Our laboratory has previously developed cell lines derived from mouse NIH 3T3 fibroblasts and C127 mammary tumor cells that stably express mouse mammary tumor virus (MMTV) long terminal repeat fusion genes in bovine papillomavirus-based episomes. Glucocorticoid hormone strongly activates transcription from episomes and induces the disruption of a single nucleosome in an array of phased nucleosomes on the MMTV promoter. Sodium butyrate inhibits the glucocorticoid hormone-dependent development of a nuclease-hypersensitive site that is due to the displacement of this nucleosome, and inhibits induction of RNA transcripts from episomes. Saturation binding studies show that butyrate treatment does not significantly affect the amount or the hormone-binding affinity of the glucocorticoid receptor. In a transient transfection assay, glucocorticoid hormone can activate transcription from a MMTV long terminal repeat-driven luciferase gene construct equivalently in untreated and butyrate-treated cells, indicating that the soluble factors necessary for transactivation of the MMTV promoter are unaffected by butyrate. The differential effect of butyrate on the induction of stable chromatin templates and transiently expressed plasmids suggests that butyrate prevents nucleosome displacement and represses transcription by inducing a modification of chromatin.The organization ofgenes into nucleosomes and higher-order chromatin structure has important implications for transcriptional regulation (for reviews see refs. 1-4). One example of this functional role of chromatin is illustrated by the regulation of mouse mammary tumor virus (MMTV) transcription by glucocorticoid hormone. A phased array of six nucleosomes is positioned on the MMTV long terminal repeat (LTR) in bovine papillomavirus (BPV)-based episomes (5) and single integrated copies (6). As an early step in the activation of the MMTV promoter by glucocorticoid hormone, the hormone-activated glucocorticoid receptor binds to glucocorticoid regulatory elements (GREs) on one of these nucleosomes (Nuc-B), initiating a process whereby Nuc-B is displaced (see Fig. 1). In vivo exonuclease III footprinting experiments have demonstrated that a consequence of this receptor-dependent chromatin reorganization is the binding of the transcription factors NF1 and TFIID to their cognate recognition sites on the MMTV promoter (7). As the apparent concentrations and specific DNA-binding activities of NF1 and TFIID are identical in extracts from hormone-treated and untreated cells (8), it has been proposed that these factors are excluded from their recognition sites by the specific positioning of Nuc-B (9).The disruption of precisely positioned nucleosomes has been shown to occur in several other systems during transcriptional activation in vivo. A yeast acid phosphatase gene (PHOS) has an array of phased nucleosomes covering its promoter when it is repressed by high Pi (10,11 transcription by regulating access of RNA polymerase and/ or transcription factors. By using DNA restriction ...
The mouse mammary tumor virus long terminal repeat (MMTV LTR) has been introduced into cultured murine cells, using the 69% transforming fragment of bovine papilloma virus type 1 (BPV). Transformed cells contain up to 200 copies of the chimeric molecules per diploid genome. The restriction endonuclease map of the acquired recombinants, as well as the physical structure of the DNA, indicates that the LTR-BPV molecules present in these cells occur exclusively as unintegrated, extrachromosomal episome. When a 72-base pair direct repeat "enhancer" element (derived from the Harvey sarcoma retrovirus) was included in the MMTV LTR-BPV chimeric plasmids, DNA acquired through transfection, with a single exception, was integrated or rearranged or both. The transcriptional potential of the episomal MMTV promoter present in these cells was tested in two ways. First, steady-state levels of MMTV-initiated RNA were measured by quantitative Si mapping. Second, the relative number of transcription complexes initiated in vivo was determined by using a subnuclear fraction highly enriched for MMTV-BPV minichromosomes in an in vitro transcription extension assay. Both approaches showed that the MMTV LTR present in the episomal state was capable of supporting glucocorticoid hormone-regulated transcription. We have therefore demonstrated the hormone response for the first time in a totally defined primary sequence environment. Significant differences both in the basal level of MMTV-initiated transcription and in the extent of glucocorticoid induction were observed in individual cell lines with similar episomal copy numbers. These phenotypic variations suggest that epigenetic structure is an important component of the mechanism of regulation.The regulation of mouse mammary tumor virus (MMTV) expression by glucocorticoids has attracted considerable interest as a model for hormone action (13, 37). The early availability of homogeneous cDNA probes from purified virion preparations led to the demonstration that an increase in newly synthesized virus-specific RNA can be detected very quickly after the addition of hormone to MMTV-producing tumor cells in culture (40, 50), suggesting that RNA levels increase as a direct result of a stimulation in the rate of transcription. The subsequent finding that hormone regulation of MMTV expression is maintained in nonmurine cells chronically infected with MMTV (38) indicated that the virus either encodes the sequences necessary for hormone responsiveness or preferentially integrates in regions of cellular DNA containing such regulatory signals.More recently, the long terminal repeat (LTR) of the provirus has been fused to coding sequences that provide a selective function in mammalian cells, and the resulting chimeras have been introduced into cultured cells by calcium phosphate-mediated transfection. These investigations implied that LTR sequences alone are sufficient to establish hormone regulation of the selectable gene product (17,24). Chimeric molecules are assimilated in these transfection exper...
