1: Biochem J. 2005 Feb 15;386(Pt 1):63-72. The expression of the human neuronal alpha3 Na+,K+-ATPase subunit gene is regulated by the activity of the Sp1 and NF-Y transcription factors. Benfante R, Antonini RA, Vaccari M, Flora A, Chen F, Clementi F, Fornasari D. Department of Pharmacology, School of Medicine, University of Milan, 32 via Vanvitelli, 20129 Milan, Italy. The Na+,K+-ATPase is a ubiquitous protein found in virtually all animal cells which is involved in maintaining the electrochemical gradient across the plasma membrane. It is a multimeric enzyme consisting of alpha, beta and gamma subunits that may be present as different isoforms, each of which has a tissue-specific expression profile. The expression of the Na+,K+-ATPase alpha3 subunit in humans is confined to developing and adult brain and heart, thus suggesting that its catalytic activity is strictly required in excitable tissues. In the present study, we used structural, biochemical and functional criteria to analyse the transcriptional mechanisms controlling the expression of the human gene in neurons, and identified a minimal promoter region of approx. 100 bp upstream of the major transcription start site which is capable of preferentially driving the expression of a reporter gene in human neuronal cell lines. This region contains the cognate DNA sites for the transcription factors Sp1/3/4 (transcription factors 1/3/4 purified from Sephacryl and phosphocellulose columns), NF-Y (nuclear factor-Y) and a half CRE (cAMP-response element)-like element that binds a still unknown protein. Although the expression of these factors is not tissue-specific, co-operative functional interactions among them are required to direct the activity of the promoter predominantly in neuronal cells. PMID: 15462673 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: 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] --------------------------------------------------------------- 3: Endocrinology. 2003 May;144(5):1675-85. Cell context-dependent differences in the induction of E2F-1 gene expression by 17 beta-estradiol in MCF-7 and ZR-75 cells. Ngwenya S, Safe S. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-4466, USA. 17 beta-Estradiol (E2) induces E2F-1 gene expression in ZR-75 and MCF-7 human breast cancer cells. Analysis of the E2F-1 gene promoter in MCF-7 cells previously showed that hormone-induced transactivation required interactions between estrogen receptor alpha (ER alpha)/Sp1 bound to upstream GC-rich sites and NFYA bound to downstream CCAAT sites within the -169 to -54 region of the promoter. This same region of the E2F-1 promoter was also E2 responsive in ER alpha-positive ZR-75 cells; however, further analysis of the promoter showed that cooperative ER alpha/Sp1/NFY interactions were not necessary for hormone-induced transactivation in ZR-75 cells. The upstream GC-rich motifs (-169 to -111) are activated independently by ER alpha/Sp1 in ZR-75 but not MCF-7 cells, and a construct (pE2F-1j(m1)) containing the -122 to -54 downstream CCAAT site that bound NFYA was also E2 responsive. E2 also induced reporter gene activity in ZR-75 cells transfected with an expression plasmid for a chimeric protein containing the DNA-binding domain of the yeast GAL4 protein fused to NFYA (pM-NFYA) and a construct containing five tandem GAL4 response elements. Subsequent studies showed that hormonal activation of pE2F-1j(m1) and pM-NFYA are dependent on nongenomic pathways in which E2 activates cAMP/protein kinase A. Hormone-dependent regulation of E2F-1 gene expression in ZR-75 and MCF-7 involves the same cis elements and interacting transcription factors but different mechanisms, demonstrating the importance of cell context on transactivation pathways, even among ER-positive breast cancer cell lines. PMID: 12697671 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Biochim Biophys Acta. 2002 Dec 12;1579(2-3):81-91. Characterization of the rat RALDH1 promoter. A functional CCAAT and octamer motif are critical for basal promoter activity. Guimond J, Devost D, Brodeur H, Mader S, Bhat PV. Laboratory of Nutrition and Cancer, Centre Hospitalier de l'Universite de Montreal-Hotel-Dieu, Montreal, Quebec, Canada Retinal dehydrogenase type 1 (RALDH1) catalyzes the oxidation of retinal to retinoic acid (RA), a metabolite of vitamin A important for embryogenesis and tissue differentiation. Rat RALDH1 is expressed to high levels in developing kidney, and in stomach, intestine epithelia. To understand the mechanisms of the transcriptional regulation of rat RALDH1, we cloned a 1360-base pair (bp) 5'-flanking region of RALDH1 gene. Using luciferase reporter constructs transfected into HEK 293 and LLCPK (kidney-derived) cells, basal promoter activity was associated with sequences between -80 and +43. In this minimal promoter region, TATA and CCAAT cis-acting elements as well as SP1, AP1 and octamer (Oct)-binding sites were present. The CCAAT box and Oct-binding site, located between positions -72 and -68 and -56 and -49, respectively, were shown by deletion analysis and site-directed mutation to be critical for promoter activity. Nuclear extracts from kidney cells contain proteins specifically binding the Oct and CCAAT sequences, resulting in the formation of six complexes, while different patterns of complexes were observed with non-kidney cell extracts. Gel shift assays using either single or double mutations of the Oct and CCAAT sequences as well as super shift assays demonstrated single and double occupancy of these two sites by Oct-1 and CBF-A. In addition, unidentified proteins also bound the Oct motif specifically in the absence of CBF-A binding. These results demonstrate specific involvement of Oct and CCAAT-binding proteins in the regulation of RALDH1 gene. PMID: 12427543 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Gene. 2001 May 16;269(1-2):141-53. Transcriptional activity of the SHP-1 gene in MCF7 cells is differentially regulated by binding of NF-Y factor to two distinct CCAAT-elements. Xu Y, Banville D, Zhao HF, Zhao X, Shen SH. Department of Animal Science, Macdonald Campus, McGill University, Ste. Anne de Bellevue, H9X 3V9, Quebec, Canada. Our previous studies have shown that SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, is expressed not only in cells of hematopoietic lineages, but also in many non-hematopoietic cells under the control of an alternative tissue-specific promoter, P1. In this study, the activity of the P1 promoter was analyzed in a region spanning 3.5 kb upstream of the major transcription start site in non-hematopoietic MCF-7 cells. Using DNA footprinting, gel retardation assays and mutational analysis, we have characterized cis-regulatory elements that are essential to confer the P1 promoter activity. An upstream Sp1 element (-126 to -118) positively regulated this TATA-box-lacking promoter. Two inverted CCAAT-elements (-332 to -328 and -66 to -62) played important roles in regulating the SHP-1 gene expression, and transcription factor NF-Y predominantly bound to the two CCAAT-elements. Binding of NF-Y to the distal CCAAT-element enhanced the transcriptional activity of the P1 promoter. In contrast, binding of NF-Y to the proximal CCAAT-element and interacting with repressor(s) inhibited the promoter activity. Furthermore, incubation of MCF7 cells with 100 ng/ml trichostatin A, an inhibitor of histone deacetylase, significantly increased the activity of the P1 promoter. Mutation in the proximal CCAAT-element, however, eliminated the activating effect of trichostatin A on the promoter. Together, our data suggest that NF-Y factor can function either as a specific positive or negative regulator of P1 promoter activity in non-hematopoietic MCF7 cells. PMID: 11376946 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------