Progesterone induction of metallothionein-IIA gene expression

Expression of the metallothionein gene is known to be induced by glucocorticoids in a variety of cells. Here we show that in human cell lines containing functional progesterone receptors, the endogenous metallothionein-IIA (hMTIIA) gene is inducible by the synthetic progestins R5020 and medroxy-progesterone acetate. That this effect reflects a direct interaction with the metallothionein gene is supported by our finding that the partially purified progesterone receptor binds to the promoter region of the gene in vitro. The limits of the DNase I footprint and the guanine residues protected in methylation studies with the progesterone receptor are similar to those previously described for the glucocorticoid receptor. Thus, the hormone regulatory element of the human metallothionein-IIA gene can mediate regulation by both glucocorticoids and progestins, as does the hormone regulatory element of mouse mammary tumor virus.     

DNA binding properties of the vitamin D3 receptor zinc finger region.

The DNA binding domains of the nuclear receptor superfamily are highly conserved and consist of residues that fold into two zinc finger-like motifs, suggesting that the structures of this region among the members of the superfamily are likely to be very similar. Furthermore, the response elements that these receptors bind to are similar in sequence and organization. Nevertheless, these receptors selectively recognize target response elements and differentially regulate linked genes. In order to study the details of receptor:DNA binding, we have overexpressed and purified the vitamin D3 receptor DNA binding domain (VDRF) and have begun characterizing its DNA binding properties. We find that the VDRF protein binds strongly and specifically to direct repeats constituting a vitamin D response element from the mouse osteopontin (Spp-1) promoter region but weakly to the human osteocalcin vitamin D response element. Unlike receptors that recognize hormone response elements oriented as inverted repeats, such as the glucocorticoid receptor (GR) and estrogen receptor, VDRF appears to bind half-sites noncooperatively, without the free energy contribution of dimerization seen when the glucocorticoid receptor DNA binding domain associates with a glucocorticoid response element. By comparing and contrasting the DNA binding properties of the vitamin D and glucocorticoid receptors, we suggest a model for how receptors that prefer direct repeats differ in their binding strategy from those that recognize inverted repeats.     

The 5'-flanking sequence and regulatory elements of the cystatin S gene.

The gene encoding rat cystatin S (Cys S), a salivary gland-specific secretory protein, has CAAT and TATA boxes upstream of the inititation codon (Cox and Shaw, 1992), and contains regions that resemble those of other hormonally responsive eukaryotic genes. The 5'-flanking sequence of the rat Cys S gene has a potential CREB/AP-1 binding site (Rupp et al., 1990; Trejo et al., 1992), two potential glucocorticoid responsive elements (GREs, Drouin et al., 1989), and a possible GR/PR (glucocorticoid/progesterone) responsive element (Forman and Samuels, 1990). One of these potential GREs is adjacent to a potential AP-2 binding site, and another is typical of the glucocorticoid and progesterone receptor binding site. In this report, we have identified three regions in the 5'-flanking region of the Cys S gene that are found in salivary gland-specific genes (Ting et al., 1992) with a GT-rich region located between conserved elements II and III. Transfection experiments described in this paper suggest that a 281-bp DNA fragment from the Cys S gene promoter region with conserved elements II and III, the GT-rich region, and a possible GR/PR responsive element contains a negative regulatory element. In addition, our experiments suggest that the GT-rich region by itself is acting as a positive regulatory element.     

Hormonal regulation of vitellogenin genes: an estrogen-responsive element in the Xenopus A2 gene and a multihormonal regulatory region in the chicken II gene

Expression of the vitellogenin genes in avian and amphibian liver is regulated by estrogens. The DNA elements mediating estrogen induction of the various vitellogenin genes of chicken and Xenopus encompass one or more copies of a 13-mer palindromic sequence called the estrogen-responsive element (ERE). Here we show that upon incubation with the purified estrogen receptor (ER) from calf uterus the Xenopus vitellogenin A2 gene yields a DNase-I footprint over the ERE between -331 and -319. This element does not mediate the response to glucocorticoids or progestins in T47D cells. The three guanine residues in each half of the palindrome are protected against methylation by dimethylsulfate after incubation with ER, but not with glucocorticoid (GR) or progesterone (PR) receptors. In contrast, the chicken vitellogenin II gene exhibits multihormonal regulation by estrogens, progestins, and glucocorticoids in T47D and MCF7 cells. Regulation is mediated by the DNA region between -721 and -591 that contains four binding sites for hormone receptors, as demonstrated by DNase-I footprints and methylation protection experiments. The two distal and most proximal binding sites are recognized by ER, GR, and PR, whereas the central binding site is only bound by ER and GR. At suboptimal concentrations, estrogens and progestins or glucocorticoids act synergistically. In experiments using a DNA fragment containing an ERE adjacent to a glucocorticoid-responsive element/progesterone-responsive element, ER and PR bind synergistically to their corresponding sites, perhaps explaining the functional synergism of both hormones. Thus, two very different regulatory elements are used to mediate estrogen induction of related genes in chickens and amphibians.     

