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.Accumulating evidence suggests that the regulation of gene expression is mediated by the interaction of cis elements and trans-acting factors (1,6,10,14,19,21,23,26). Many sequence elements, including the TATA box, upstream promoter elements, enhancers, and silencers, have been identified as important for the expression of a large number of eucaryotic genes transcribed by RNA polymerase II. Similarly, a number of trans-acting factors which bind to these regulatory elements have been identified. Nevertheless, a critical question remains as to the molecular mechanism by which these trans-acting factors regulate transcription. A clear understanding requires the purification and characterization of these factors.The insulin gene is expressed only in pancreatic ,B cells, thus providing a good model system for the study of tissuespecific gene expression. In rats, there are two nonallelic insulin genes which are highly homologous in flanking as well as coding regions (17) and are expressed at similar levels (2, 3). It is therefore likely that these two genes are controlled by similar transcriptional regulatory mechanisms.The DNA sequences important for the expression of the rat insulin genes have been approximately determined. With insulin-CAT (chloramphenicol acetyltransferase) fusion genes, Walker et al. (32) showed that the -300 to +51 fragment of the rat insulin I gene is sufficient for tissuespecific expression in transfected insulinoma cells. Subsequently, Edlund et al. (7) demonstrated that this fragment can be divided into two distinct sequence elements, the promoter and the enhancer, each of which can direct tissuespecific expression. For the rat insulin II gene, Hanahan (13) demonstrated that 520 base pairs of 5'-flanking sequences linked to the simian virus 40 (SV40) T-antigen gene induced P-cell-specific tumor formation in transgenic mice. A similar fragment is sufficient for tissue-specific expression in cultured cells, as shown by Episkopou et al. (8). In addition to the positive elements, sequences at 2 to 4 kilobases (15) and 250 base pairs (22) upstream from the cap site inhibit expression of the rat insu...
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