Preferential binding of estrogen-receptor complex to a region containing the estrogen-dependent hypomethylation site preceding the chicken vitellogenin II gene.

Abstract

DNA-cellulose competition binding assays were used to measure the ability of cloned DNA fragments of the chicken vitellogenin H gene to displace the estrogen-receptor complex from total chicken DNA coupled to cellulose. The DNA fragment that gave the highest competition is situated in the upstream region of the gene between nucleotides -458 and -725. This DNA fragment has four small clusters of A+T-rich sequences and contains the estrogen-dependent hypomethylation site. In vitro methylation of the Msp I site does not change the capacity of the DNA fragment to compete for estrogenreceptor complex, whereas cleavage of the C-C-G-G (Msp I site) results in a complete loss of competition of this fragment for estrogen-receptor complex. These results, combined with deoxyribonuclease I protection experiments, suggest that the most probable binding site for estrogen-receptor complex is.. This sequence has 73% homology with the core enhancer sequence of simian virus 40, .. .One mechanism in the regulation of specific gene expression by steroid hormones is the binding of the steroid receptor complex to the chromatin (1, 2). Given the large number of receptor molecules translocated to the nucleus and the relatively small number of genes that are stimulated, Yamamoto and Alberts (3, 4) proposed a two-site model of DNA binding in which the selective action of the steroid-receptor complex is achieved through interaction of a small number of highaffinity binding sites and a large number of low-affinity sites. This binding of steroid receptor to high-affinity binding sites could be, for example, responsible for the hypomethylation of a Msp I site situated at -611 base pairs (bp) in the upstream region of the vitellogenin II gene (5-7). Similarly, an interaction of the steroid-receptor complex with the DNA in this region of the gene may explain the presence of a deoxyribonuclease I-hypersensitive site situated approximately 700 bp upstream from the gene (8). The appearance of deoxyribonuclease I (DNase I) hypersensitivity at -700 bp and the hypomethylation of the Msp I site take place only in the presence of estrogen and are both observed in the estrogentreated liver and the oviduct. The common denominator of these two phenomena in liver and oviduct is the requirement of estrogen and therefore of nuclear receptor complex by which the effect of estrogen is expressed. We therefore formulate the working hypothesis that the steroid-receptor complex binds to the 5' upstream region of the gene where both hypomethylation and DNase I hypersensitivity are observed. We have tested this hypothesis and demonstrate that nuclear estrogen-receptor complex has a preferential binding to a DNA fragment situated upstream from the 5' end of the gene containing the Msp I hypomethylation site.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. MATERIALS AND METHODSPreparation of Nuclear R...

Genomic actions of estrogen receptor α: what are the targets and how are they regulated?

“…Classically, target genes have been identified using 'single gene' experiments. The egg-white proteins in the chicken oviduct and the Xenopus laevis vitellogenin gene are among the first ERa target genes to be identified (Hayward et al 1982, Lai et al 1983, Chambon et al 1984, Jost et al 1984. Later, by comparing cDNA libraries of nontreated and E 2 -treated MCF-7 human breast cancer cells several other ERa-responsive genes were identified such as the classical and intensively studied ERa target gene pS2/TFF1 (Brown et al 1984, Jakowlew et al 1984.…”

Building a metal-responsive promoter with synthetic regulatory elements.

“…Several regulatory proteins that recognize specific nucleotide sequences have been described recently; for example, steroid hormone receptors have been shown to bind to specific recognition sequences in the DNA (17,19,29,32,41). Similarly, binding of a protein upstream of heat shock genes appears to mediate their response (26,39,40), and a specific transcription factor that binds to the simian virus 40 early promoter has been partially purified (10).…”

The orphan receptors NGFI-B and steroidogenic factor 1 establish monomer binding as a third paradigm of nuclear receptor-DNA interaction.

“…This term may still be inadequate, since molecular cloning techniques have revealed many proteins that share these structural features but whose function is unknown (the orphan receptors), which may serve as transcription factors regulated by other means (27,41). At least one protein of this class, NGFI-B (also called nur77), is closely regulated at the transcriptional level by the actions of growth factors and membrane depolarization, but once expressed NGFI-B can activate transcription without exogenously added ligand (6,16,38,43,61,64,65).The first demonstrations of DNA binding by nuclear receptors were made by examining the promoters of known glucocorticoid-and estrogen-regulated genes (19,20,44). The identified response elements function as inverted repeats of protein-specific 6-bp elements (termed half-sites) separated by three nonconserved base pairs, each half-site interacting with one molecule of a steroid receptor dimer (23, 55).…”

“…The first demonstrations of DNA binding by nuclear receptors were made by examining the promoters of known glucocorticoid-and estrogen-regulated genes (19,20,44). The identified response elements function as inverted repeats of protein-specific 6-bp elements (termed half-sites) separated by three nonconserved base pairs, each half-site interacting with one molecule of a steroid receptor dimer (23, 55).…”