The 5'-flanking region of the rat insulin II gene (-448 to +50) is sufficient for tissue-specific expression. To further determine the tissue-specific cis-acting element(s), important sequences defined by linker-scanning mutagenesis were placed upstream of a heterologous promoter and transfected into insulin-producing and -nonproducing cells. Rat insulin promoter element 3 (RIPE3), which spans from -125 to -86, was shown to confer jI-cell-specific expression in either orientation. However, two subregions of RIPE3, RIPE3a and RIPE3b (defined by linker-scanning mutations), displayed only marginal activities. These results suggest that the two subregions cooperate to confer tissue specificity, presumably via their cognate binding factors.The insulin gene is expressed exclusively in pancreatic I cells in normal adults and is therefore a good model system for the study of tissue-specific gene expression. Previous studies have shown that less than 600 base pairs (bp) of the 5'-flanking sequences are sufficient for tissue specificity of the insulin gene in both cultured cells (6, 23) and transgenic mice (10). Therefore, the interaction between upstream sequence elements and trans-acting factors is important for regulation of insulin gene expression. Upstream flanking sequences of the mammalian insulin genes are homologous up to 500 bp (22). Furthermore, interspecies gene transfer experiments indicate that the molecular mechanism underlying the tissue specificity of the insulin gene is conserved among mammals.There are two nonallelic insulin genes in rats, the rat insulin I (rInsI) and rat insulin II (rInsII) genes (3, 23). These two genes have more than 80% sequence similarity in both the coding and flanking sequences, and they are coordinately expressed (8). The rInslI gene is more similar in structure than the rInsI gene to the other mammalian insulin genes, and it is proposed that rlnsl gene was duplicated from the ancestral rInsIl gene (21). To better define which sequences within the 5'-flanking region of the rInsIl gene are important for gene activity, linker-scanning (LS) mutagenesis was carried out in our laboratory (4). Mutation of seven regions (referred to as the rat insulin promoter elements [RIPEs]) resulted in significant decreases in gene activity, suggesting that the insulin gene control region is composed of several functional domains or cis elements. These cis elements may cooperate through their cognate trans-acting factors to achieve high levels of transcription. One of the trans-acting factor is the COUP (chicken ovalbumin upstream promoter) transcription factor (18,19), which binds to RIPE1 (-53 to -46) and is likely to play an important role in insulin promoter function (11). Interestingly, the COUP transcription factor contacts the insulin and ovalbumin promoter quite differently (12), the physiological significance of which remains unknown.These mutagenesis results, however, did not define the control element(s) for tissue specificity. It is possible that one or a combination of the LS-define...
The estrogen receptor (ER) is a transcription factor involved in steroid hormone signal transduction in higher eukaryotes. The receptor also functions as a ligand-dependent transcriptional activator when introduced into Saccharomyces cerevisiae (baker's yeast), which suggests that at least some of the components of the signal transduction pathway are conserved between yeast and mammalian cells, and, moreover, allows the possibility of using this simple eukaryotic organism to dissect receptor function. However, whether the ER actually activates transcription in a mechanistically similar fashion in yeast and mammalian cells is unclear, since it has been reported that the transactivation function within the hormone binding domain (TAF-2) does not function in yeast. In this report, we have characterized the activity of the transactivation functions of the ER in yeast. Our results indicate that both TAF-2 and the N-terminal transactivation region (TAF-1) are functional in yeast and contribute synergistically to the receptor's total activity. These results are consistent with those obtained in mammalian cells. Furthermore, we show that in yeast the antagonistic effects of the antiestrogen nafoxidine arise from a modulation of the synergistic interactions of TAF-1 and TAF-2, and not simply from an inactivation of TAF-2 by antihormone. Finally, we characterize the effect of ER deletion mutants on chromatin structure in yeast. Our data lend support to the view that the formation of competent transcriptional initiation complexes requires a precise disruption of chromatin structure.
Band-shifting and DNase I-footprinting assays have been used to study the trans-acting factor(s) binding to an important promoter element (-53 to -46 relative to the transcription start) of the rat insulin II gene. A binding activity which footprints a region between -60 and -40 was found in both HIT, a hamster insulinoma cell line, and HeLa cells. A mutation within this region which drastically decreases promoter activity in vivo also greatly reduces binding activity in vitro. This binding activity was purified from HeLa cells and identified by competition and renaturation analyses as being the same as the COUP (chicken ovalbumin upstream promoter) transcription factor, a DNA-binding protein required for efficient transcription of the ovalbumin gene in vitro. Interestingly, the binding sequences of the COUP transcription factor in the ovalbumin and the insulin promoters have only limited similarities.
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