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.     

Synergism of closely adjacent estrogen-responsive elements increases their regulatory potential

Recent gene transfer experiments have shown that an estrogen-responsive DNA element (ERE) GGTCANNNTGACC mediates the estrogen inducibility of the Xenopus laevis vitellogenin A1 and A2 genes as well as the chicken vitellogenin II gene. We report here on experiments that explain the estrogen regulation of the Xenopus vitellogenin B1 and B2 genes. In these genes, two ERE homologues, which have only low, if any, regulatory capacity on their own, act synergistically to achieve high estrogen inducibility. Furthermore we show that synergism of EREs is most efficient, when the two elements are closely adjacent and that it is lost when the synergistic elements are separated by 125 basepairs. In-vitro estrogen receptor binding experiments indicate that co-operative binding of estrogen receptors to closely adjacent EREs is not essential for synergism of ERE homologues that have no intrinsic regulatory capacity. Functional synergism of EREs is observed in the human estrogen-responsive MCF-7 cell line as well as in mouse fibroblasts (Ltk-) cotransfected with estrogen receptor expression vectors. Even expression of a truncated receptor protein lacking 178 amino acid residues of the amino-terminal end allows synergism, suggesting that the amino-terminal end preceding the DNA-binding domain of the estrogen receptor is not required.     

The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction.

Sequences within the long terminal repeat region (LTR) of mouse mammary tumour virus (MMTV) confer progestin inducibility to either the tk-promoter or the MMTV-promoter in T47D cells, a human mammary tumour cell line which possesses high constitutive levels of progesterone receptor. In a clone of MCF7 cells, another human mammary tumour cell line with a low level of progesterone receptor, as well as in rat fibroblasts, glucocorticoid but not progestin induction is observed. The effect of the progesterone analogue R5020 is much more pronounced than the effect of dexamethasone, and at the concentrations required for maximal induction, R5020 does not significantly compete with binding of dexamethasone to the glucocorticoid receptor. In conjunction with previous results on the DNA binding of the glucocorticoid and progesterone receptors, these data show that two different steroid hormones, acting through their respective receptors, can mediate the induction of gene expression by interacting with the same DNA sequences. Our results suggest that the hormone regulatory element of MMTV may primarily be a progesterone-responsive element in mammary cells.     

Genetic regulation of beta 2-adrenergic receptors in 3T3-L1 fibroblasts.

The beta 2-adrenergic receptor from mouse 3T3-L1 cells is up-regulated through genetic mechanisms by glucocorticoids and butyrate. To study the genetic regulation of these receptors, we sequenced a 5 kb region of genomic DNA from 3T3-L1 cells, containing the beta-adrenergic receptor gene and approx. 1.5 kb of both 5' and 3' flanking sequences. The sequence contained one copy of an 8 bp consensus sequence which can confer phorbol ester-responsiveness to genes. Phorbol esters attenuated the up-regulation of beta 2-adrenergic receptors by glucocorticoids but not by butyrate. This effect was probably due to a phorbol ester-induced decrease in glucocorticoid receptor number. Using methylation-sensitive restriction enzymes, we examined the methylation of a CG-rich region occurring 5' to the gene and did not detect any changes in methylation of this region upon dexamethasone or butyrate treatment. A total of 16 putative glucocorticoid response elements were found which may mediate the glucocorticoid-induced increase in beta 2-adrenergic receptors. A comparison of the regulatory sequences of the two beta-adrenergic receptor subtypes from human and mouse confirms the observed physiological controls of receptor subtype expression and offers an explanation as to why the subtypes differ in genetic regulation.