Analysis of the cis-acting transcriptional regulatory sequences associated with mouse mammary tumor virus proviral DNA

Abstract

The transcriptional activity of the mouse mammary tumor virus (MMTV) promoter is positively regulated by the action of glucocorticoid hormones. This action is thought to be mediated, at least in part, by the binding of the glucocorticoid hormone receptor complex (HRC) to specific DNA sequences, collectively called the glucocorticoid responsive enhancer element (GRE), located within each of the proviral long terminal repeats (LTRs). Other sequences within the proviral genome have also been shown to specifically bind the HRC in vitro, but their function as GREs has not been tested in a defined sequence context. These observtions suggest the possibility that the overall levels of glucocorticoid hormone-mediated transcription from the MMTV promoter may be modulated by the number of GREs associated in cis with this promoter. This possibility was tested utilizing transient transfection experiments, in which promoter activity was monitored either by way of expression of the bacterial chloramphenicol acetyltransferase gene or by direct analysis of RNA transcripts. The results indicate that the overall levels of transcription from the MMTV promoter is modulated in an additive way by the number of functional GREs derived from the MMTV LTR. Other sequences within the MMTV genome which bind the glucocorticoid hormone receptor complex in vitro, were not able to mediate such additive effects. In addition, neither these, nor any other sequences from within the coding region of the provirus, were functional when directly tested for their ability to serve as GREs, indicating that they do not act in such a manner in the intact MMTV provirus. However, sequences located on a 1.8 kilobase DNA fragment from within the proviral genome, were able to act as a constitutive enhancer element when linked to a region of the MMTV LTR which contains the MMTV promoter. RNA analysis indicate that this constitutive enhancer element may affect a previously unrecognized promoter within the MMTV LTR. The sequences on this 1.8 kilobase fragment were also able to activate the promoter of the herpes simplex virus thymidine kinase gene. There are other sequences within the MMTV LTR which, in addition to the GRE sequences, are thought to be necessary for maximal levels of hormone-mediated transcription from the MMTV promoter...