1: J Biol Chem. 2005 Oct 10; [Epub ahead of print] PPARgamma suppresses proximal alpha 1(I) collagen promoter via inhibition of p300-facilitated NF-I binding to DNA in hepatic stellate cells. Yavrom S, Chen L, Xiong S, Wang J, Rippe RA, Tsukamoto H. Pathology Dept., Keck School of Medicine of USC, Los Angeles, CA 90089-9141. Depletion of peroxisome proliferator-activated receptor gamma (PPARgamma) represents one of the key molecular changes that underlie transdifferentiation (activation) of hepatic stellate cells (HSC) in the genesis of liver fibrosis (Miyahara T, et al. J Biol Chem 275: 35715-22; Hazra S, et al. J. Biol Chem 279:11392-401, 2004). In support of this notion, treatment with PPARgamma ligands or ectopic expression of PPARgamma suppresses HSC activation markers, most notably expression of alpha1(I) procollagen. However, the mechanisms underlying this anti-fibrotic effect are largely unknown. The present study utilized deletion-reporter gene constructs of proximal 2.2kb alpha1(I) procollagen promoter to demonstrate that a region proximal to -133bp is where PPARgamma exerts its inhibitory effect. Within this region, two DNase footprints with overlapping NF1 and Sp1 sites exist. NF-I independently stimulates the promoter activity and synergistically promotes Sp1-induced activity. PPARgamma inhibits NF-I binding to the most proximal footprint (-97/-85bp) and inhibits its transactivity. The former effect is mediated by PPARgamma's ability to inhibit p300-facilitated NF1 binding to DNA as demonstrated by chromatin immunoprecipitation assay. PMID: 16216869 [PubMed - as supplied by publisher] --------------------------------------------------------------- 2: J Biol Chem. 2005 Sep 23;280(38):32676-82. Epub 2005 Jul 29. Differentiation-dependent up-regulation of intestinal thiamin uptake: cellular and molecular mechanisms. Nabokina SM, Reidling JC, Said HM. Veteran Affairs Healthcare System, Long Beach, California 90822, USA. Differentiation of intestinal epithelial cells is associated with up-and-down regulation of expression of a variety of genes including those involved in nutrient uptake. Nothing is known about possible differentiation-dependent regulation of the intestinal thiamin uptake process and the cellular and molecular mechanisms involved in such regulation. Using as models human-derived intestinal epithelial Caco-2 cells and crypt/villus epithelial cells isolated from wild-type and transgenic mice carrying promoters for human thiamin transporter-1 and -2 (hTHTR-1 and hTHTR-2), we addressed this issue. Our results showed that differentiation of Caco-2 cells is associated with a significant up-regulation in carrier-mediated thiamin uptake. Up-regulation was associated with a significant increase in the level of expression of hTHTR-1 and hTHTR-2 protein and mRNA as well as in activity of the corresponding transfected human thiamin transporter-1 (SLC19A2) and -2 (SLC19A3) promoters. Deletion analysis identified the differentiation-responsive region to be at position -356 to -275 bp for the SLC19A2 promoter and at position -77 to -13 bp for the SLC19A3 promoter. In addition, a critical and specific role in the differentiation-mediated effects for an NF1 binding site (-348 to -345 bp) in the SLC19A2 promoter and a SP1/GC-box binding site (-48 to -45 bp) in the SLC19A3 promoter were established using mutational analysis. The physiological relevance of in vitro findings with Caco-2 cells was confirmed in wild-type and transgenic mice by demonstrating that thiamin uptake and mRNA levels of the mouse THTR-1 and THTR-2, as well as activity of human SLC19A2 and SLC19A3 promoters expressed in transgenic mice, were all significantly higher in intestinal villus compared with crypt epithelial cells. These studies demonstrate for the first time that differentiation of intestinal epithelial cells is associated with an up-regulation in thiamin uptake process and that this up-regulation appears to be mediated via transcriptional regulatory mechanisms that involve the SLC19A2 and SLC19A3 genes. PMID: 16055442 [PubMed - in process] --------------------------------------------------------------- 3: Biochim Biophys Acta. 2005 Jan 11;1681(2-3):88-98. Epub 2004 Nov 24. Functional analysis of the mouse Fcgrt 5' proximal promoter. Tiwari B, Junghans RP. Biotherapeutics Development Lab, Harvard Institutes of Human Genetics and Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA. The Brambell receptor (FcRB, FcRn) mediates transmission of immunity from mother to young perinatally and plays a central role in IgG protection and homeostasis throughout life. Developmental and tissue specific expression is via transcriptional regulation of the gene for the receptor heavy chain, Fcgrt. In neonatal mouse intestine, expression is high to absorb IgG from mother's milk, but then nearly absent in adult intestine and in most other tissues. In this initial functional characterization, reporter gene assays revealed at least two upstream promoter regions (-372/-140 and -105/-1), each with repressor and activator functions, and a downstream activator domain (+1/+78). The gene carries no upstream TATA element or Inr sequence, and an apparent downstream promoter feature (DPE) lacks typical context for an active element, rendering uncertain the nature of the organizing feature for the transcription initiation complex. Electrophoretic mobility shift analysis of the proximal upstream region identified transcription factor (TF) binding to motifs for NF1, Sp1 (GT box) and Ets. Binding to the GT box and Ets motif was observed only with mature cell sources, and not with neonatal enterocyte extracts. Site-directed mutagenesis confirmed that TF binding to the GT box up-regulates promoter activity in adult (NIH3T3) cells, whereas binding to the Ets motif represses activity. Binding of NF1 protein to the Fcgrt promoter was confirmed in nuclear extracts of NIH3T3 cells and in adult mouse enterocytes, whereas an apparently different TF bound uniquely to the NF1 site in neonatal enterocyte extracts as the sole identified candidate for an expected developmental-specific high-level activator in this tissue. These data indicate regions of potential importance in the Fcgrt proximal promoter and additionally suggest that the selective temporal and spatial availability of specific TFs may contribute to the developmental and tissue-specific regulation of Fcgrt expression. PMID: 15627500 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Eur J Cancer. 2004 Dec;40(18):2820-8. Methylation analysis of the neurofibromatosis type 1 (NF1) promoter in peripheral nerve sheath tumours. Harder A, Rosche M, Reuss DE, Holtkamp N, Uhlmann K, Friedrich R, Mautner VF, von Deimling A. Institute of Neuropathology, Charite-University Medicine Berlin, Augustenburger Platz 1, Berlin 13353, Germany. anja.harder@charite.de Peripheral nerve sheath tumours are hallmarks of neurofibromatosis type 1 (NF1). Development of plexiform neurofibromas to malignant peripheral nerve sheath tumours (MPNST) is common. The NF1 gene promoter harbours a hypomethylated CpG island. Thus, methylation changes may be involved in the development of different types of neurofibromas and malignant transformation. We investigated NF1-associated dermal (n=9) and plexiform neurofibromas (n=7), MPNST (n=5) and non-NF1 leucocyte samples (n=20) for their methylation pattern by bisulphite genomic sequencing. We could not find global hypermethylation in the NF1 promoter in our series. Nevertheless, site-specific methylation, involving transcription factor binding sites for SP1, CRE (-10), and AP-2, was observed. One region of the 5'-UTR (untranslated region) overlapping with a putative AP-2 binding site was methylated at 30-100% in 4/20 control samples. In conclusion, we did not find hypermethylation in NF1-associated tumours. Instead, low level methylation could parallel a global genomic hypomethylation in malignancy. PMID: 15571966 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: J Clin Invest. 2004 Oct;114(8):1146-57. Epigenetic regulation of 11 beta-hydroxysteroid dehydrogenase type 2 expression. Alikhani-Koopaei R, Fouladkou F, Frey FJ, Frey BM. Department of Nephrology and Hypertension, University Hospital of Berne, Berne UNK 3010, Switzerland. The enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) is selectively expressed in aldosterone target tissues, where it confers aldosterone selectivity for the mineralocorticoid receptor by inactivating 11 beta-hydroxyglucocorticoids. Variable activity of 11 beta HSD2 is relevant for blood pressure control and hypertension. The present investigation aimed to elucidate whether an epigenetic mechanism, DNA methylation, accounts for the rigorous control of expression of the gene encoding 11 beta HSD2, HSD11B2. CpG islands covering the promoter and exon 1 of HSD11B2 were found to be densely methylated in tissues and cell lines with low expression but not those with high expression of HSD11B2. Demethylation induced by 5-aza-2'-deoxycytidine and procainamide enhanced the transcription and activity of the 11 beta HSD2 enzyme in human cells in vitro and in rats in vivo. Methylation of HSD11B2 promoter-luciferase constructs decreased transcriptional activity. Methylation of recognition sequences of transcription factors, including those for Sp1/Sp3, Arnt, and nuclear factor 1 (NF1) diminished their DNA-binding activity. Herein NF1 was identified as a strong HSD11B2 stimulatory factor. The effect of NF1 was dependent on the position of CpGs and the combination of CpGs methylated. A methylated-CpG-binding protein complex 1 transcriptional repression interacted directly with the methylated HSD11B2 promoter. These results indicate a role for DNA methylation in HSD11B2 gene repression and suggest an epigenetic mechanism affecting this gene causally linked with hypertension. PMID: 15489962 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Mol Endocrinol. 2004 May;18(5):1144-57. Epub 2004 Feb 26. GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1. Fluck CE, Miller WL. Department of Pediatrics, Building MR IV, Room 207, University of California San Francisco, San Francisco, California 94143-0978, USA. Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C (nuclear factor 1-C) to the first 227 bp of 5'flanking DNA (-227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express -227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at -64/-58 and an SF-1 site at -58/-50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated -227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in -227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at -196/-188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected -227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells. PMID: 14988427 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Gene. 2004 Jan 21;325:145-55. Genomic organization and differential splicing of the mouse and human Pcyt2 genes. Poloumienko A, Cote A, Quee AT, Zhu L, Bakovic M. Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1, Canada. CTP: ethanolaminephosphate cytidylyltransferase (Pcyt2) is an important regulatory enzyme in phosphatidylethanolamine and plasmalogen biosynthesis. We cloned the mouse gene mPcyt2 and established its relationship with the human homolog PCYT2. The two genes share similar size and contain two conserved catalytic domains but exhibit different exon/intron organization. An internal region could be alternatively spliced producing a longer mouse transcript, mPcyt2 alpha, and a shorter human transcript, PCYT2 beta. The spliced region is entirely made from mPcyt2 Exon 7 and encodes the peptide PPHPTPAGDTLSSEVSSQ, located upstream of the second catalytic motif HIGH. Mouse and human proteins also differ in amino acid composition at the C-terminus due to an additional splicing between Exons 13 and 14 in PCYT2.The 5' RACE analyses and subsequent cloning of the promoter regions demonstrated that the mPcyt2 and PCYT2 promoters are located immediately upstream of the first exon. There is no sequence homology between the two promoters but they are both TATA-less, have conserved CAAT boxes at a matching distance (-85/-70 bp) from the transcription start site and contain cis-elements for transcription factors of the CAAT, Sp1 and NF1 family, all in accordance with ubiquitous expression of both genes. The mPcyt2 gene is highly expressed in liver, brain, adipose tissues, heart, skeletal muscle, spleen, lungs and kidney. In THP-1 and U937 cells, PCYT2 expression could vary with the stage of cell differentiation. Luciferase reporter analyses show that the Pcyt2 and PCYT2 promoters are strong promoters similar to other ubiquitous promoters, such as those of Pcyt1 and SV-40. PMID: 14697519 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Oncogene. 2004 Jan 15;23(2):330-9. Characterization of functional elements in the neurofibromatosis (NF1) proximal promoter region. Zou MX, Butcher DT, Sadikovic B, Groves TC, Yee SP, Rodenhiser DI. London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada. An essential requirement to understand how genes contribute to genetic disease is the thorough knowledge of the transcriptional regulation of gene expression. Here, we have characterized transcription factor binding sites within the type 1 neurofibromatosis (NF1) proximal regulatory region, and addressed the molecular mechanisms that regulate NF1 transcription. Overlapping regions of the NF1 proximal promoter have been cloned and characterized for use in luciferase reporter assays. These assays have identified a 500 bp region displaying activities up to 80-fold higher than control reporter levels. Mutations at putative CRE and SP1-binding sites immediately 5' to the transcription start site have dramatic effects that lead to a 70-90% decrease in reporter activity in all cell lines tested. Gelshift assays confirm binding of CREB and SP1/KLF family members to their putative recognition sequences, and provide the first evidence identifying functional sites likely involved in regulating NF1 transcription. These assays have also revealed a putative repressor region within the NF1 promoter region corresponding to CCCTC-rich sequences between the transcription and translation start sites. This work provides new information concerning the transcriptional regulation of the NF1 gene, and is the most thorough attempt to date to map functionally relevant regions within the NF1 proximal promoter region. PMID: 14647436 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Genes Chromosomes Cancer. 2003 Oct;38(2):157-67. Site-directed mutagenesis of the ATM promoter: consequences for response to proliferation and ionizing radiation. Gueven N, Keating K, Fukao T, Loeffler H, Kondo N, Rodemann HP, Lavin MF. Queensland Cancer Fund Research Laboratory, The Queensland Institute of Medical Research, Royal Brisbane Hospital, Brisbane, Queensland, Australia. Although ATM, the protein defective in ataxia-telangiectasia (A-T), is activated primarily by radiation, there is also evidence that expression of the protein can be regulated by both radiation and growth factors. Computer analysis of the ATM promoter proximal 700-bp sequence reveals a number of potentially important cis-regulatory sequences. Using nucleotide substitutions to delete putative functional elements in the promoter of ATM, we examined the importance of some of these sites for both the basal and the radiation-induced activity of the promoter. In lymphoblastoid cells, most of the mutations in transcription factor consensus sequences [Sp1(1), Sp1(2), Cre, Ets, Xre, gammaIre(2), a modified AP1 site (Fse), and GCF] reduced basal activity to various extents, whereas others [gammaIre(1), NF1, Myb] left basal activity unaffected. In human skin fibroblasts, results were generally the same, but the basal activity varied up to 8-fold in these and other cell lines. Radiation activated the promoter approximately 2.5-fold in serum-starved lymphoblastoid cells, reaching a maximum by 3 hr, and all mutated elements equally blocked this activation. Reduction in Sp1 and AP1 DNA binding activity by serum starvation was rapidly reversed by exposure of cells to radiation. This reduction was not evident in A-T cells, and the response to radiation was less marked. Data provided for interaction between ATM and Sp1 by protein binding and co-immunoprecipitation could explain the altered regulation of Sp1 in A-T cells. The data described here provide additional evidence that basal and radiation-induced regulation of the ATM promoter is under multifactorial control. Copyright 2003 Wiley-Liss, Inc. PMID: 12939743 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: J Biol Chem. 2003 Aug 15;278(33):30624-33. Epub 2003 May 30. Repression of the human adenine nucleotide translocase-2 gene in growth-arrested human diploid cells: the role of nuclear factor-1. Luciakova K, Barath P, Poliakova D, Persson A, Nelson BD. Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden. Katarina.Luciakova@savba.sk Adenine nucleotide translocase-2 (ANT2) catalyzes the exchange of ATP for ADP across the mitochondrial membrane, thus playing an important role in maintaining the cytosolic phosphorylation potential required for cell growth. Expression of ANT2 is activated by growth stimulation of quiescent cells and is down-regulated when cells become growth-arrested. In this study, we address the mechanism of growth arrest repression. Using a combination of transfection, in vivo dimethyl sulfate mapping, and in vitro DNase I mapping experiments, we identified two protein-binding elements (Go-1 and Go-2) that are responsible for growth arrest of ANT2 expression in human diploid fibroblasts. Proteins that bound the Go elements were purified and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as members of the NF1 family of transcription factors. Chromatin immunoprecipitation analysis showed that NF1 was bound to both Go-1 and Go-2 in quiescent human diploid cells in vivo, but not in the same cells stimulated to growth by serum. NF1 binding correlated with the disappearance of ANT2 transcripts in quiescent cells. Furthermore, overexpression of NF1-A, -C, and -X in NIH3T3 cells repressed expression of an ANT2-driven reporter gene construct. Two additional putative repressor elements in the ANT2 promoter, an Sp1 element juxtaposed to the transcription start site and a silencer centered at nucleotide -332, did not appear to contribute to growth arrest repression. Thus, enhanced binding of NF1 is a key step in the growth arrest repression of ANT2 transcription. To our knowledge, this is the first report showing a role for NF1 in growth arrest. PMID: 12777383 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Thromb Res. 2003 Feb 15;109(4):207-15. Characterization and functional analysis of TFPI-2 gene promoter in a human choriocarcinoma cell line. Hube F, Reverdiau P, Iochmann S, Cherpi-Antar C, Gruel Y. Laboratoire d'Hemostase, EA 3249 Cellules Hematopoietiques, Hemostase et Greffe, IFR 120 Faculte de Medecine, 2 bis Bd Tonnelle, 37032 Tours Cedex, France. Tissue factor pathway Inhibitor-2 (TFPI-2) is associated with extracellular matrices and plays a major role in cell migration and tumor invasion. In this study, a 4.8-kb human TFPI-2 gene 5'-flanking region was isolated, cloned and sequenced. Promoter region analysis revealed a high GC-rich content without canonical TATA and CAAT boxes but three transcription initiation sites were identified. Moreover, several putative binding sites for transcription factors were identified (MyoD, LYF1, NF-Y, GATA, oct-1, AP-1, Sp1, NF1, NF-kappa B and egr-1). To characterize potential regulatory regions, TFPI-2/luciferase promoter constructs were then transfected in human choriocarcinoma JEG-3 cells. We first showed that the minimal TFPI-2 promoter is located between -166 and -111 from the translation start site. Luciferase activity consistently increased after stimulation of JEG-3 cells by phorbol 12-myristate 13-acetate indicating that NF1, NF-kappa B and egr-1/Sp1 binding sites are crucial in inducible TFPI-2 expression. Moreover, negative regulatory regions included AP-1 binding sites were identified. This study demonstrates that the TFPI-2 gene promoter exhibits typical features of a housekeeping gene. PMID: 12757776 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: Nucleic Acids Res. 2003 Jan 15;31(2):562-9. Basal transcriptional regulation of human damage-specific DNA-binding protein genes DDB1 and DDB2 by Sp1, E2F, N-myc and NF1 elements. Nichols AF, Itoh T, Zolezzi F, Hutsell S, Linn S. Department of Molecular and Cell Biology, Barker Hall, University of California, Berkeley, CA 94720-3202, USA. The human DDB1 and DDB2 genes encode the 127 and 48 kDa subunits, respectively, of the damage-specific DNA-binding protein (DDB). Mutations in the DDB2 gene have been correlated with the hereditary disease xeroderma pigmentosum group E. We have investigated the proximal promoters of the DDB genes, both of which are G/C-rich and do not contain a TATA box. Transient expression analysis in HeLa cells using a luciferase reporter system indicated the presence of core promoters located within 292 bp (DDB1) and 220 bp (DDB2) upstream of the putative transcription initiation sites. Both core promoters contain multiple active Sp1 sites, with those of DDB1 at -123 to -115 and of DDB2 at -29 to -22 being critical determinants of promoter activity. In addition, an N-myc site at -56 to -51 for DDB1 is an essential transcription element, and mutations in a DDB1 NF-1 site at -104 to -92, a DDB2 NF-1 site at -68 to -56 and a DDB2 E2F site at +36 to +43 also reduce promoter activity. Taken together, these results suggest a regulation of basal transcription typical of cell cycle-regulated genes, and therefore support conjectures that the DDB heterodimer and/or its subunits have functions other than direct involvement in DNA repair. PMID: 12527763 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Gene. 2002 Jul 10;294(1-2):259-68. Structural and functional characterization of the human PAX7 5'-flanking regulatory region. Syagailo YV, Okladnova O, Reimer E, Grassle M, Mossner R, Gattenlohner S, Marx A, Meyer J, Lesch KP. Department of Psychiatry and Psychotherapy, University of Wurzburg, Fuchsleinstrasse 15, 97080, Wurzburg, Germany. The human PAX7 gene is a member of the paired box containing gene family of transcription factors implicated in development of the skeletal muscle of the trunk and limbs as well as elements of the central nervous system. To understand the molecular mechanisms involved in its expression, we have localized the transcription start sites in adult skeletal muscle and functionally characterized the 5'-flanking regulatory region responsible for PAX7 expression in this tissue. The major transcription start was identified 664 bp upstream from the ATG codon using primer extension and 5'-rapid amplification of cDNA ends (5'-RACE). Analysis of the 5'-flanking sequence revealed the absence of a TATA-box and the presence of an inverted CCAAT-box. Several consensus sites for common transcriptional regulators including Oct-1, NF1, AP2, AP4, CREB, Sp1, Nkx2.5, and MyoD are present in the promoter region. To determine the sites critical for the function of the PAX7 promoter, a series of deletion fragments of the 5'-flanking region were cloned adjacent to luciferase reporter gene and expressed in RD, Cos-7 and JAR cell lines. The maximal promoter activity was achieved by a fragment extending from the position -403 to +373. No strong positive or negative regulatory elements were discovered by adding of further sequences (up to 2.97 kb). A polymorphic (CCT)(n) repeat sequence was found 107 bp upstream of the transcription initiation site. PCR-based systematic screening for length variations in 227 unrelated individuals of a Caucasian population showed a bimodal distribution of three alleles containing 8, 10 or 11 repeat units. When different variants of this PAX7 gene-linked polymorphic region (PAX7-LPR) were fused to a luciferase reporter gene and transfected into RD cells, the variant with 11 repeat units revealed higher transcriptional efficiency compared to the 8 or 10 repeat alleles. PMID: 12234688 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Int J Dev Neurosci. 2002 Apr;20(2):103-11. Cyclic AMP inducibility of the myelin basic protein gene promoter requires the NF1 site. Clark RE Jr, Miskimins WK, Miskimins R. Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, 414 E. Clark St., Vermillion 57069, USA. In the central nervous system oligodendrocyte differentiation is accompanied by the activation of a specific transcriptional program responsible for the synthesis of myelin genes. One of the signals leading to the expression of myelin components, such as the myelin basic protein (MBP) gene is cyclic AMP (cAMP). Previous work using a cell line in which the endogenous MBP gene can be induced by increased cAMP levels (D6P2T) showed that the region of the MBP gene that was required for induction of the gene by cAMP lay between -248 and -105 in the 5' flanking region. This region contains numerous transcription factor binding sites, including sites for NF1, Sp1, and MEBA. In order to determine if the NF1 site itself was specifically responsible for the cAMP responsiveness of the MBP promoter, stably transfected cells carrying MBP promoter deletion constructs were used. Deletion of just the NF1 site caused loss of responsiveness to cAMP levels. Furthermore, site-specific mutations in the NF1 site that interfere with NF1 protein binding, in the context of the full length promoter, abolished cAMP responsiveness and caused derepression of the promoter. Analysis of protein binding to the NF1 site showed that the mutation resulted in loss of binding to the site and that the proteins binding at the site are modified in the presence of cAMP elevating agents. These results demonstrate that the NF1 site is indispensable for cAMP responsiveness of the MBP promoter and, together with other DNA elements, plays a role in controlling MBP gene expression. PMID: 12034141 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Biochim Biophys Acta. 2002 Mar 19;1574(2):187-92. Characterization of the 5' regulatory region of the human sodium-dependent multivitamin transporter, hSMVT. Dey S, Subramanian VS, Chatterjee NS, Rubin SA, Said HM. Veterans Affairs Medical Center, Los Angeles, CA 90073, USA. We cloned and functionally characterized the 5' regulatory region of the human sodium-dependent multivitamin transporter (hSMVT) gene, a remarkably versatile carrier responsible for uptake of biotin, pantothenic acid and lipoate. Two potential transcriptional start sites were determined by 5'-RACE and found to be at -4603, and -4303. Two distinct promoters (P1 and P2) were identified. Both putative promoter sequences were TATA-less, CAAT-less, contained highly GC-rich sites, and had multiple putative regulatory cis-elements (e.g., AP1, AP2, C/EBP, SP1, NF1, and GATA). The activities of the putative promoters were confirmed using a firefly luciferase reporter gene assay system following transient transfection into three cultured human cell lines: Caco-2, HEK293, and vascular smooth muscle cells. The minimal region required for basal activity of the hSMVT promoter was also determined by generating a series of deletion constructs and found to be encoded by a sequence between -5846 to -5313 for P1 and between -4417 to -4244 for P2 relative to the translation initiation codon. These results demonstrate the first molecular characterization of the regulatory region of this important human gene. PMID: 11955628 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: J Virol. 2002 Apr;76(8):4113-8. Nucleoprotein structure of immediate-early promoters Zp and Rp and of oriLyt of latent Epstein-Barr virus genomes. Niller HH, Salamon D, Uhlig J, Ranf S, Granz M, Schwarzmann F, Wolf H, Minarovits J. Institut fur Medizinische Mikrobiologie und Hygiene, Universitat Regensburg, D-93053 Regensburg, Germany. Hans-Helmut.Niller@klinik.uni-regensburg.de Genomic footprints across Rp, Zp, and oriLyt of Epstein-Barr virus (EBV) have been conducted in a panel of latently infected B-cell lines. Close protein-base contacts were found about 360 nucleotides upstream of the Zp initiation site. Gel shifts and transient transfection assays indicated that an Sp1-NF1 locus may serve as a repressive transcriptional element against Zp induction from latent EBV genomes. PMID: 11907252 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: EMBO J. 2002 Feb 1;21(3):334-43. NF1/X represses PDGF A-chain transcription by interacting with Sp1 and antagonizing Sp1 occupancy of the promoter. Rafty LA, Santiago FS, Khachigian LM. Centre for Thrombosis and Vascular Research, Department of Pathology, The University of New South Wales, Sydney, Australia. The regulatory mechanisms mediating basal and inducible platelet-derived growth factor (PDGF)-A expression have been the focus of intense recent investigation, but repression of PDGF-A expression is largely unexplored. Here we isolated a nuclear factor that interacts with the proximal region of the PDGF-A promoter using bulk binding assays and chromatography techniques. Peptide mass fingerprint and supershift analysis revealed this DNA-binding protein to be NF1/X. NF1/X repressed PDGF-A promoter-dependent transcription and endogenous mRNA expression, which was reversible by oligonucleotide decoys bearing an NF1/X-binding site. Mutation in the DNA-binding domain of NF1/X abolished its repression of PDGF-A promoter. NF1/X antagonized the activity of a known activator of the PDGF-A chain, Sp1, by inhibiting its occupancy of the proximal PDGF-A promoter. NF1/X physically and specifically interacts with Sp1 via its subtype-specific domain and blocks Sp1 induction of the promoter. NF1/X residues 311-416 mediated NF1/X suppression of basal PDGF-A transcription, whereas residues 243-416 were required for NF1/X repression of Sp1-inducible promoter activity. These findings demonstrate that repression of PDGF-A gene transcription is governed by interplay between NF1/X and Sp1. PMID: 11823426 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: J Clin Virol. 2001 Dec;23(1-2):65-77. Intratype HPV16 sequence variation within LCR of isolates from asymptomatic carriers and cervical cancers. Schmidt M, Kedzia W, Gozdzicka-Jozefiak A. Department of Molecular Virology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Miedzychodzka 5, 60-371 Poznan, Poland. mschmidt@amu.edu.pl BACKGROUND: HPV16 is a predominant type of virus identified in genital lesions and strongly associated with the development of genital cancers. Infection with the virus is considered to be the main risk factor in the development of cervical cancer. Based on HPV16 DNA isolated from invasive cancers, a classification of intratype genetic variants was established and the strains were designated according to geographical regions. The HPV16 variants classification was based on isolates derived from cancers. OBJECTIVES: Analysis of HPV16 LCR variants isolated from asymptomatic carriers for comparison with cervical cancer isolates to examine whether a correlation can be found between cervical epithelium state and variant of HPV16 it carries. MATERIALS AND METHODS: The HPV16 LCR fragments were amplified by PCR using DNA isolated from cervical swabs and tissue sections then screened for nucleotide changes by SSCP. Polymorphic sites were analysed for regulatory protein binding properties by EMSA. RESULTS: Comparison of the two groups revealed that isolates from cervical cancers predominantly carry changes in sequences of YY1 binding sites (especially at nucleotide 7519), while variants from asymptomatic carriers contained nucleotide changes within or close to transcription binding sites for AP-1, Oct-1, NF1, Tef-1, Tef-2, Sp1, YY1 and viral E2. EMSA study showed that sequence changes in the segment alter binding and formation of transcriptional complexes in quantitative and/or qualitative manner and so they may inflict viral activity. CONCLUSION: The results of our study show that there might be HPV16 variants of decreased oncogenic potential