Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.

Abstract

In mouse Ltk-cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.The expression of mouse mammary tumor virus (MMTV) in mice as well as in tissue culture cells is regulated by steroid hormones (18,23,25,28,38). Studies utilizing homologous and heterologous cell lines revealed that the strong stimulation of viral RNA synthesis could be attributed to the action of glucocorticoids alone. The regulation is mediated by a hormone-receptor complex; it is very rapid and independent of simultaneous protein synthesis (30, 34). The hormone-receptor complex binds specifically to sequences in the long terminal repeat (LTR) of MMTV DNA (9,11,26,27,32). In transfection experiments it was shown that the same DNA region is involved in glucocorticoidregulated transcription (7,13,16,17,19). Upon deletion of some of these sequences, the hormonal effect on MMTV transcription is lost (2,7,21). It was demonstrated that the sequences required for the hormone response reside between -100 and -200 nucleotides upstream from the initiation site for viral RNA synthesis (2, 21). Larger DNA fragments containing this sequence but lacking the MMTV promoter were shown to confer hormone inducibility to other, normally non-hormone-responsive promoters (3, 21). As a result of hormonal treatment, the rate of viral RNA synthesis increases, as measured by pulse labeling (12,29,36,37,40), and higher levels of stable mRNA accumulate. From these studies it was deduced that the rate of initiation of viral RNA synthesis is increased, but they could not exclude the possibility of rapid metabolic effects of glucocorticoids, which may alter for example the stability of the MMTV mRNA. To further study the molecular events which are caused by hormone-receptor binding to DNA, it was therefore necessary to apply an approach which looks more directly at initiation of transcription. In the experiments presented here we measured the number of RNA polymerase II molecules transcribing MMTV DNA with and without hormone treatment. With the sa...

The dose dependence of glucocorticoid-inducible gene expression results from changes in the number of transcriptionally active templates.

“…Although the phenomenon that steroid hormones induce gene expression levels in a dose dependent manner is well known (Ringold et al, 1977;Young et al, 1977;Ucker et al, 1981Ucker et al, , 1983Groner et al, 1983;Firzlaff and Diggelmann, 1984), there have been no published reports that distinguish whether this dose dependence is a response of individual templates or of the mass of the templates. Since typical eukaryotic cells have just two templates in a single cell and the phenotypes of the cells are determined at a single cell level, a differentiation between these two possibilities seems to be fundamentally important.…”

“…Possible regulatory steps directly related to heterogeneous gene induction It is well established that the dose dependent increase of the gene expression level by the MTV enhancer/promoter occurs at the transcription level (Ringold et al, 1977;Young et al, 1977;Ucker et al, 1981Ucker et al, , 1983Groner et al, 1983). More directly, Firzlaff and Diggelmann (1984) showed that this is caused by a specific dose dependent increase in the RNA polymerase II loading after hormone treatment. Thus, it is reasonable to infer that the current phenomenon, a dose dependent increase in responding cells, occurs at the transcription level.…”

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

“…Transcription from the proviral DNA of mouse mammary tumor virus (MMTV) is controlled by steroid hormones (1,2) through their binding to receptor proteins and the interaction of hormone-receptor complexes with regulatory DNA sequences, thereby causing an increase of the number of active RNA polymerase II molecules on the MMTV promoter (3). The hormone-responsive sequences are located in the long terminal repeat (LTR) of the provirus (4,5,6,7), in a region of approximately 200 bp upstream of the transcription start site (8,9,10).…”

Use of a glucocorticoid-inducible promoter for expression of herpes simplex virus type 1 glycoprotein gC1, a cytotoxic protein in mammalian cells.

“…transfected cells leads to an increase in the level of RNA polymerase II molecules, initiating transcription at the MMTV LTR promoter (16), and to an approximately 100-fold accumulation of the corresponding specific mRNA (6).…”