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K F H Grandien, A Berkenstam, S Nilsson, and J-Å Gustafsson


The human oestrogen receptor (hER) gene has recently been shown to be transcribed from two different promoters. In order to study the hER gene promoter region we initially isolated and sequenced 3·3 kb of the 5′ region of the hER gene. DNase I hypersensitivity experiments with nuclei from the ER-expressing cell line MCF-7 mapped three different hypersensitive sites, located in the vicinity of the two hER promoters previously identified. However, in another ER-positive cell line, ZR-75-1, DNase I hypersensitivity was observed only in the region of the downstream promoter, suggesting differences between the two cell lines in promoter usage. Polymerase chain reaction-mediated detection of mRNAs transcribed from the two different promoters demonstrated transcripts from the downstream promoter in both the cell lines studied. However, mRNAs transcribed from the upstream promoter could only be detected in MCF-7 cells. Thus, the pattern of DNase I hypersensitivity correlates with the transcriptional activity of the two promoters in the hER gene. Based on these data, we suggest that differential promoter usage might be a novel level of regulation of the hER gene.

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K Alheim, J Corness, MK Samuelsson, LG Bladh, T Murata, T Nilsson, and S Okret

Glucocorticoids are known regulators of the cell cycle, normally exerting an anti-proliferative effect. We have previously shown that glucocorticoids stimulate expression of p57(Kip2), a member of the Cip/Kip family of cyclin-dependent kinase inhibitors which, in some cell types, may account for the anti-proliferative responses seen after glucocorticoid treatment. The induction of p57(Kip2) involves primary transcriptional effects where no de novo protein synthesis is necessary, suggesting a direct interaction of the glucocorticoid receptor with the p57(Kip2) gene. In this study we have identified a functional glucocorticoid response element (GRE), located 5 kilo bases (kb) upstream of the transcription start site in the human p57(Kip2) promoter. This GRE was functional also when isolated, suggesting a direct transcriptional effect of the glucocorticoid receptor. Furthermore, mutation of this GRE abolished glucocorticoid induction of the reporter gene, whereas mutation of a nearby Sp1 site did not. Using electrophoretic mobility shift assays, we have shown that the -5 kb p57(Kip2) promoter GRE was able to compete with a well-known GRE for glucocorticoid receptor binding. Sequence comparisons with the mouse genome showed that this GRE is highly conserved, further strengthening the biological importance of this site. All these data emphasize the involvement of this GRE in the glucocorticoid-mediated induction of p57(Kip2) expression.

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L Lundholm, S Moverare, KR Steffensen, M Nilsson, M Otsuki, C Ohlsson, JA Gustafsson, and K Dahlman-Wright

Estrogens reduce adipose tissue mass in both humans and animals. The molecular mechanisms for this effect are, however, not well characterized. We took a gene expression profiling approach to study the direct effects of estrogen on mouse white adipose tissue (WAT). Female ovariectomized mice were treated for 10, 24 and 48 h with 17beta-estradiol or vehicle. RNA was extracted from gonadal fat and hybridized to Affymetrix MG-U74Av2 arrays. 17beta-Estradiol was shown to decrease mRNA expression of liver X receptor (LXR) alpha after 10 h of treatment compared with the vehicle control. The expression of several LXRalpha target genes, such as sterol regulatory element-binding protein 1c, apolipoprotein E, phospholipid transfer protein, ATP-binding cassette A1 and ATP-binding cassette G1, was similarly decreased. We furthermore identified a 1.5 kb LXRalpha promoter fragment that is negatively regulated by estrogen. Several genes involved in lipogenesis and lipolysis were identified as novel targets that could mediate estrogenic effects on adipose tissue. Finally, we show that ERalpha is the main estrogen receptor expressed in mouse white adipose tissue (WAT) with mRNA levels several hundred times higher than those of ERbeta mRNA.