Abstract
Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) and hepatocyte nuclear factor-4 (HNF-4) are orphan members of the steroid/thyroid receptor superfamily and exhibit ubiquitous and liver-enriched tissue distribution, respectively. The gene for rat ornithine transcarbamylase (OTC), an ornithine cycle enzyme, is mainly expressed in the liver and is under the control of the promoter and the 11-kilobase upstream enhancer, both of which are liver-selective. Two sites of the promoter region and two sites of the enhancer region of the OTC gene, as well as the ovalbumin promoter site, were recognized by both HNF-4 and COUP-TF, showing that these two factors have closely related binding specificities. Since HNF-4 activated expression from the OTC promoter in cotransfection analysis, this factor appears to participate in liver-selective activation of the OTC gene. On the other hand, COUP-TF repressed the expression from the OTC promoter, whereas it activated expression from several other promoters. Therefore, COUP-TF plays a dual regulatory role depending on the promoter context. Repression of a tissue-specific promoter by a ubiquitous transactivator and derepression by a related tissue-enriched transactivator is potentially an important mechanism for tissue-specific activation of a gene.
Highlights
The plasmid expressing the COUP-cTDFNA wasconstructed and transfected intoCOS7 cells. Another orphan receptorsuperfamilymemberHNF-4(Sladek et al, 1990), which is enriched in the liver and intestine and recognizes DNA sequences similar to chicken ovalbumin upstream promoter-trantion factor (COUP-TF) binding sites,was expressedin COS7cells, using the HNF-4 isoform cDNA (Hata et al.,1992).Nuclear extracts preparefdrom transfected cells were subjected to gel shift analysisusing OTC promoter andenhancersitesasprobes (Fig. 4)
112 18 observed with HNF-4,in addition to COUP-TF(Fig. 5).These results show that COUP-TF and HNF-4 havcelosely related DNA bindingspecificities
Insertion of one reporter constructs containing thBe site-tk promoter and the copy of the B site led to no remarkable change in the basal SV40 early promoter, confirming that COUP-TF can act as chloramphenicol acetyltransferase (CAT) activity, insertion of the two copies resulted in a 2.5fold reduction of thebasalactivity.B sitesarranged in tandem can function as a negative element in the context of the tk promoter, in spite of original identification of this site asa positive element of the OTC promoter
Summary
1fmol of the probe (about 2 X lo4dpm), and 5 pg of nuclear extract protein. Incompetition analysis, 1pmol of the competitor oligonucleotide was mixed before the addition of nuclear extracts. Gel Shift Assay-Nuclear extracts from the liver, brain, and spleen of Wistar rats were prepared as described (Gorski et al, 1986).COS7 cells weregrown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and transfected by the calcium phosphate precipitation method (Chen and Okayama, 1987) with 15 pg of the COUP-TF or HNF-4 expression plasmid. To characterize factors binding to these promoter elements, we carried out gel shift analysis, using synthetic oligonucleotides for B and C sites as probes (Fig. 1B). Bands of group 1 were the most prominent with liver nuclear extracts compared with otherextracts, whereas bands of group 2 were the most prominent with brainextracts.Theseresults suggest that there are multiple forms of factors binding to site B andthat their contents incells vary from one tissue to another.Using probe C, two groups of bands were detected. In foregoing work (Murakami et al, 1990), we found that factors binding to sites I1and 111are related to aliver-enriched transcription factor C/EBP
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
