Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region

Payvar, Farhang; DeFranco, Donald B.; Firestone, Gary L.; Edgar, Bruce A.; Wränge, Örjan; Okret, Sam; Gustafsson, Jan Åke; Yamamoto, Keith R.

doi:10.1016/0092-8674(83)90171-x

Cited by 697 publications

(312 citation statements)

References 30 publications

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“…DNA mobility shift assays were performed as originally described by Sen and Baltimore [45]. The MMTV GRE [46] consists of base pairs from −191 to −159 of the MMTV-LTR and has the following sequence: 5 -GTT, TAT, GGT, TAC, AAA, CTG, TTC, TTA, AAA, CAA, GGA-3 . The GRE oligonucleotides were end-labeled with [␥-32 P] using T4-polynucleotide kinase.…”

Section: Electrophoretic Mobility Shift Assay (Emsa)mentioning

confidence: 99%

Vanadate increases glucocorticoid receptor-mediated gene expression: a novel mechanism for potentiation of a steroid receptor

Calzi

Periyasamy

et al. 2002

The Journal of Steroid Biochemistry and Molecular Biology

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Transition metal oxyanions, such as molybdate, tungstate and vandadate, have been shown to prevent in vitro hormone-induced activation of the glucocorticoid receptor (GR) by blocking dissociation of the GR/heat shock protein heterocomplex. In this work, we report a novel effect of vanadate: in vivo potentiation of GR-mediated gene expression. In cells stably-transfected with complex (mouse mammary tumor virus (MMTV)) or minimal GR-regulated CAT reporters, treatment with 500 M vanadate caused CAT gene expression to dramatically increase, even at saturating concentrations of dexamethasone; while no such effect was seen in response to RU486 antagonist. Similar treatment with molybdate had no effect on GR activity, suggesting that the response to vanadate was not a general property of transition metal oxyanions. Treatment with vanadate after hormone-induced nuclear translocation of the GR also caused potentiation, demonstrating that vanadate was acting on a post-transformation event, perhaps by affecting the transactivation function of DNA-bound GR. Paradoxically, vanadate caused an apparent but temporary "loss" of GR protein immediately after treatment (as measured by loss of reactivity to BuGR2 antibody and of hormone-binding capacity) that returned to normal at approximately 8 h post-treatment, suggesting that potentiation of GR transactivation function (as measured by our CAT assays) was probably occurring during the later stages (8-24 h) of this assay. However, gel shift analyses revealed that vanadate could induce binding of the hormone-free GR to glucocorticoid response element (GRE)-containing oligonucleotides immediately after treatment. Thus, the rapid vanadate-induced "loss" of GR was not due to degradation of GR protein. Yet, vanadate in the absence of hormone had no effect on CAT reporter expression, demonstrating that this form of the GR still requires agonist for its enhanced transcriptional activity. As an indication of the potential mechanism of vanadate action, vanadate was found to dramatically stimulate the mitogen-activated protein kinases, ERK-1 and ERK-2. In addition, vanadate potentiation of GR reporter gene expression was completely blocked by the tyrosine kinase inhibitor herbimycin A. Taken as a whole, our results suggest that vanadate can have dramatic and complex effects on GR structure and function, resulting in hormone-free activation of GR DNA-binding function, as well as alterations to the BuGR2 epitope and hormone-binding domains-while at the same time stimulating tyrosine phosphorylation pathways controlling GR-mediated gene transcription.

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Section: Electrophoretic Mobility Shift Assay (Emsa)mentioning

confidence: 99%

Vanadate increases glucocorticoid receptor-mediated gene expression: a novel mechanism for potentiation of a steroid receptor

Calzi

Periyasamy

et al. 2002

The Journal of Steroid Biochemistry and Molecular Biology

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“…It is precisely within this segment that one finds the GRE defined by the transfection experiments discussed above. DNAbound receptors were visualized by electron microscopy as clusters of putative tetramers occupying discrete regions along the DNA molecule (Payvar et al, 1983).…”

Section: Glucocorticoids Modulate Mrna Concentrationmentioning

confidence: 99%

“…In addition to the receptor-binding region in the LTR upstream from the cap site, four binding regions were detected within transcribed sequences between 4 and 8 kbp downstream from the cap site. The functional significance of these binding sites is unknown (Payvar et al, 1983). The actual DNA sequences recognized by the receptor were identified by 'footprinting' experiments.…”

Section: Glucocorticoids Modulate Mrna Concentrationmentioning

confidence: 99%

Control of gene expression by glucocorticoid hormones

Rousseau¹

1984

Biochemical Journal

142

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Glucocorticoids control the expression of a small number of transcriptionally active genes by increasing or decreasing mRNA concentration. Either effect can result from a transcriptional or a post-transcriptional mechanism. Induction of mouse mammary tumour virus RNA results from a stimulation of transcription initiation and depends on the presence of defined regions in proviral DNA. These regions bind the glucocorticoid receptor and behave functionally as proto-enhancers. Glucocorticoid-inducible genes can retain their sensitivity to the hormone after transfer to a heterologous cell by transfection techniques. Non-inducible genes can become inducible when linked to the promoter region of an inducible gene. The mechanisms by which the receptor-steroid complex stimulates or inhibits transcription or influences mRNA stability are unknown. Receptor binding to nucleic acids appears to be a necessary but not sufficient condition. It is likely that the receptor also interacts with chromatin proteins. This might lead to a catalytic modification of these proteins, resulting in a modulation of gene expression. Development of glucocorticoid-sensitive, biochemically defined, cell-free transcription systems should provide a tool to delineate the molecular determinants of this essential regulatory mechanism.

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“…While steroids like progestins and androgens have recently been shown to also affect MMTV transcription in mammary cells (11,12,13,14,15), it is the response to glucocorticoids in cultured fibroblasts that has been studied in greatest detail (16,17,18,19). In vitro binding of purified glucocorticoid receptor to MMTV DNA has been demonstrated, showing several binding sites within the glucocorticoid regulatory region (20,21,22).…”

Section: Introductionmentioning

confidence: 99%

Dual function of a nuclear factor I binding site in MMTV transcription regulation

Buetti¹,

Kühnel

Diggelmann

1989

Nucl Acids Res

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Using linker-scanning mutagenesis we had previously identified four elements within the MMTV LTR which are necessary for transcriptional stimulation by glucocorticoid hormones. Two of them overlapped with regions to which the glucocorticoid receptor binds in vitro. The third element contained a NF-I binding site, and the fourth the TATA box. Here we show that mutations that abolish in vitro binding of NF-I had a negative effect also on the basal activity of the MMTV promoter of LTR-containing plasmids stably integrated in Ltk-fibroblasts. The analysis of double mutants altered in the NF-I plus either one of the receptor binding elements further demonstrated that the NF-I site functionally cooperated with the proximal (-120) element, which alone was extremely inefficient in stimulation. The stronger distal (-181/-172) element was independent of NF-I and showed functional cooperativity with the proximal hormone-binding element.

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Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region

Cited by 697 publications

References 30 publications

Vanadate increases glucocorticoid receptor-mediated gene expression: a novel mechanism for potentiation of a steroid receptor

Vanadate increases glucocorticoid receptor-mediated gene expression: a novel mechanism for potentiation of a steroid receptor

Control of gene expression by glucocorticoid hormones

Dual function of a nuclear factor I binding site in MMTV transcription regulation

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