1: J Hypertens. 2003 Nov;21(11):2111-24. Comment in: J Hypertens. 2003 Nov;21(11):2013-4. Regulation of the major isoform of human endothelin-converting enzyme-1 by a strong housekeeping promoter modulated by polymorphic microsatellites. Funke-Kaiser H, Thomas A, Bremer J, Kovacevic SD, Scheuch K, Bolbrinker J, Theis S, Lemmer J, Zimmermann A, Zollmann FS, Herrmann SM, Paul M, Orzechowski HD. Institute of Clinical Pharmacology and Toxicology, Charite - Campus Benjamin Franklin, Berlin, Germany. BACKGROUND: Human endothelin-converting enzyme (ECE)-1, the key enzyme in endothelin biosynthesis, shows broad cell and tissue expression within the cardiovascular system. Expression of ECE-1c, which represents the major ECE-1 isoform, is directed by an alternative promoter, but the mechanisms of ECE-1c promoter regulation are largely unknown. As ECE-1c transcription is initiated from several start sites, we hypothesized that the ECE-1c promoter functions as a housekeeping promoter. OBJECTIVE: To investigate the putative housekeeping function of the ECE-1c promoter in vascular endothelial cells, which represent a main site of its expression. RESULTS: Using promoter reporter assays, gel shift and supershift assays, we have demonstrated, in human endothelial EA.hy926 cells, functionality of cis-acting elements for binding of the CAAT-box binding protein NF-YB, GATA-2) E2F-2, and a GC-box binding factor, which are spatially associated with transcriptional start sites of ECE-1c. In the more upstream promoter region we have identified three highly polymorphic dinucleotide repeats, 5'-(CA)n, (CG)n and 3'-(CA)n, which strongly affected promoter function in endothelial EA.hy926 cells (2.7-fold activation comparing the most active to the least active allele) and, in a similar manner, in human neuronal KELLY cells. Finally, by in-vitro methylation, we were able to achieve strong suppression of the ECE-1c promoter activity in endothelial cells. CONCLUSION: Our results provide a molecular explanation for constitutive expression of ECE-1c mRNA. Modulation by genetic and epigenetic mechanisms as revealed in our study may account for interindividual variation of the constitutive endothelin system activity in humans and thus influence individual predisposition to cardiovascular disease. PMID: 14597855 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Biochem Biophys Res Commun. 2002 Mar 15;291(5):1138-45. Transcriptional regulation of the human tumor suppressor p14(ARF) by E2F1, E2F2, E2F3, and Sp1-like factors. Parisi T, Pollice A, Di Cristofano A, Calabro V, La Mantia G. Department of Genetics, General and Molecular Biology, University of Naples Federico II, via Mezzocannone 8, 80134 Naples, Italy. The human ARF/INK4a locus encodes two cell cycle inhibitors, p16(INK4a) and p14(ARF), by using separate promoters. A variety of mitogenic stimuli upregulate ARF but a direct modulation at the transcriptional level has been reported only for E2F-1. We show here that the ARF promoter is strongly responsive also to E2F2 and E2F3, thus providing a strong support to their suggested role in the induction of apoptosis. Through the usage of both deletion mutants and/or site-directed mutants, we surprisingly found that none of the four putative E2F consensus sites is strictly necessary for the upregulation of ARF expression, as a minimal deletion mutant, lacking all the putative E2F binding sites, is still transactivated by E2F. Moreover, our data suggest that the ARF promoter is regulated by E2F through both direct binding to the promoter sequences and indirectly, probably by being tethered to the ARF promoter by Sp1-like factors. (C)2002 Elsevier Science (USA). PMID: 11883935 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Oncogene. 1998 Dec 31;17(26):3463-9. Activation and repression of p21(WAF1/CIP1) transcription by RB binding proteins. Gartel AL, Goufman E, Tevosian SG, Shih H, Yee AS, Tyner AL. Department of Molecular Genetics, University of Illinois at Chicago, 60607, USA. The Cdk inhibitor p21(WAF1/CIP1) is a negative regulator of the cell cycle, although its expression is induced by a number of mitogens that promote cell proliferation. We have found that E2F1 and E2F3, transcription factors that activate genes required for cell cycle progression, are strong activators of the p21 promoter. In contrast, HBP1 (HMG-box protein-1), a novel retinoblastoma protein-binding protein, can repress the p21 promoter and inhibit induction of p21 expression by E2F. Both E2Fs and HBP1 regulate p21 transcription through cis-acting elements located between nucleotides -119 to +16 of the p21 promoter and the DNA binding domains of each of these proteins are required for activity. Sequences between -119 and -60 basepairs containing four Sp1 consensus elements and two noncanonical E2F binding sites are of major importance for E2F activation, although E2F1 and E2F3 differ in the extent of their ability to activate expression when this segment is deleted. The opposing effects of E2Fs and HBP1 on p21 promoter activity suggest that interplay between these factors may determine the level of p21 transcription in vivo. PMID: 10030670 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Mol Cell Biol. 1996 Apr;16(4):1659-67. Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F. Karlseder J, Rotheneder H, Wintersberger E. Institut Fur Molekularbiologie, Universitat Wien, Austria, USA. Within the region around 150 bp upstream of the initiation codon, which was previously shown to suffice for growth-regulated expression, the murine thymidine kinase gene carries a single binding site for transcription factor Sp1; about 10 bp downstream of this site, there is a binding motif for transcription factor E2F. The latter protein appears to be responsible for growth regulation of the promoter. Mutational inactivation of either the Sp1 or the E2F site almost completely abolishes promoter activity, suggesting that the two transcription factors interact directly in delivering an activation signal to the basic transcription machinery. This was verified by demonstrating with the use of glutathione S-transferase fusion proteins that E2F and Sp1 bind to each other in vitro. For this interaction, the C-terminal part of Sp1 and the N terminus of E2F1, a domain also present in E2F2 and E2F3 but absent in E2F4 and E2F5, were essential. Accordingly, E2F1 to E2F3 but not E2F4 and E2F5 were found to bind sp1 in vitro. Coimmunoprecipitation experiments showed that complexes exist in vivo, and it was estabilished that the distance between the binding sites for the two transcription factors was critical for optimal promoter activity. Finally, in vivo footprinting experiments indicated that both the sp1 and E2F binding sites are occupied throughout the cell cycle. Mutation of either binding motif abolished binding of both transcription factors in vivo, which may indicate cooperative binding of the two proteins to chromatin-organized DNA. Our data are in line with the hypothesis that E2F functions as a growth- and cell cycle regulated tethering factor between Sp1 and the basic transcription machinery. PMID: 8657141 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------