We have stably introduced a reporter gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) into human T47D breast cancer cells to study the action of the progesterone receptor (PR) on transcription from a chromatin template. Unexpectedly, the chromatin organization of the MMTV LTR in these human breast cancer cells differed markedly from what we have observed previously. The region adjacent to the transcription start site (؊221 to ؊75) was found to be constitutively hypersensitive to restriction enzyme cleavage in the absence of hormone. This region is normally encompassed within the second nucleosome of a phased array of six nucleosomes that is assembled when the MMTV LTR is stably maintained in mouse cells. Characteristically, in these rodent cells, the identical DNA sequences show increased restriction enzyme cleavage only in the presence of glucocorticoid. The increased access of restriction enzymes observed in the human PR ؉ cells was not observed in adjacent nucleosomes and was unaffected by treatment with the progesterone antagonist RU486. In addition, exonuclease III-dependent stops corresponding to the binding sites for nuclear factor 1 and the PR were observed before and after hormone treatment. These results indicate that MMTV chromatin replicated in these cells is organized into a constitutively open architecture and that this open chromatin state is accompanied by hormone-independent loading of a transcription factor complex that is normally excluded from uninduced chromatin.Mouse mammary tumor virus (MMTV) is a well-developed model system with which to evaluate the regulation of transcription by steroid hormones in the eukaryotic nucleus. It represents one of a handful of promoters for which there is extensive knowledge of the cis control elements and transacting proteins and for which a well-defined chromatin structure has been delineated (26). When stably maintained in rodent cells, the MMTV long terminal repeat (LTR) is established into a phased array of six nucleosomes (41), with the hormone-responsive elements (HREs) and other transcription factor binding sites falling within the confines of the second nucleosome (Nuc-B, Ϫ221 to Ϫ75). As a consequence of this organization, ubiquitous transcription factors such as nuclear factor 1 (NF1) and the octamer transcription factor (OTF) are excluded from the promoter (8,17,30). Treatment with glucocorticoids results in the rapid induction of transcription and a concomitant remodelling of the chromatin, as demonstrated by the appearance of a hypersensitive region spanning 41,45,50). Further, in vivo footprinting experiments demonstrate that in the presence of hormone, the transcription factors NF1, OTF, and the TATA-binding protein are able to load onto the promoter. This led to the proposal that the chromatin organization of the promoter prevents transcription factor binding, and chromatin remodelling is required for the formation of the transcription initiation complex (5). In contrast to stably replicated templates...
The POMC gene is expressed predominantly in the anterior pituitary. The high level of POMC transcription in this tissue is modulated by peptide hormones and repressed by glucocorticoids. In this present study we have investigated promoter elements required for the high basal transcription and glucocorticoid repression using transient transfection and in vitro transcription assays. We first determined that the region between -77 to -51 of the promoter, which has previously been shown to harbor a glucocorticoid receptor-binding site, is required for high basal expression both in vivo and in vitro. This promoter domain is also required for glucocorticoid repression of transcription in vivo. Two site-directed mutants within this area both decreased basal transcription, but were fully repressed by glucocorticoids, implying that the -77 to -51 region is a complex regulatory region harboring separable basal and glucocorticoid-repressible elements. Electrophoretic mobility shift and exonuclease III footprinting analysis revealed the existence of two factors that bind in this region. We also examined the effect of broad promoter deletions on basal expression and glucocorticoid repression. These experiments revealed that the region between -480 and -320 is also required for glucocorticoid repression. Taken together, the data suggest a model in which high basal transcription is generated by direct interaction of factors binding between -480 to -320 and -77 to -51. Glucocorticoid repression could occur by direct receptor disruption of these interactions.
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