ABSTRACT The transcription initiation site of the Saccharomyces cerevisiae PRS1 gene was mapped at −179 bp. Measurement of β-galactosidase activity of the successively deleted PRS1promoter linked to lacZ and integrated at theura3 locus defined three DNA regions involved in the control of PRS1 expression. Gel shift analysis confirmed the data.

The transcription of the fatty acid synthase (FAS) gene is regulated by the sterol status of the cell via cleavage of the sterol regulatory element-binding protein (SREBP). When human HepG2 hepatoma cells were cotransfected with an expression plasmid for mature SREBP-1a together with FAS promoter/reporter constructs significant increases in reporter activity were observed. Deletion analysis of the FAS promoter between -151 and -52 relative to the transcription start site pinpoint two cis-elements important in sterol regulation of the FAS gene. One element, FIRE3, between -71 and -52 can bind in vitro translated and transcribed SREBP-1a whereas the other element, the inverted CCAAT element ICE(-97/-92), binds the trimeric transcription factor NF-Y/CBF as shown with rat liver extract and reconstituted, recombinant NF-Y. The results clearly show that the coactivator for SREBP-1a in this cell line is NF-Y. This finding was confirmed by using a dominant negative form of NF-YA, NF-YAm29, which interferes with the effect of ectopically expressed SREBP-1a on FAS reporter activity.

AbstractAs a first step towards understanding the mechanism underlying the differential gene expression of the two variants of the rat proteinase-inhibitor α1-Inhibitor 3 (α1I3) corresponding genomic clones were isolated. The 100% similarity between the sequence of one genomic clone and that of the α1-I3 variant I cDNA strongly suggested that its 5′-sequence represented the upstream region of the corresponding gene. Several putativecis-regulatory elements were identified as well as a polypyrimidine tract located between the transcription start site of the α1-I3 variant I mRNA and the AUG codon. The polypyrimidine tract functions as a positivecis-element in a heterologous promoter. By electrophoretic mobility shift assays (EMSA) we have shown that a GST (glutathione S-transferase) fusion of the rat polypyrimidine tract binding protein (PTB) has a high affinity for the pyrimidine-rich sense strand but not for the complementary sequence of the 5′-untranslated region of the α1-I3 variant I gene.

Several putative insulin-responsive elements (IRE) in the fatty acid synthase (FAS) promoter have been identified and shown to be functional in adipocytes and hepatocytes. Here we report on the insulin-responsiveness in the rat hepatoma cell line H4IIE of four cis-elements in the FAS promoter: the FAS insulin-responsive elements, FIRE2 and FIRE3; the inverted CCAAT element, ICE; and the insulin/glucose-binding element, designated hepatic FIRE element, hFIRE, originally identified in rat hepatocytes. Using electrophoretic mobility shift assay (EMSA) competition experiments together with supershifts and in vitro transcription/translation we show that FIRE3 (-68/-58) binds not only the upstream stimulatory factors USF-1/USF-2 but also the CCAAT-binding factor CBF, also known as the nuclear factor Y, NF-Y. The putative IRE FIRE2, which shows sequence similarity to FIRE3, is located between -267 and -249. Gel retardation experiments indicate that USF-1 and USF-2 also bind to this element, which contains an imperfect E-box motif. Using the same approach we have shown that hFIRE binds the stimulatory proteins Sp1 and Sp3 in addition to CBF. Transient transfection experiments using FAS promoter constructs deleted for FIRE2 and FIRE3 demonstrate that neither of these elements mediates the insulin response of the FAS promoter in the rat hepatoma cell line H4IIE, however, ICE at -103/-87 is responsible for mediating the effect of the insulin antagonist cAMP. The hFIRE element located at -57/-34, in spite of its role in the glucose/insulin response in primary rat hepatocytes, is apparently not involved in the insulin regulation of the rat FAS promoter in H4IIE cells. The fact that the topology of the promoters of the FAS genes in rat, human, goose and chicken is conserved regarding CBF-binding sites and USF-binding sites implies an important role for these ubiquitously expressed transcription factors in the regulation of the FAS promoter.

Using EMSA competition experiments together with supershifts and in vitro transcription/translation we show that the basal transcription factor NF-Y or a related factor binds to the cAMP-responsive inverted CCAAT box recently identified in the rat fatty acid synthase (FAS) gene from nucleotide -99 to -92 relative to the transcription start site of the FAS mRNA. This result indicates a putative novel role for NF-Y in the cAMP-dependent gene regulation in a small class of genes such as FAS and tryptophan hydroxylase. Since NF-Y is a constitutively produced factor, not surprisingly, no differences in the specific DNA/protein complex with the CCAAT(FAS) box and nuclear proteins from H4IIE cells treated with cAMP and/or insulin or not could be observed. This implies that NF-Y might be modified in response to cAMP or might interact with another factor whose properties are altered by cAMP.

Screening of two rat liver cDNA libraries, one of which was constructed using an α1‐inhibitor 3 (α1‐I3) specific primer, yielded overlapping cDNA clones which correspond to the full length cDNA for α1‐I3 mRNA. On the basis of sequence microheterogeneity existing throughout the cDNA sequence we identified two α1‐I3 mRNA species whose sequences are so grossly different in their bait regions that the amino acid homology therein is only 30%. Using oligonucleotide probes derived from their respective bait regions we investigated the regulation of the two α1 I3 mRNA species and demonstrated that only one of them, α1‐I3 variant I, is regulated pretranslationally following experimentally induced inflammation.