatin structure can influence the response of a promoter to activation of the cAMP signalling pathway.The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been used extensively as a model for both steroid-induced transcription and the effects of chromatin structure on transcription. The MMTV promoter can be activated by glucocorticoids, progestins, androgens, and mineralocorticoids through their individual receptors (8,18,21,55). In chromatin, the MMTV LTR exists as an ordered array of six nucleosomes, one of which (Nuc-B) is associated with the promoter region containing binding sites for glucocorticoid receptor (GR) and nuclear factor 1 (NF1) (54). In the absence of activated GR, the MMTV chromatin template is found to be in a ''closed'' architecture, with the transcription factors NF1 and OTF1 largely occluded from their high-affinity binding sites (20,35). Upon treatment with glucocorticoids, the Nuc-B region undergoes a structural transition which results in an opening of chromatin structure, characterized by increased nuclease accessibility and the binding of NF1, OTF1, and the basal transcriptional machinery (4,20,35,54). These observations suggested a bimodal model of MMTV transcriptional activation (6), in which nucleoprotein structure limits the access of transcription factors to their sites on MMTV chromatin in the absence of hormone. This repression is ...

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Interaction of the TGGCA-binding protein with upstream sequences is required for efficient transcription of mouse mammary tumor virus.

Miksicek¹,

Borgmeyer²,

Nowock³

1987

The EMBO Journal

169

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A high-affinity binding site for the TGGCA-binding protein, also known as nuclear factor I, has previously been shown to reside within the mouse mammary tumor virus (MIMTV) long terminal repeat. We have introduced mutations into this binding site to test the importance of this ubiquitous nuclear protein in MMTV transcription. Mutations which abolish the binding of the TGGCA protein in vitro are shown to impair strongly glucocorticoid-induced transcription from this promoter in vivo. These data demonstrate that the TGGCA-binding protein is a multifunctional DNA-binding protein, capable of serving a transcriptional role in the case of MMTVTY, in addition to its known involvement in the replication of adenovirus.

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Distinct sequence elements involved in the glucocorticoid regulation of the mouse mammary tumor virus promoter identified by linker scanning mutagenesis

Cited by 163 publications

References 38 publications

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

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

Nucleoprotein Structure Influences the Response of the Mouse Mammary Tumor Virus Promoter to Activation of the Cyclic AMP Signalling Pathway

Interaction of the TGGCA-binding protein with upstream sequences is required for efficient transcription of mouse mammary tumor virus.

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