Estrogen and progesterone receptor-binding sites on the chicken vitellogenin II gene: synergism of steroid hormone action.

Abstract

The chicken vitellogenin II gene is transcriptionally activated by estrogens. In transient transfection experiments in human T47D cells that contain receptors for various steroids, we showed estradiol, progestin, and androgen responses of a chimeric chicken vitellogenin II construct. This construct consists of DNA sequences from -626 to -590 upstream of the start of transcription of the chicken viteliogenin gene linked to the herpes simplex virus thymidine kinase promoter driving the transcription of the bacterial chloramphenicol acetyltransferase gene. Treatment of the transfected T47D cells with a combination of estradiol and the progestin R5020 led to a superinduction of chloramphenicol acetyltransferase activity, showing a synergistic action of these two steroids. This synergism was not observed upon treatment of the transfected cells with estradiol and the androgen dihydrotestosterone. Using point mutations in the vitellogenin gene fragment, we showed in functional and in in vitro DNase I footprinting assays with a purified progesterone receptor that, for the synergistic action of estradiol and R5020 to occur, the progesterone receptor must be bound to the vitellogenin gene fragment. The progesterone receptor-binding site was localized at -610 to -590, close to the consensus sequence (-626 to -613) for estrogen receptor binding and function. We therefore demonstrate here that two different steroid hormones can be functionally synergistic through the interaction of their corresponding receptors with two different binding sites adjacent to one another.The regulation of gene expression by steroid hormones is thought to occur through the interaction of steroid hormone receptor complexes with discrete nucleotide sequences in the neighborhood of the promoters of inducible genes (for reviews, see references 2 and 35). Short perfect or imperfect palindromic sequences that mediate steroid hormone action have been identified near the promoters of a number of steroid hormone-regulated genes. For example, 13-base-pair (bp) palindromic elements that mediate the estrogen response have been identified near the promoters of the Xenopus and chicken vitellogenin genes (4,17,18,19,22). Similarly, 15-bp perfect or imperfect palindromes that encompass the hexanucleotide motif 5'-TGTT/CCT-3' have been shown to mediate glucocorticoid and progestin responses in a number of genes (5,6,29,30). The delimitation of these short nucleotide sequences was achieved through gene transfer studies with either in vitro mutagenesis of known hormone receptor-binding sites (6) or different chimeras containing synthetic oligonucleotides (13,17,30). In a number of hormone response elements, such as that in mouse mammary tumor virus DNA (3, 6), the tyrosine aminotransferase gene (13), and the Xenopus vitellogenin Bi and chicken vitellogenin II (VTG II) genes (4,16,22,27) sites function by cooperating with the binding sites for other transcriptional factors that lie in their immediate vicinity.In the VTG II gene, a gene whose transcription is known t...

A mutant androgen receptor from patients with Reifenstein syndrome: identification of the function of a conserved alanine residue in the D box of steroid receptors.

“…The indicator plasmid pHCwt and the plasmids in the ERE+ARE+ series (originally ERE+PRE+) have already been described (3,5). The construct ARE2 TATA CAT was derived from (PRE)2pLovTATA (24) (6).…”

“…Immediately after transfection and a 2-min shock with 15% glycerol in phosphate-buffered saline, the transfected cells were treated with hormones and cultured for 2 days. Thereafter, the cells were harvested and disrupted and a chloramphenicol acetyltransferase (CAT) assay was performed as described previously (3,5). Transfection of the AR for gel retardation experiments was carried out in Cos-7 cells by electroporation as previously described by Klocker et al (25).…”

Different regions of the estrogen receptor are required for synergistic action with the glucocorticoid and progesterone receptors.

“…Steroid hormone receptor binding sites when placed near the binding sites of other transcription factors also functioned synergistically with these binding sites (35,37). How functional synergism is achieved is not clear, although protein-protein interactions of the receptors are among the many suggestions put forward to explain this phenomenon (7).…”

“…In some cases, multiple copies of these sequences mediate the hormone response through the concerted action of the individual receptor binding sites (8)(9)(10). In experiments in which chimeric constructs consisting of two or more receptor binding sites were placed in front of a hormone-insensitive promoter, the functional activity of the receptor binding sites was determined to be more than the additive responses of the individual elements (2, 7,22,37). This effect was described as synergism of steroid hormone action.…”

“…In this study, we examined the regions of the progesterone, glucocorticoid, and estrogen receptors involved in the synergistic action of these receptors described previously by Cato et al (7) …”