We have previously demonstrated that an array of six nucleosomes are phased on the mouse mammary tumor virus (MMTV) long terminal repeat (1,2). In this study, we devised a new assay to measure the translational positions of specific nucleosomes on the MMTV promoter. Nucleosome core particles were purified and shown to contain A and B nucleosomal DNA by Taq polymerase primer extension with nucleosome-specific primers. The 5' and 3' boundaries of A and B nucleosomes were measured by extending to the end of the core DNA with internal primers. This approach yielded results consistent with major translational positions of -23 to +123 and -221 to -75 for A and B nucleosomes, respectively. The micrococcal nuclease cleavage patterns of A and B nucleosome regions in isolated nuclei are conserved at base-pair resolution in multiple murine cell lines containing either stable MMTV-reporter chimeras or endogenous proviruses. As the refined nucleosome positions place important transcription factor binding sites at the 3' edge of the B nucleosome and in the nucleosome A/B linker, we propose that linker histone depletion and chromatin unfolding may be required to expose these cis-elements during steroid hormone-induced transcription initiation.
There has been exciting progress in the understanding of the mechanism of action of steroid hormones. The structures and functions of the various receptor protein domains as well as the various domains of steroid-regulated genes, including steroid response elements, are currently being elucidated. The roles of transcription factors in the steroid-altered regulation of gene transcription are just being defined. The nature of the nuclear acceptor sites, i.e. nuclear-binding sites, for steroid receptors are under investigation. The composition, location, and function of these nuclear acceptor sites for steroid receptors is crucial for understanding the mechanism of steroid regulation of gene expression. Possible roles of specific, DNA-binding, chromatin proteins in these acceptor sites have been suggested. A very rapid action of steroids on the expression of proto-oncogenes that code for nuclear regulatory proteins has recently been described. Using this information, we have proposed a novel steroid action model utilizing "regulatory genes" whereby the steroids would rapidly alter the expression of regulatory genes (early genes) whose protein products would return to the nucleus to regulate the expression of structural genes (late genes). The latter would occur via binding of these regulatory proteins (e.g. transcription factors) to the steroid-regulatory elements neighboring these late genes. This model would explain many of the characteristics reported from many laboratories on the action of steroid hormones on gene expression.
The glucocorticoid dexamethasone (DEX) causes a rapid, reversible reduction in c-myc mRNA level in the oviducts of estrogen-treated, immature chickens. The c-myc mRNA level begins to decrease by 5 min after injection of 0.5 mg DEX, reaches a minimum of 10% of the control value by 30 min, and returns to 30-40% of the control value by 4 h post injection. This rapid effect of DEX on the c-myc mRNA level occurs in both diethylstilbestrol-stimulated and diethylstilbestrol-withdrawn oviducts. The effect is dose dependent, with reduction of the c-myc mRNA measured with as little as 10 micrograms DEX injection (0.03 micrograms/g BW). The effect of the steroid is gene specific with H2B histone mRNA displaying a significantly reduced response. The effect is also tissue specific with liver displaying an increase of 170% of control values in c-myc mRNA level by 30 min after injection of 0.5 mg DEX. The reduction of avian oviduct c-myc mRNA levels by DEX may play a role in glucocorticoid inhibition of cell proliferation in this tissue. The rapidity of the steroid effects on c-myc expression makes it likely that the steroid-induced reduction of c-myc mRNA levels represents a direct primary action of the steroid-receptor complex on the c-myc gene expression.
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