therefore infection with such variants can recede. PMID: 11595585 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Biochim Biophys Acta. 2001 Jul 30;1520(1):85-8. Cloning and functional characterization of the human heterogeneous nuclear ribonucleoprotein type I promoter. Romanelli MG, Faggioli L, Lorenzi P, Morandi C. Department of Mother and Child, Biology and Genetics, University of Verona, Italy. romane@borgoroma.univr.it We have cloned and functionally characterized a portion of the human hnRNP I (heterogeneous nuclear ribonucleoprotein type I) gene containing the promoter elements. HnRNP I is an alternative splicing modulator of tissue-specific transcripts that is expressed in three different isoforms. The DNA sequence at the transcription start site, identified by 5'-rapid amplification of cDNA ends, shows a high 'GC' content, lacks canonical TATA sequences and contains multiple putative Sp1 and NF1 transcription factor-binding sites, a GATA box and a CAAT box. By means of a chloramphenicol acetyltransferase reporter construct and deletion analyses, we have identified two regions between -770 bp and -206 bp that had a positive effect on expression activity in HeLa cells. PMID: 11470163 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Biochim Biophys Acta. 2001 Feb 16;1517(3):416-23. Characterization of the human UDP-galactose:ceramide galactosyltransferase gene promoter. Tencomnao T, Yu RK, Kapitonov D. Department of Biochemistry and Molecular Biophysics, Medical College of Virginia Commonwealth University, Richmond 23298-0614, USA. UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthesis of galactocerebroside, the most abundant glycosphingolipid in the myelin sheath. An 8 kb fragment upstream from the transcription initiation site of CGT gene was isolated from a human genomic DNA library. Primer extension analysis revealed a single transcription initiation site 329 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start site; however, this region contains a high GC content and multiple putative regulatory elements. To investigate the transcriptional regulation of CGT, a series of 5' deletion constructs of the 5'-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in the human oligodendroglioma (HOG) and human neuroblastoma (LAN-5) cell lines, we found that the CGT promoter functions in a cell type-specific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at -292/-256 which contains the potential binding sites for known transcription factors (TFs) such as Ets and SP1 (GC box), a distal region at -747/-688 comprising a number of binding sites such as the ERE half-site, NF1-like, TGGCA-BP, and CRE, and a third positive cis-acting region distally localized at -1325/-1083 consisting of binding sites for TFs such as nitrogen regulatory, TCF-1, TGGCA-BP, NF-IL6, CF1, bHLH, NF1-like, GATA, and gamma-IRE. A negative cis-acting domain localized in a far distal region at -1594/-1326 was also identified. Our results suggest the presence of both positive and negative cis-regulatory regions essential for the cell-specific expression in the TATA-less promoter of the human CGT gene. PMID: 11342220 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: J Biol Chem. 2001 Jun 8;276(23):20766-73. Epub 2001 Mar 7. Nuclear factor 1 interferes with Sp1 binding through a composite element on the rat poly(ADP-ribose) polymerase promoter to modulate its activity in vitro. Laniel MA, Poirier GG, Guerin SL. Oncology and Molecular Endocrinology Research Center and the Unit of Health and Environment, CHUL Research Center, Ste-Foy, Quebec G1V 4G2, Canada. Poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the rapid and extensive poly(ADP-ribosyl)ation of nuclear proteins in response to DNA strand breaks, and its expression, although ubiquitous, is modulated from tissue to tissue and during cellular differentiation. PARP-1 gene promoters from human, rat, and mouse have been cloned, and they share a structure common to housekeeping genes, as they lack a functional TATA box and contain multiple GC boxes, which bind the transcriptional activator Sp1. We have previously shown that, although Sp1 is important for rat PARP1 (rPARP) promoter activity, its finely tuned modulation is likely dependent on other transcription factors that bind the rPARP proximal promoter in vitro. In this study, we identified one such factor as NF1-L, a rat liver isoform of the nuclear factor 1 family of transcription factors. The NF1-L site on the rPARP promoter overlaps one of the Sp1 binding sites previously identified, and we demonstrated that binding of both factors to this composite element is mutually exclusive. Furthermore, we provide evidence that NF1-L has no effect by itself on rPARP promoter activity, but rather down-regulates the Sp1 activity by interfering with its ability to bind the rPARP promoter in order to modulate transcription of the rPARP gene. PMID: 11278663 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Eur J Hum Genet. 2000 Dec;8(12):939-45. Microsatellite instability and promoter methylation as possible causes of NF1 gene inactivation in neurofibromas. Luijten M, Redeker S, van Noesel MM, Troost D, Westerveld A, Hulsebos TJ. Department of Human Genetics, Academic Medical Center, University of Amsterdam, The Netherlands. Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder. One of the characteristic features of this disease is the development of neurofibromas. Since the NF1 gene is supposed to be a tumour suppressor gene, these neurofibromas should develop upon inactivation of both NF1 alleles. So far, mutation and deletion have been found to be involved in NF1 gene inactivation. However, these inactivating mechanisms explain the development of only a limited fraction of analysed neurofibromas. In this study, we investigated microsatellite instability (MSI) and promoter methylation as potential contributors to NF1 gene inactivation. As site-specific methylation in the NF1 promoter inhibits binding of transcription factors Sp1 and CREB, we studied the methylation status of their binding sites in particular. We analysed 20 neurofibromas and three neurofibrosarcomas, but did not find evidence for microsatellite instability or NF1 promoter methylation in any of the tumours. Thus, our data suggest that both microsatellite instability and promoter methylation are unlikely to be the major causes of NF1 gene inactivation in these tumours. PMID: 11175282 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Mol Endocrinol. 2001 Jan;15(1):93-111. SF-1 (steroidogenic factor-1), C/EBPbeta (CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells. Aigueperse C, Val P, Pacot C, Darne C, Lalli E, Sassone-Corsi P, Veyssiere G, Jean C, Martinez A. UMR Centre National de la Recherche Scientifique 6547 Physiologie Comparee et Endocrinologie Moleculaire, Universite Blaise Pascal Clermont II, Complexe Universitaire des Cezeaux 63177 Aubiere cedex, France. The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1. PMID: 11145742 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Hepatology. 2000 Dec;32(6):1377-85. The promoter of the long variant of collagen XVIII, the precursor of endostatin, contains liver-specific regulatory elements. Lietard J, Theret N, Rehn M, Musso O, Dargere D, Pihlajaniemi T, Clement B. Detoxication and Tissue Repair Unit, INSERM U-456, Universite de Rennes I, France. The endostatin precursor collagen XVIII is expressed at high levels in human livers, the main source being hepatocytes. We have studied the regulatory elements in the promoter 2 of the Col18a1 gene that directs the transcription of the NC1-517 variant of collagen alpha1(XVIII), which is the main form expressed in the liver. The 5'-flanking region of Col18a1 gene was cloned, and a series of 5'-deletions from -3286 bp to +285 bp were linked to the luciferase reporter gene. Transfection experiments in HepG2 cells allowed to identify a silencer-like element containing putative HNF1 and HNF3 sites and activator elements containing stretches of GC-rich sequences. Another putative HNF3 site in close apposition to a NF1/CTF site was localized upstream of the silencer-like element. Cotransfection experiments showed that the Col18a1 promoter 2 was transactivated by Sp1 and HNF3alpha. Gel-shift analyses showed that HNF3, NF1/CTF, and Sp1-like sites specifically recognized nuclear factors. Super-shift experiments indicated that HNF3beta was the major form of HNF3 interacting with the HNF3/NF1 site. The well-differentiated hepatoma cell line mhATFS315 transfected with a truncated form of HNF3beta, which competitively blocks HNF3 transactivating activity, expressed the Col18a1gene at a very low level. Taken together, these data strongly suggest that Col18a1 is a liver-specific gene. Furthermore, gel-shift analyses performed with nuclear factors prepared from well-differentiated hepatocellular carcinomas showed increased HNF3/NF1 binding activity compared with normal livers. Consequently, the precursor of endostatin might be differently expressed according to the differentiated and/or transformed state of hepatocytes. PMID: 11093745 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Biochim Biophys Acta. 2000 Oct 31;1488(1-2):149-58. Transcriptional regulation of inflammatory secreted phospholipases A(2). Andreani M, Olivier JL, Berenbaum F, Raymondjean M, Bereziat G. CNRS-associated research unit of Pierre and Marie Curie University (Paris VI), ESA 7079, 7 quai Saint Bernard, 75005, Paris, France. Secreted phospholipases A(2) is a family of small molecular weight and calcium-dependent enzymes of which the members list is presently growing. Among these enzymes, the synovial type IIA and the type V phospholipases A(2) are involved in inflammation. Although their actual mechanism is still a subject of debate, new therapeutic strategies can result from the knowledge of the regulations of their gene expression. The human genes of the type IIA and type V phospholipases A(2) are located on the chromosome 1 at close positions and transcribed in reverse orientations. These genes can therefore be regulated by common elements but only the regulation of the type IIA phospholipase A(2) gene expression has been extensively studied. Pro-inflammatory cytokines upregulate while the growth factors downregulate the type IIA phospholipase A(2) gene expression. Interleukin-6 and interleukin-1beta exert their effects at least partially at the transcriptional level. The transcriptional regulation of the type IIA phospholipase A(2) gene is cell- and species-specific. The activity of the human promoter is controlled by the CAAT-enhancer binding protein (C/EBP) factors while that of the rat promoter is regulated by nuclear factor kappaB (NF-kappaB) and