Specific aspects of glucocorticoid-regulated transcription of the proviral genes of mouse mammary tumor virus (MMTV) will be investigated to address the long range goals of understanding the molecular mechanisms by which steroid hormones affect gene expression. A combination of molecular and genetic approaches will be taken with continuous tissue culture cell lines. Preliminary evidence has indicated that the MMTV long terminal repeat (LTR) contains a negative regulatory element that decreases transcription from the MMTV promoter in the absence of glucocorticoids; the function of this element may be to maintain regulated genes in an inactive state in the absence of hormone. The DNA sequences required for this negative element will be defined, and the element will be characterized with regard to its position, orientation and promoter specificity and its requirement for specific trans-acting factors utilizing transient expression assays. A number of endogenous MMTV proviral genes have been isolated by molecular cloning; these endogenous genes are normally not transcriptionally active. The ability of the LTRs of these isolated genes to function as regulated promoters will be compared in a defined experimental context to assess the relative extent to which DNA sequence defects and chromosomal position effects contribute to the lack of endogenous gene expression. The function of the LTRs in vivo will be correlated with the DNA sequence of regulatory elements and with glucocorticoid receptor binding in vitro. The potential role of DNA methylation in controlling the potential for steroid-regulated transcription will be investigated. Variants in glucocorticoid-responsive gene expression will be selected that take advantage of selectable markers expressed from the MMTV promoter. The proposed experiments will explore the molecular basis of regulatory and developmental phenomena that are potentially related to many human diseases. In addition, the increased understanding of transcriptional regulatory elements within the MMTV LTRs may aid in elucidating the mechanism of viral tumorigenesis.
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