To study the influence of various transactivators and the role of nucleosomal structure in gene regulation by steroid hormones, we have introduced mouse mammary tumor virus (MMTV) promoter sequences along with expression vectors for the glucocorticoid receptor (GR) and nuclear factor I (NFI) in Saccharomyces cerevisiae, an organism amenable to genetic manipulation. Both in the context of an episomal multicopy vector and in a centromeric single-copy plasmid, the MMTV promoter was virtually silent in the absence of inducer, even in yeast strains expressing GR and NFI. Induction was optimal with deacylcortivazol and required both GR and NFI. The transactivation function AF1 in the N-terminal half of GR is required for ligand-dependent induction and acts constitutively in truncated GR lacking the ligand binding domain. A piece of the MMTV long terminal repeat extending from -236 to +111 is sufficient to position a nucleosome, B, over the regulatory region of the promoter from -45 to -190 and another nucleosome over the transcription start region. The rotational orientation of the DNA on the surface of nucleosome B is the same as that previously found in animal cells and in reconstitution experiments. This orientation is compatible with binding of GR to two sites, while it should preclude binding of NFI and hence be responsible for constitutive repression. Upon ligand induction, there is no major chromatin rearrangement, but the proximal linker DNA, including the TATA box, becomes hypersensitive to nucleases. The transcriptional behavior of the MMTV promoter was unaffected by deletions of the genes for zuotin or SIN1/SPT2, two proteins which have been claimed to assume some of the functions of linker histones. Thus, despite the lack of histone H1, yeast cells could be a suitable system to study the contribution of nucleosomal organization to the regulated expression of the MMTV promoter.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Chávez S, Candau R, Truss M, Beato M

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