Abstract
The potential of the CREM family of proteins to activate transcription of the genes encoding the testis-specific isozyme of angiotensin converting enzyme (ACET) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (EC 4.1.1.32) were investigated. Both CREM tau and CREM alpha bind efficiently to the putative cyclic AMP response element (CRE) present in the ACET gene (CRET) and to the CRE in the PEPCK gene. In HepG2 cells, the CRE was required for the strong stimulation by CREM tau of the expression of a chimeric PEPCK (-210 to +73)-chloramphenicol acetyl transferase (CAT) gene. The CRE could be mutated to the CRET sequence without losing the stimulatory effects of CREM tau. However, a similar chimeric gene driven by the regulatory region of the ACET gene, which contains the CRET site, could only be stimulated by CREM tau when its imperfect TATA element was mutated to an authentic TATA. Surprisingly, CREM alpha, an alleged inhibitor of CRE-mediated transcription, stimulated the expression of both PEPCK-CAT and ACET-CAT genes in HepG2 cells, a process which required the presence of the CRE and the CRET sites, respectively. In contrast, when the same CRE elements were used to drive the transcription of a chimeric gene containing the thymidine kinase promoter linked to the CAT structural gene, CREM alpha inhibited its expression in HepG2 and JEG3 cells. The expression of the same chimeric gene, however, was stimulated by CREM alpha in F9 embryonal carcinoma cells. These results demonstrated that the nature of the transcriptional effects of CREM isoforms on CRE-mediated transcription depends on the specific gene, the specific cell type and the promoter context of the CRE site.
Highlights
Angiotensin-converting enzyme (ACE)l is the key enzyme of the renin-angiotensin system which regulates blood pressure [1]
We have analyzed the potential role of CREMT and CREMa in the regulation of PEPCK gene transcription in HepG2 cells and have compared the results of this analysis with the induction of transcription of the gene for ACE T · Since the cyclic AMP response element (CRE)-like site may be involved in sperm-specific regulation of ACE T expression, we were interested in determining the trans-acting factors which are preferentially expressed in sperm cells and bind to this regulatory element
Our studies demonstrated that CREMT stimulated transcription via both the CRE of the PEPCK gene and the CRE-like site from the ACET promoter (CRET) of the ACE T gene
Summary
Dept. of Molecular Biology, The Cleveland Clinic Foundation, 9500 Euclid Ave., NC20, Cleveland, OH 44195. We have analyzed the potential role of CREMT and CREMa in the regulation of PEPCK gene transcription in HepG2 cells and have compared the results of this analysis with the induction of transcription of the gene for ACE T · Since the CRE-like site may be involved in sperm-specific regulation of ACE T expression, we were interested in determining the trans-acting factors which are preferentially expressed in sperm cells and bind to this regulatory element One such family of proteins, the CREM proteins, has been recently described [20]. Further experiments demonstrated that the ability of CREMa to stimulate or repress gene transcription depends on the promoter context of the CRE sequence and the cell type
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