C/EBPs. Furthermore, the human promoter is constitutively repressed in hepatocytes by single strand DNA binding proteins whose effects are relieved by C/EBP factors while the glucocorticoid receptor interacts with C/EBPs in chondrocytes to achieve full basal and interleukin-1beta-stimulated transcription activity. Other factors like CTF/NF1 and Sp1 might be involved in the regulation of both the rat and human promoter. Peroxisome proliferator-activated receptors could contribute to the stimulation of the rat promoter by NF-kappaB in vascular smooth muscle cells. The study of the coactivators and coinhibitors associated to these transcription factors will give a better understanding of the diversity and complexity of the transcriptional regulations of the type IIA phospholipase A(2) gene. Publication Types: Review Review, Tutorial PMID: 11080684 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: J Biol Chem. 2000 Jul 28;275(30):22686-94. Induction of secreted type IIA phospholipase A2 gene transcription by interleukin-1beta. Role of C/EBP factors. Massaad C, Paradon M, Jacques C, Salvat C, Bereziat G, Berenbaum F, Olivier JL. UPRES-A CNRS 7079, UFR Saint Antoine, UPRES-A CNRS 7079, Universite Pierre et Marie Curie, 7 quai Saint Bernard 75252 Paris Cedex 05, France. Secreted type IIA phospholipase A(2), which is involved in arachidonic acid release, is abundantly produced by chondrocytes and secreted in the synovial fluids of patients affected by rheumatoid arthritis. Transfection experiments showed that interleukin-1beta stimulates the phospholipase A(2) [-1614; +20] promoter activity by 6-7-fold and that the [-210; -176] fragment is critical for this stimulation. CAAT enhancer-binding protein (C/EBP) beta and C/EBPdelta transcription factors bind to this element as shown by bandshift experiments. Interleukin-1beta increased the levels of C/EBPdelta mRNA as soon as 2 h and up to 24 h without affecting those of C/EBPbeta. Higher amounts of C/EBPdelta proteins correlate with the stimulation of C/EBPdelta mRNA. Mutations or 5' deletions in the upstream [-247; -210] region reduced by 2-fold the basal and interleukin-1beta-stimulated transcription activities. Two types of factors bind to overlapping sequences on this fragment: NF1-like proteins and the glucocorticoid receptor. The glucocorticoid receptor is responsible for a moderate stimulation of the promoter activity by dexamethasone and may interact with C/EBP factors to achieve a full transcription activity in basal conditions and in the presence of interleukin-1beta. A [-114; -85] proximal regulatory element forms three complexes in bandshift experiments, the slowest mobility one involving the Sp1 zinc finger factor. Mutation of this sequence reduced to 2-fold the stimulation of the promoter activity by interleukin-1beta or the C/EBP factors. Induction of the transcription of secreted type IIA phospholipase A(2) gene by interleukin-1beta in chondrocytes absolutely requires C/EBPbeta and C/EBPdelta factors but does not involve NF-kappaB. PMID: 10791956 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: J Biol Chem. 2000 Jan 28;275(4):2295-304. Uroporphyrinogen III synthase. An alternative promoter controls erythroid-specific expression in the murine gene. Aizencang GI, Bishop DF, Forrest D, Astrin KH, Desnick RJ. Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029, USA. Uroporphyrinogen III synthase (URO-synthase, EC 4.2.1.75) is the fourth enzyme of the heme biosynthetic pathway and is the defective enzyme in congenital erythropoietic porphyria. To investigate the erythroid-specific expression of murine URO-synthase, the cDNA and approximately 24-kilobase genomic sequences were isolated and characterized. Three alternative transcripts were identified containing different 5'-untranslated regions (5'-UTRs), but identical coding exons 2B through 10. Transcripts with 5'-UTR exon 1A alone or fused to exon 1B were ubiquitously expressed (housekeeping), whereas transcripts with 5'-UTR exon 2A were only present in erythroid cells (erythroid-specific). Analysis of the TATA-less housekeeping promoter upstream of exon 1A revealed binding sites for ubiquitously expressed transcription factors Sp1, NF1, AP1, Oct1, and NRF2. The TATA-less erythroid-specific promoter upstream of exon 2A had nine putative GATA1 erythroid enhancer binding sites. Luciferase promoter/reporter constructs transfected into NIH 3T3 and mouse erythroleukemia cells indicated that the housekeeping promoter was active in both cell lines, while the erythroid promoter was active only in erythroid cells. Site-specific mutagenesis of the first GATA1 binding site markedly reduced luciferase activity in K562 cells (<5% of wild type). Thus, housekeeping and erythroid-specific transcripts are expressed from alternative promoters of a single mouse URO-synthase gene. PMID: 10644678 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Oncogene. 1999 Jul 15;18(28):4108-19. Site-specific DNA methylation in the neurofibromatosis (NF1) promoter interferes with binding of CREB and SP1 transcription factors. Mancini DN, Singh SM, Archer TK, Rodenhiser DI. Molecular Medical Genetics Program, Child Health Research Institute, London Health Sciences Centre, Department of Paediatrics, University of Western Ontario, Canada. Tumour suppressor genes and growth regulatory genes are frequent targets for methylation defects that can result in aberrant expression and mutagenesis. We have established a methylation map of the promoter region of the neurofibromatosis (NF1) gene and demonstrated functional sensitivity for methylation at specific sites for the SP1 and CRE binding (CREB) proteins in the NF1 regulatory region. We evaluated the methylation status of CpG dinucleotides within five promoter subregions in the human and mouse homologues of the neurofibromatosis (NF1) genes. Three 5' subregions were found to be consistently methylated in all the tissues analysed. In contrast, DNA methylation was absent in the vicinity of the transcription start site bounded by SP1 recognition sequences. Gelshift assays showed that methylation specifically inhibits the CREB transcription factor from binding to its recognition site at the NF1 transcription start site. Furthermore, SP1 elements within the NF1 promoter are methylation sensitive, particularly when methylation is present on the antisense strand. We propose that for NF1 as with several other tumour suppressor genes, CpG methylation occurs in a complex, site-specific manner with the maintenance of a methylation-free promoter region bounded by SP1 binding sites that allow an accessible promoter to be retained. When these SP1 boundaries are breached, methylation can sweep in, rendering the promoter inaccessible for specific methylation-sensitive transcription factors and leading to a loss of functional integrity of the methylation-free CpG island. PMID: 10435592 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: J Med Virol. 1999 Aug;58(4):413-9. Identification of a new control region in the genome of the DDP strain of BK virus isolated from PBMC. Degener AM, Pietropaolo V, Di Taranto C, Jin L, Ameglio F, Cordiali-Fei P, Trento E, Sinibaldi L, Orsi N. Department of Cellular and Developmental Biology, University "La Sapienza", Rome, Italy. The various strains of human polyomavirus BK (BKV) show a marked heterogeneity in the non-coding control region (NCCR), which includes the origin of replication and the regulatory region for early and late transcription. A new BKV strain (DDP, U91605) was identified by direct detection and sequencing of PCR products of BKV-NCCR DNA obtained from PBMC samples of HIV-positive or -negative subjects. The DDP strain NCCR sequence showed an organisation not described previously in vivo with the maximum homology with the archetypal strain (WW) (M34048), as compared with those collected in GenBank. Structurally, P68, Q39, and S68 boxes were perfectly conserved, whereas the R63 box was completely deleted. This deletion involves the loss of sequences able to bind cellular factors essential for the DNA transcription, such as NF1 binding sites, normally present twice in the R box and the modification of SP1. It is possible that these rearrangements represent a cause of the loss of the VP1 region observed in 9/22 PBMC samples and never observed in urine isolates, which are similar to the WW strain. PMID: 10421410 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: Arch Virol. 1998;143(10):1967-83. Regulation of the Epstein-Barr viral immediate early BRLF1 promoter through a distal NF1 site. Glaser G, Vogel M, Wolf H, Niller HH. Institut fur Medizinische Mikrobiologie und Hygiene, Universitat Regensburg, Germany. The immediate early BRLF1 and BZLF1 promoters of Epstein-Barr virus are crucial for triggering the replicative cycle of the virus. To better understand the cell type dependence of the lytic cycle we conducted an analysis of the BRLF1-promoter in the epithelial cell line HeLa and the lymphoid cell line IM9. To analyze promoter activities, transient transfections with 5'-deletions of the BRLF1-promoter in front of luciferase as reporter gene were conducted. Besides the already known cis-acting elements of the promoter close to the TATA-box, more distal elements were located and functionally tested. A nuclear factor 1 consensus site was found to act positively in HeLa cells, but did not in lymphoid IM9 cells. The NF1 site was shown to bind protein by electrophoretic mobility shift assays, antibody-supershifts and in vitro footprinting. Thus, a protein belonging to the nuclear factor 1 family of proteins was identified as additional cellular trans-acting factor for the BRLF1-promoter besides the already described factors Sp1, Zta and Zif268. PMID: 9856084 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: J Cell Sci. 1998 Dec;111 ( Pt 23):3487-96. Regulation of a hair follicle keratin intermediate filament gene promoter. Dunn SM, Keough RA, Rogers GE, Powell BC. Department of Animal Science, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia. During hair growth, cortical cells emerging from the proliferative follicle bulb rapidly undergo a differentiation program and synthesise large amounts of hair keratin proteins. To identify some of the controls that specify expression of hair genes we have defined the minimal promoter of the wool keratin intermediate filament gene K2.10. The region of this gene spanning nucleotides -350 to +53 was sufficient to direct expression of the lacZ gene to the follicle cortex of transgenic mice but deletion of nucleotides -350 to -150 led to a complete loss of promoter activity. When a four base substitution mutation was introduced into the minimal functional promoter at the binding site for lymphoid enhancer factor 1 (LEF-1), promoter activity in transgenic mice was decreased but specificity was not affected. To investigate the interaction of trans-acting factors within the minimal K2.10 promoter we performed DNase I footprinting analyses and electrophoretic mobility shift assays. In addition to LEF-1, Sp1, AP2-like and NF1-like proteins bound to the promoter. The Sp1 and AP2-like proteins bound sequences flanking the LEF-1 binding site whereas the NF1-like proteins bound closer to the transcription start site. We conclude that the LEF-1 binding site is an enhancer element of the K2.10 promoter in the hair follicle cortex and that factors other than LEF-1 regulate promoter tissue- and differentiation-specificity. PMID: 9811563 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Eur J Biochem. 