Glucocorticoid induction of mouse mammary tumor virus (MMTV) is short lived, returning to base levels within 24 h despite the continued presence of hormone. MMTV DNA sequences assembled as chromatin require hormone for binding by nuclear factor 1 (NF1) and octamer proteins (OCT). However, in the same cells, NF1 and OCT factors are bound to transiently introduced DNA in the absence of hormone. In contrast, recruitment of the TATA-binding protein and a novel DNA-binding protein, which we have designated FDT, for factor downstream of the TATA-binding protein, is hormone dependent for both stable and transient templates. Furthermore, transient DNA templates, but not nucleosomal templates, retain these transcription factors over the course of 24 h. This finding suggests that MMTV chromatin structure contributes to activation and cessation of transcription in vivo.The regulation of transcription requires the complex interaction of sequence-specific transactivating proteins as well as the extensive array of basal transcription factors without sequence-specific binding capacity (24, 58). These interactions are knit together by an increasingly large number of coactivator proteins which appear to facilitate contact between the enhancer or repressing proteins on this basal machinery (16,30,40,59). In the nucleus of eukaryotes, this process is further complicated by the fact that the target sites of trans-acting factors are often assembled as chromatin (18,51). Not surprisingly, this compaction of DNA sequences produced by the association of histones has been shown to be relevant to the regulation of transcription for several eukaryotic genes in vivo (17,18,51). A particularly well studied group are the inducible promoters as exemplified by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) (6, 57), the PH05 gene in Saccharomyces cerevisiae (1), and the tyrosine aminotransferase gene in liver (12,42). While recent experiments analyzing the Drosophila HSP26 and the chicken vitellogenin genes have suggested a positive potentiation for chromatin (29,44), in most cases its effect has been to inhibit transcription (1,6,50).The significance of a repressive function of chromatin structure has been amply demonstrated with genetic and biochemical experiments (15,19,37). Genetic studies demonstrated that production of yeast strains deleted of specific histones and/or with specific mutations to prevent posttranslational modifications results in profound effects on regulated gene expression. In one case, Simpson and colleagues demonstrated that the ability of certain transactiving factors such as the a2 repressor to influence nucleosomal positions * Corresponding author. Mailing address:
Transcriptional activation of the mouse mammary tumor virus (MMTV) promoter by ligand-bound glucocorticoid receptor (GR) is transient. Previously, we demonstrated that prolonged hormone exposure results in displacement of the transcription factor nuclear factor 1 (NF1) and the basal transcription complex from the promoter, the dephosphorylation of histone H1, and the establishment of a repressive chromatin structure. We have explored the mechanistic link between histone H1 dephosphorylation and silencing of the MMTV promoter by describing the putative kinase responsible for H1 phosphorylation. Both in vitro kinase assays and in vivo protein expression studies suggest that in hormone-treated cells the ability of cdk2 to phosphorylate histone H1 is decreased and the cdk2 inhibitory p21 protein level is increased. To address the role of cdk2 and histone H1 dephosphorylation in the silencing of the MMTV promoter, we used potent cdk2 inhibitors, Roscovitine and CVT-313, to generate an MMTV promoter which is associated predominantly with the dephosphorylated form of histone H1. Both Roscovitine and CVT-313 block phosphorylation of histone H1 and, under these conditions, the GR is unable to remodel chromatin, recruit transcription factors to the promoter, or stimulate MMTV mRNA accumulation. These results suggest a model where cdk2-directed histone H1 phosphorylation is a necessary condition to permit GR-mediated chromatin remodeling and activation of the MMTV promoter in vivo.DNA in eukaryotic nuclei is highly packaged into chromatin by two molecules each of the core histone proteins H2A, H2B, H3, and H4 and one molecule of linker histone H1 (44). In addition, to the intrinsic stearic considerations of wrapping DNA around the histone octamer, the posttranslation modification of the core histones have come under increased scrutiny (22,44). Numerous studies support a strong link between transcriptional regulation and the remodeling of chromatin structure through the acetylation of core histones H3 and H4 (20, 40, 46). The acetylation of core histones in vivo is presumed to play a role in increasing the accessibility of transcription factors to the promoters of target genes (23). More recently, the Mi-2 ATPase complex, which contains chromatin remodeling activity, has been linked to both DNA methylation and histone deacetylation (39, 47).The role of histone H1 in the regulation of transcription is less clear, but there is evidence that histone H1 interacts differentially with transcriptionally active and inactive regions of chromatin (29). Indeed, studies in Xenopus and Tetrahymena thermophila have ruled out an exclusive role for histone H1 phosphorylation in chromatin condensation (31,36). However, other studies in mammals and T. thermophila have found a correlation between transcriptional activation and decreased amounts of histone H1 (9,12,14). Thus, it is plausible, given the role of histone H1 in the packaging of the nucleosome, that posttranslational modifications of this protein may also be involved in transcript...
The Mouse Mammary Tumor Virus (MMTV) contains sequences in its proximal promoter region to which both glucocorticoid and progesterone receptors can bind. In transient transfection experiments both hormones are able to stimulate transcription from reporter plasmids containing either native or consensus hormone response elements (glucocorticoid response element/progesterone response element). Previous experiments have demonstrated that the MMTV long terminal repeat is reproducibly assembled into a phased array of nucleosomes when stably introduced into cells. Stimulation by glucocorticoids of endogenous templates led to a rapid but transient increase in transcription initiation and mRNA accumulation that can be correlated with increased sensitivity to restriction enzymes. In contrast, experiments using progesterone or a truncated glucocorticoid receptor failed to elicit a similar increase in mRNA levels as dexamethasone from stable chromatin templates. In an attempt to understand this differential response, we have compared the responsiveness of the MMTV promoter to glucocorticoids and progesterone when it is organized in either stable chromatin or in transiently acquired plasmids. Our results demonstrate that the native chromatin structure prevents activation of this locus by progesterone, but permits stimulation by glucocorticoids.
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