Differential gene activation by glucocorticoids and progestins through the hormone regulatory element of mouse mammary tumor virus

The hormone regulatory element (HRE) of mouse mammary tumor virus can mediate activation of an adjacent promoter by glucocorticoids and progestins. A detailed comparison of the DNA binding of receptors for both hormones using DNAase I footprinting and methylation protection detects clear differences in their interactions with the HRE region between positions -130 and -100. Binding studies and gene transfer experiments with a variety of mutants covering the entire HRE demonstrate differences in the relevance of the individual sequence motifs for induction by each hormone. The influence of changes in the angular orientation of receptor binding sites is also different for glucocorticoid and progesterone induction. In transfection experiments with mutated HREs, we find a functional cooperation between the receptor binding sites that does not correlate with variations in the in vitro affinity of the receptors for the corresponding DNA fragment.     

Hormone-induced changes in the in vitro DNA-binding activity of the chicken progesterone receptor

Previous analyses have indicated that steroid hormone receptors undergo an allosteric change in structure upon binding by the steroid ligand. This structural change was envisioned as an intramolecular unmasking of the protein's DNA-binding domain, thus allowing the receptor to function in gene regulation. We report an analysis of the effect of hormone on the DNA-binding activity of the chicken progesterone receptor. Using an isocratic elution of DNA affinity columns we show that unliganded receptor (aporeceptor) can bind a 23-basepair progesterone response element with high affinity and a high degree of sequence preference. Hormone causes a 1.5-fold increase in affinity for the PRE sequence and a 2-fold decrease in affinity for non-specific DNA. Kinetic analysis of the off-rate of receptor-DNA complexes is consistent with this minor effect of hormone. In addition, gel retardation analysis of receptor-progesterone response element complexes further substantiates that hormone is not required for sequence-specific DNA binding. These results indicate that hormone is not necessary for the progesterone receptor to fold into a conformation that recognizes specific gene regulatory sequences.     

The chicken ovalbumin promoter is under negative control which is relieved by steroid hormones.

Steroid hormone regulation of activity of the chicken ovalbumin promoter was studied by microinjection of chimeric genes into the nuclei of primary cultured oviduct tubular gland cells. The chimeric genes contained increasing lengths of ovalbumin gene 5'-flanking sequences fused to the sequence coding for the SV40 T-antigen. Promoter activity was estimated by monitoring synthesis of T-antigen. The activity of the ovalbumin promoter is cell-specifically repressed in these oviduct cells and the repression is relieved upon addition of steroid hormones. The -132 to -425 region of the ovalbumin promoter which is responsible for this negative regulation behaves as an independent functional unit containing the regulatory elements necessary for both repression (in the presence of steroid hormone antagonists) and induced derepression (in the presence of steroid hormones) of linked heterologous promoters.     

Multiple protein binding sites within the ovalbumin gene 5''-flanking region: isolation and characterization of sequence-specific binding proteins.

To understand the molecular basis of the steroid hormone regulated expression of the ovalbumin gene, we sought to identify and isolate nuclear factors from chicken oviduct which interact specifically with the ovalbumin promoter. Using DNase I footprinting and mobility shift assays, we have defined at least four distinct protein binding sites, OV-150, OV-220, OV-250 and OV-330, in the promoter region between -100 to -400. Binding competition and protein fractionation studies revealed the existence of two distinct proteins, each recognizing two promoter sites: Both OV-330 and OV-250 are recognized by one protein factor which is distinct from the one binding to both OV-220 and OV-150. The location of the DNase I footprints coincides with those of in vivo chromatin hypersensitive sites. The OV-330 site is located in a sequence area required for the repression of the gene in the absence of hormone. The factor binding to OV-330 has been substantially purified and renaturation experiments indicate that the binding activity is associated with a polypeptide(s) of Mr 40K.     

Ecdysone regulation of the Drosophila Sgs-4 gene is mediated by the synergistic action of ecdysone receptor and SEBP 3.

The steroid hormone 20-hydroxyecdysone controls both induction and repression of the Drosophila 'intermolt gene' Sgs-4. We show here that the ecdysone receptor binds to two sites, element I and element II, in the regulatory region of Sgs-4. A functional analysis revealed that element II appears to be of no importance for Sgs-4 expression, while element I proved to be an ecdysone response element that is necessary, but not sufficient, for induction of Sgs-4 expression. Our results provide no evidence that repression of Sgs-4 expression is mediated by one of the two receptor binding sites. In the close vicinity of elements I and II, we detected two binding sites of secretion enhancer binding protein 3 (SEBP 3). Like receptor element I, one of these sites also proved to be necessary, but not sufficient, for expression of Sgs-4. Therefore, induction of Sgs-4 requires binding of both ecdysone receptor and SEBP 3 to a complex hormone response unit, which also contains binding sites for a third factor, SEBP 2. The SEBP 2 sites coincide with binding sites of products of the Broad-Complex locus, which has been implicated recently with transduction of the hormonal signal. Thus, the available data suggest that induction of Sgs-4, and possibly other 'intermolt genes', is a combination of a primary and a secondary response to the hormone.