1998 Oct 1;257(1):62-8. Structure of the promoter and complete sequence of the gene coding for the rabbit translationally controlled tumor protein (TCTP) P23. Thiele H, Berger M, Lenzner C, Kuhn H, Thiele BJ. Institute of Biochemistry, University Clinics Charite, Humboldt-University Berlin, Germany. holger.thiele@rz.hu-berlin.de We have isolated genomic recombinants containing the complete gene coding for the rabbit translationally controlled tumor protein (TCTP), also known as histamine-releasing factor (HRF) P23. The gene is organized into five introns and six exons and its total length amounts to 3819 nucleotides. All intron/exon boundaries are in accordance with the GT/AG rule. Transcription of the gene generates two mRNAs of 843 and 1163 nucleotides differing in the length of their 3'-untranslated regions. They are formed by alternative polyadenylation. The transcription initiation site has been determined by comparison of sequences of the gene and several processed TCTP pseudogenes. The full-length 5'-untranslated region comprises 116 nucleotides and starts with an oligopyrimidine tract important for translational regulation. Additionally 1.2 kb of the 5'-flanking promoter region has been sequenced. The promoter contains a TATA box at -30 and potential binding sites for transcription factors such as stimulating protein 1 (Sp1), nuclear factor 1 (NF1), activator protein 1 (AP1), c-Ets1, cAMP-response element (CP2), myeloid-specific zinc finger protein 1 (MZF1) and others. For functional analysis 5'-flanking sequences up to -918 were fused to the chloramphenicol acetyltransferase (CAT) gene and tested using a rabbit aortic smooth-muscle cell line by cell transfection and CAT assays. The results confirm that the analyzed gene is the actively transcribed TCTP gene. PMID: 9799103 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: J Neurosci. 1998 Aug 15;18(16):6186-94. Neuronal expression of the 5HT3 serotonin receptor gene requires nuclear factor 1 complexes. Bedford FK, Julius D, Ingraham HA. Department of Physiology, University of California at San Francisco, San Francisco, California 94143-0444, USA. The 5HT3 receptor (5HT3R) is a serotonin-gated ion channel whose expression is restricted to a subset of cells within the central and peripheral nervous systems. In vitro analysis shows that a small proximal region of the TATA-less 5HT3R promoter is sufficient to direct neuronal-specific reporter gene expression. Three potential regulatory elements conserved between the mouse and human genes were identified within this proximal promoter, two of which are known sites for the ubiquitously expressed factors Sp1 and nuclear factor 1 (NF1). Surprisingly, mutation of the NF1 binding site abolished all reporter activity in cell transfection studies, suggesting that this element is essential for neuronal-specific transcriptional activity of the 5HT3R. Furthermore, a complex of neuronal proteins that includes a member(s) of the NF1 family binds to this site, as shown by gel mobility super shift and DNaseI footprinting analyses. Although NF1 has been proposed to mediate basal transcription of many ubiquitously expressed genes, our data suggest that a member of the NF1 transcription factor family participates in neuronal-specific gene expression by promoting interactions with other regulatory factors found in sensory ganglia. PMID: 9698312 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: J Biochem (Tokyo). 1998 Jul;124(1):23-7. The human PTFgamma/SNAP43 gene: structure, chromosomal location, and identification of a VNTR in 5'-UTR. Maeng JH, Yoon JB. Department of Biochemistry, College of Science, Yonsei University, Seoul, 120-749, Korea. PTF/SNAPc is a multisubunit complex which specifically recognizes the PSEs of small nuclear RNA genes and activates transcription by RNA polymerase II or III. Here we describe the isolation and characterization of genomic clones encoding the human PTFgamma/SNAP43 gene. The gene spans approximately 29 kilobases, and is composed of 9 exons and 8 introns. A major transcription initiation site was identified at the position 58 base pairs upstream of the AUG translation initiator codon on primer extension analysis with HeLa mRNA. The 5' flanking region lacks a typical TATA box but contains many putative binding sites for various transcription factors, such as Sp1, Oct1, NF1, AP1, E2F, and USF. Immediately downstream of the transcription start site, we found a VNTR of a 17-bp sequence rich in (G+C). Four different alleles with two to five copies of the tandem repeat were identified in 10 individuals examined, indicating a high degree of variation at the PTFgamma/SNAP43 locus. In addition, the PTFgamma/SNAP43 gene was mapped to human chromosome 14q22 by fluorescence in situ hybridization. PMID: 9644240 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Gene. 1998 Apr 14;210(2):315-24. Molecular cloning, characterization and alternative splicing of the human cytoplasmic serine hydroxymethyltransferase gene. Girgis S, Nasrallah IM, Suh JR, Oppenheim E, Zanetti KA, Mastri MG, Stover PJ. Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA. The human cytoplasmic serine hydroxymethyltransferase (CSHMT) gene was isolated, sequenced and its expression characterized in human MCF-7 mammary carcinoma and SH_5Y5Y neuroblastoma cells. The 23-kb gene contains 12 introns and 13 exons; all splice junctions conform to the gt/ag rule. The open reading frame is interrupted by 10 introns, two of which are positionally conserved within the human mitochondrial SHMT gene. The gene is expressed with 330 nucleotides of 5' untranslated message within three exons. The 5' promoter region does not contain a consensus TATA, and primer extension and 5'-RACE studies suggest that transcription initiation occurs at multiple sites. Consensus motifs for several regulatory proteins, including SP1, mammary and neuronal-specific elements, NF1, a Y-box, and two steroid hormone response elements, are present within the first 408 nucleotides of the 5' promoter region. The human gene is expressed as multiple splice variants in both the 5' untranslated region and within the open reading frame, all due to exon excision. The splicing pattern is cell-specific. At least six CSHMT mRNA splice forms are present in MCF-7 cells; the gene is expressed as a full-length message as well as splice forms that lack exon(s) 2, 9 and 10. In 5Y cells, the predominant form of the message lacks exon 2, which encodes part of the 5' untranslated region, but does not contain deletions within the open reading frame. Western analysis suggests that the CSHMT gene is expressed as a single full-length protein in 5Y cells, but as multiple forms in MCF-7 cells. Multiple tissue Northern blots suggest that the CSHMT message levels and alternative splicing patterns display tissue-specific variations. PMID: 9573390 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Biochem Biophys Res Commun. 1998 Apr 17;245(2):370-7. Identification of conserved potentially regulatory sequences of the SRY gene from 10 different species of mammals. Margarit E, Guillen A, Rebordosa C, Vidal-Taboada J, Sanchez M, Ballesta F, Oliva R. Hospital Clinic, Institut de Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Barcelona, Spain. We have sequenced the 5' region of the SRY gene from human, chimpanzee, sheep, and mouse and from four additional mammalian species, not previously characterized (gorilla, gazelle, rat, and guinea pig). In order to identify conserved DNA elements potentially involved in the regulation of the SRY gene, the newly determined sequences were analyzed and compared to all mammalian SRY promoter sequences available at present. Ten highly conserved potential regulatory elements have been identified in all 10 species (AP1, Barbie, GATA, Gfi1, cMyb, vMyb, NF1, Oct1, Sp1, and SRY). The known function of several of these regulatory elements fits well with the known expression of the SRY gene. However, except for the highly conserved coding HMG motif, only a short region close to the initiation of transcription in the human SRY is conserved in the exact position along the gene in all the species analyzed. This lack of sequence identity at the orthologous positions is consistent with the suggested rapid evolution of the SRY gene. This relative lack of homology contrasts with a high sequence identity of the putative regulatory sequences found within each taxonomic group of species (primates, bovids, and rodents), which supports a common mechanism of SRY expression and possibly also a similar function. PMID: 9571157 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: Biochem Cell Biol. 1997;75(4):427-34. A nuclear factor other than Sp1 binds the GC-rich promoter of the gene encoding rat poly(ADP-ribose) polymerase in vitro. Laniel MA, Bergeron MJ, Poirier GG, Guerin SL. Laboratory of Molecular Endocrinology, CHUL Research Center, Ste-Foy, QC, Canada. Poly(ADP-ribose) polymerase is a nuclear enzyme that has been shown to exert a key role in many important cellular functions, including DNA repair. Its activity was shown to vary substantially between tissues; the testis and the thymus expressed the highest levels of PARP whereas the liver and the kidney (as well as a few other tissues) expressed only low levels of PARP proteins in vivo. The GC-rich nature of its upstream gene promoter, along with the lack of TATA and CAAT boxes, a feature common to most housekeeping genes, is consistent with a major regulatory function played by the positive transcription factor Sp1 in rat PARP gene transcription. Sp1 was indeed recently shown to interact with five distinct GC or GT boxes present in the rat PARP promoter. However, the observation that PARP activity was lower in rat liver than in other tissues was shown not to be the result of reduced Sp1 activity in liver cells but rather suggests the interplay of nuclear proteins other than Sp1 that are required to restrict PARP expression in this organ and maybe in others (such as the kidney). In this study, we investigated this possibility further by defining whether other nuclear proteins might bind the PARP promoter to modulate its transcription in liver cells. As a result, we identified a nuclear factor distinct from Sp1 that binds the PARP promoter at a site overlapping the F2 Sp1 element previously identified. Our results suggest that this protein likely belongs to the CTF-NF1 family of transcription factors. PMID: 9493965 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: J Biol Chem. 1998 Jan 23;273(4):2277-87. Characterization of transcriptional regulatory elements in the promoter region of the murine blood coagulation factor VII gene. Stauffer DR, Chukwumezie BN, Wilberding JA, Rosen ED, Castellino FJ. Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA. To identify the 5' sequences of the murine coagulation factor VII (fVII) gene that resulted in its efficient transcription, a variety of 5'-flanking sequences up to 7 kilobase pairs upstream of the translation ATG initiation codon were fused to the reporter gene, bacterial chloramphenicol acetyltransferase, and relative expression levels of this gene in mouse Hepa 1-6 cells were determined. It was found that the 5' region extending approximately 85 base pairs (bp) upstream of the transcriptional initiation site served as the minimal DNA region that provided full relative promoter activity for chloramphenicol acetyltransferase expression. This region of the gene also contains consensus sequences for liver-enriched transcription factors, C/EBP beta and HNF4, as well as for the ubiquitous protein factors, AP1, H4TF1, NF1, and Sp1. In vitro DNase I footprinting of the 200-bp proximal region of the promoter with a murine Hepa 1-6 cell nuclear extract revealed a clear footprint of a region corresponding to -80 to -28 bp of the murine fVII gene, suggesting that liver factors interact with this region of the DNA. Competitive gel shift and supershift assays with different synthetic oligonucleotide probes demonstrate that proteins contained in the nuclear extract, identified as C/EBP beta, H4TF1, and HNF4, bind to a region of the murine fVII DNA from 85 to 32 bp upstream of the transcription start site. Purified Sp1 also interacts with this region of the DNA at a site that substantially overlaps, but is not identical to, the H4TF1 binding locus. Binding of Sp1 to the mouse DNA was not observed with the nuclear extract as the source of the transcription factors, suggesting that Sp1 is likely displaced from its binding site by H4TF1 in the crude extract. In vivo dimethyl sulfate footprint analysis confirmed the existence of these sites and additionally revealed two other binding regions slightly upstream of the CCAAT/enhancer-binding protein (C/EBP) binding locus that are homologous to NF1 binding sequences. The data demonstrate that appropriate transcription factor binding sites exist in the proximal promoter region of the murine fVII gene that are consistent with its strong liver-based expression in a highly regulated manner. PMID: 9442072 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: DNA Cell Biol. 1997 Dec;16(12):1477-82. Estrogen receptor diminishes DNA-binding activities of chicken GATA-1 and CACCC-binding proteins. Holth LT, Sun JM, Coutts AS, Murphy LC, Davie JR. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada. The estrogen receptor (ER) repressed erythroid differentiation and erythroid-specific gene expression. In this study, we investigated the effect of ER alpha (referred to throughout as ER) on DNA-binding activities of transcription factors involved in regulating the expression of erythroid-specific genes, and, in particular, the histone H5 gene. Using electrophoretic mobility shift assays, we found that in the presence of rabbit reticulocyte lysate, human ER reduced the binding activities of chicken immature erythrocyte nuclear extracted proteins to GATA and CACCC sites in the H5 promoter and enhancer. In contrast, the binding activities of NF1 and Sp1 were not affected by ER. Binding of ER to an estrogen response element was enhanced by addition of rabbit reticulocyte lysate. This lysate was also necessary for ER to diminish the DNA-binding activity of GATA-1. These results suggest that additional factor(s) are necessary for full ER function. Both GATA-1 and CACCC-binding proteins are critical for the developmentally regulated expression of erythroid-specific genes. We hypothesize that interference in DNA-binding activities of GATA-1 and CACCC-binding proteins is the mechanism by which the ER inhibits regulation of these genes. PMID: 9428796 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: J Gen Virol. 1997 Nov;78 ( Pt 11):3029-38. Characterization of the hepatitis B virus major surface antigen promoter hepatocyte nuclear factor 3 binding site. Raney AK, McLachlan A. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA. Transcription of the HBV 2.1 kb RNAs is regulated by the major surface antigen promoter. Previously, transient transfection analysis identified regulatory sequence elements in this promoter located between -189 and +1 which govern the level of transcription from this promoter and appear to bind only ubiquitous transcription factors including NF1, Sp1 and NF-Y. However, in vivo transcription analysis in transgenic mice has demonstrated that the expression of the HBV 2.1 kb RNAs is largely restricted to hepatocytes. In this study, the presence of a functional HNF3 transcription factor binding site located between -231 and -240 in the major surface antigen promoter suggests that the in vivo liver-restricted expression of the 2.1 kb RNAs may be governed by this liver-enriched transcription factor. The identification of a functional HNF3 binding site upstream of the DNA polymerase open reading frame also supports the contention that transient transfection analysis may fail to detect all of the cis-acting regulatory sequence elements involved in modulating the level of transcription from the viral promoters. PMID: 9367390 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Mol Cell Endocrinol. 1997 Sep 19;132(1-2):13-23. Ubiquitous transcription factors NF1 and Sp1 are involved in the androgen activation of the mouse vas deferens protein promoter. Darne CH, Morel L, Claessens F, Manin M, Fabre S, Veyssiere G, Rombauts W, Jean CL. Laboratoire Reproduction et Developpement, Centre National de la Recherche Scientifique Unite de Recherche Associee 1940, Universite Blaise Pascal-Clermont-Ferrand II, Aubiere, France. Transcription of the mouse vas deferens protein (MVDP) gene is stimulated by androgens and we have previously shown that in a 162 bp fragment, located at position -121 to +41, a TGAAGTtccTGTTCT sequence functions as an androgen-dependent enhancer. To determine which factors are involved in the hormonally regulated MVDP gene transcription, we have used DNase I footprinting and band-shift assays to examine in vitro binding of proteins to the enhancer and promoter sequences and have determined the functional significance of the recognized DNA sequences in transient transfection assays. Studies using recombinant proteins such as the DNA binding domain of the androgen receptor (AR-DBD) and Sp1, and crude cellular extracts from T47D and vas deferens epithelial cells (VDEC) showed that in addition to AR-DBD, the transcriptional activators NF1 and Sp1 interact with the -121/+41 fragment in a specific manner. Transient transfection assays revealed that site-directed mutations in the NF1 and Sp1 binding elements strongly reduced (NF1) or abolished (Sp1) androgen induced expression. The results demonstrate that the -121/+41 sequence is a composite site for the androgen receptor mediated transactivation of the MVDP gene. PMID: 9324042 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: J Virol. 1997 Nov;71(11):8832-40. Transcription activities of human papillomavirus type 11 E6 promoter-proximal elements in raft and submerged cultures of foreskin keratinocytes. Zhao W, Chow LT, Broker TR. Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294-0005, USA. Human papillomaviruses (HPVs) replicate only in differentiated squamous epithelia in warts and in epithelial raft cultures grown at the medium-air interface. Virus-encoded and host transcription factors are thought to be responsible for repressing the viral enhancer and promoter located within the upstream regulatory region (URR) in the undifferentiated basal and parabasal cells while up-regulating their activities in the differentiated spinous cells. Using recombinant retroviruses, we acutely transduced neonatal foreskin keratinocytes (PHKs) with a lacZ reporter gene driven by the wild-type URR of the low-risk HPV type 11 or by a URR with individual mutations in seven promoter-proximal elements, some of which have not been characterized previously. Beta-galactosidase activities were detected in the submerged, proliferating PHKs and also in the differentiated spinous cells, but not in the steady-state proliferating basal cells, of stratified raft cultures. In particular, mutation of an Oct1, an Sp1, or a previously unknown promoter-proximal AP1 site severely reduced the reporter activity, whereas mutation of either of two NF1 sites flanking the Oct1 site had no effect. These results demonstrate changes in cellular transcription factor profiles under different culture conditions and begin to characterize the naturally differentiation-dependent activation of the URR. They provide one molecular explanation for the patterns of HPV expression in warts and help validate epithelial raft cultures as an important experimental system for genetic dissection of HPV regulatory elements. PMID: 9343243 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: Eur J Biochem. 