Abstract
At least two genes encode isoenzymes of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Alternative splicing of one of these genes generates a skeletal muscle-specific transcript from an upstream promoter and a liver-specific transcript from a downstream promoter. A potent glucocorticoid response element was identified in the first intron of the gene, i.e. between liver exon I and exon II. The element is approximately 3.5 kilobase pairs (kb) downstream of the liver isoenzyme transcription start site and 13 kb upstream of exon II of the gene and confers dexamethasone-sensitive expression of chloramphenicol acetyltransferase (CAT) activity from a heterologous thymidine kinase promoter and from both homologous 5'-flanking regions of the gene. This glucocorticoid response element also exhibits androgen- but not estrogen-sensitive expression of CAT activity in HeLa cells cotransfected with the appropriate receptor expression vector. DNase footprint and sequence analysis revealed that the element is comprised minimally of two adjacent 15-mer glucocorticoid receptor dimer binding sites situated in opposite orientations. Glucocortcoid regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in liver and skeletal muscle is mediated by a single complex glucocorticoid response element located in the first intron of the skeletal muscle/liver gene.
Highlights
At least two genes encode isoenzymesof rat 6-phos- notransferase (TAT)’ is induced by dexamethasone (11, and phofructo-2-kinase/fructose-2,6-bisphosphatase.Al- investigations of the regulation of this and other genes by ternative splicing of one of these genes generates a glucocorticoids led tothe identification of DNA response skeletal muscle-specific transcript from an upstream elements (GREs) which bind ina site-specific manner to promoter and aliver-specific transcript from a down- glucocorticoid hormone-receptor complexes (2,3)
Muscle is mediated by a single complex glucocorticoid Rat liver 6-PF-2-K/Fru-2,6-P2ase is encoded by a gene response element located in the first introonf the skel- whose expression is subject to a complex pattern of hormonal etal muscle/liver gene
Dexamethasone induction of specific proteins has been determined by twodimensional high resolution electrophoresis in two hepatoma cell lines (1).It hasbeen demonstrated that theexpression of a small subsetof the total liver proteins is affected by the hormone
Summary
Isolation andStructure of Rat Liver 6-PF-2-K/Fru-2,6Prase Gene-In order to identify the glucocorticoid response element of the skeletal muscle/liver 6-PF-2-K/Fru-2,6-P2ase gene it was necessary to isolate the gene and characterize its genomic organization. Three restriction fragments from the 5”flanking region of the liver first exon were used 1)a 2700-bp HindIII-HinfI fragment; 2) a 800-bp HinfI-HinfI fragment, and 3) a 500-bp AccI-HinfI fragment All of these restriction fragmentshave the same 3’ end, 133 bp3’ of the transcription startsite reported by Darville et al (23). This position is 212 bp 3‘ of the transcription startsite determined in this paper (see above) When these constructs were transfected into FAO, FTOSB, or DDTcells, their CAT activity was very low and no greater than that of the “promoterless” pBLCAT3 parent construct (data not shown). As shown, when single and multiple copies of the SV40 enhancer 72-bp repeat were cloned upstream of the liver 500-bp AccI-HinfI 5”flanking region inthe pBLCAT3 construct, CAT activity was seen in transfected FA0 cells.
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