1997 Sep 15;248(3):676-83. Characterisation of the rat tissue-type plasminogen activator gene promoter -- identification of a TAAT-containing promoter element. Leonardsson G, Ny T. Department of Medical Biochemistry and Biophysics, Umea University, Sweden. Tissue-type plasminogen activator (tPA) activates plasminogen to the active protease plasmin and is implicated in many biological processes that require extracellular proteolysis. In rat ovarian cells, gonadotropins induce the tPA gene by a cAMP-dependent pathway and this induction correlates with the time of follicular rupture. We have previously identified several promoter elements within the first 621 bp of the rat tPA promoter that are important for constitutive and cAMP-induced expression of the gene, including a cAMP responsive element (CRE), a nuclear factor 1 (NF1) element, a SP1-binding site and a G+C-rich box. In this report we have extended our study by analysing promoter constructs, ranging in size from 7.7 kb to 135 bp fused to the luciferase reporter gene. Transient transfection analysis of rat granulosa cells and human 293 cells, reveal that the proximal 268 bp of the promoter is enough to confer high basal and cAMP-induced expression of the gene. At position -162 to -172, between the previously identified CRE and NF1 sites, a novel TAAT-containing promoter element was identified. Mutational inactivation of the TAAT motif indicates that this element is important for both constitutive and cAMP-induced expression of the gene, and for the binding of a presumably novel nuclear factor that we have termed tPA promoter factor-1 (tPF-1). PMID: 9342217 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: EMBO J. 1997 Sep 15;16(18):5722-9. Three classes of mammalian transcription activation domain stimulate transcription in Schizosaccharomyces pombe. Remacle JE, Albrecht G, Brys R, Braus GH, Huylebroeck D. Department of Cell Growth, Differentiation and Development (VIB-07), Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium. jre@sgi.celgen.kuleuven.ac.be Representatives of three distinct classes of mammalian protein domain activating RNA polymerase II were fused to the yeast GAL4p DNA-binding domain. The resulting fusion proteins were tested in the fission yeast Schizosaccharomyces pombe for their ability to activate transcription of different reporter constructs containing GAL4-binding sites in positions close to or far from the TATA box. The acidic-rich activation domain of VP16 stimulates transcription in S.pombe from proximal and distal positions, suggesting that the mechanism of activation is conserved from man to budding and fission yeasts. Unlike in Saccharomyces cerevisiae, the glutamine-rich activation domains of Sp1, Oct1 and Oct2 activate transcription in S. pombe when tested in a proximal TATA box context. Similarly to mammalian cells, these domains are inactive or weakly active when tested in a distal position. Moreover, the proline-rich activation domains of AP-2 and CTF/NF1 display strong transcriptional activities from a TATA box-proximal position, and weak activities when tested in a remote position. Consequently, proline-rich and glutamine-rich activation domains act differently in S.cerevisiae and mammalian cells, but similarly in S.pombe and mammalian cells. PMID: 9312030 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: Gene. 1997 Aug 22;195(2):121-6. Cloning and characterization of the human mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase gene. Boukaftane Y, Mitchell GA. Service de genetique medicale, Hopital Ste-Justine, Universite de Montreal, Quebec, Canada. We report the characterization of lambda and P1 phage clones containing the entire human mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (mHS) gene. The human mHS locus (HMGCS2) on chromosome 1p12-13 spans 25 kb and contains 10 exons. Exon 1 contains most of the mitochondrial leader, consistent with a recent hypothesis of the evolution of the ketogenic pathway. By primer extension and cDNA amplification (RACE-PCR) we localized the transcription start point (tsp) to 60 bp upstream of the initiation codon. Nine blocks of conserved sequence were identified by comparing the 5' flanking regions of the mHS genes of human and rat. The 5' flanking region contains potential binding sites for TATA-binding protein, Sp1, nuclear factor 1 (NF1), CAAT-box binding protein (C/EBP), hepatocyte nuclear factors 1 and 5 (HNF1, HNF5) and activator proteins 1 and 2 (AP1, AP2). PMID: 9305755 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: J Biol Chem. 1997 Sep 12;272(37):23144-50. Sp1-mediated transcriptional activation from the dominant promoter of the rat alpha1B adrenergic receptor gene in DDT1MF-2 cells. Chen J, Spector MS, Kunos G, Gao B. Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA. In the rat liver, NF1 and CP1 bind to the major P2 promoter of the alpha1B adrenergic receptor gene to generate footprint II. Here we show that, in DDT1MF-2 smooth muscle cells, the major protein bound to footprint II is not NF1 but Sp1, which binds to the 5'-portion of the footprint II sequence (footprint IIb). Mutational analyses demonstrate that the CCCGCG sequence in footprint IIb is critical for Sp1 binding and P2 promoter activity. A second GC box in the P2 promoter also binds the Sp1 protein and contributes to the P2 promoter activity. Gel shift assays indicate that footprint II can bind Sp1, NF1, and CP1, and that the binding of these 3 proteins is mutually exclusive. This is also indicated by the results of functional cotransfection experiments, where transient overexpression of NF1 and Sp1 together caused a similar increase in the activity of a P2/CAT reporter construct as overexpression of either Sp1 or NF1 alone, indicating lack of additivity. The preferential interaction of footprint II with Sp1 in DDT1MF-2 cells and NF1 in liver appears to be due to low levels of NF1 expression in DDT1MF-2 cells and low levels of Sp1 in liver. These observations suggest that NF1 and Sp1 are the major transcription factors involved in controlling the P2 promoter in liver versus DDT1MF-2 cells, respectively, which may be one of the mechanisms responsible for the complex tissue-specific regulation of the expression of the alpha1B adrenergic receptor gene. PMID: 9287317 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: J Gen Virol. 1997 Jun;78 ( Pt 6):1487-95. The hepatitis B virus MHBst167 protein is a pleiotropic transactivator mediating its effect via ubiquitous cellular transcription factors. Caselmann WH, Renner M, Schluter V, Hofschneider PH, Koshy R, Meyer M. Klinikum Grosshadem, Department of Medicine II, Ludwig-Maximilians-University, Munich, Germany. CASELMANN@Unl-Bonn.de C-terminally truncated surface proteins of hepatitis B virus (HBV) are frequently translated from genomically integrated viral sequences. They may be relevant for hepatocarcinogenesis by stimulating gene expression. First, we examined the transactivating potential of middle hepatitis B surface protein truncated at amino acid (aa) position 167 (MHBst167) on the HBV regulatory element. In transient cotransfection assays using Chang liver or HepG2 cell lines and chloramphenicol acetyltransferase (CAT) reporter constructs only the HBV enhancer I, but no other HBV regulatory elements like the X promoter, the S1 or S2 promoter or the enhancer II/core promoter could be stimulated by MHBst167. Since there is no evidence for a direct interaction of MHBst167 with DNA, we subsequently analysed whether cellular transcription factors were involved in mediating transactivation. This was tested both with isolated transcription-factor-binding sites and in the natural context of viral and cellular promoter elements. Deletion analysis and electrophoretic mobility shift assays revealed that Sp1, AP1 and NF-kappa B can mediate transactivation by MHBst167. No involvement of CREB, NF1 or the liver-specific factor C/EBP was found. These data indicate that MHBst167 is a pleiotropic, non-liver-specific transactivator which exerts its effect via ubiquitous cellular transcription factors that are also involved in the regulation of expression of cellular genes relevant for proliferation and inflammation. PMID: 9191947 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: Virus Res. 1997 Feb;47(2):155-66. Identification of cis-regulatory elements in the upstream regulatory region of human papillomavirus type 59. Rho J, Lee S, de Villiers EM, Choe J. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea. Human papillomavirus type 59 (HPV-59) was cloned from a vulvar intraepithelial neoplasia and the complete nucleotide sequence was determined. This virus is closely related to HPV-18 and -39 (60% homology in nucleotide sequence) and is grouped with the genital HPV types. In the present paper, we demonstrate that the HPV-59 E2 transactivator represses its E6 promoter-mediated transcription. We have also analyzed cis-regulatory elements in the upstream regulatory region (URR) of HPV-59 using chloramphenicol acetyl transferase assays as well as electrophoresis mobility shift assays (EMSA). The results allow for a subdivision of the HPV-59 URR into three regions of activity: distal (nt 7149-7493), central (nt 7493-7742), and proximal (nt 7742-7748). In particular, the 250 bp (nt 7493-7742) of the central region plays an important role as a constitutive enhancer element for the maximal transcription of the E6 promoter. Our results suggest that the transcription factors AP1, Oct1, SP1 and unidentified factors bind to the HPV-59 E6 promoter region, whereas NF1, GRE and TFIID fail to bind despite the presence of putative binding sites in the DNA sequence. PMID: 9085547 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Mol Reprod Dev. 1997 Jan;46(1):39-44; discussion 44-5. Transcriptional regulation of the mouse CSF-1 gene. Harrington M, Konicek BW, Xia XL, Song A. Indiana University, Indianapolis 46202, USA. Research in our laboratory is aimed at understanding the cellular and molecular mechanisms that govern colony stimulating factor-1 (CSF-1) gene expression. Our hypothesis is that a basal set of trans-acting factors is bound to the CSF-1 gene during fibroblast proliferation, resulting in constitutive CSF-1 gene expression. Modulation of CSF-1 gene transcription by growth-arrest (decrease) or stimulation of growth-arrested fibroblasts (re-initiate) is mediated by changes in the basal set of factors bound and/or by the addition of stimulus-specific factors. We have extended our hypothesis to include other cell types (monocytes) to determine if mechanisms used to control CSF-1 gene expression in fibroblasts are unique or represent common nontissue-specific regulatory mechanisms. Analysis of CSF-1-CAT reporter constructs in transiently transfected fibroblasts and monocytes was used to identify CSF-1 genomic sequences that affect transcriptional activity. DNase I protection, electrophoretic mobility shift, and methylation interference assays were used to identify the putative cis-acting elements. Results of our study suggest multiple trans-acting factors may regulate CSF-1 gene expression; some may be tissue specific, while others, such as AP1, CTF/NF1, Sp1, and Sp3, are shared in common. Publication Types: Review Review, Tutorial PMID: 8981362 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: FEBS Lett. 1996 Dec 9;399(1-2):14-20. Cloning and functional analysis of the hematopoietic cell-specific phospholipase C(gamma)2 promoter. Kang JS, Kohlhuber F, Hug H, Marme D, Eick D, Ueffing M. Mount Sinai Medical School, Department of Biochemistry, New York, NY 10029, USA. Phospholipase C(gamma)2 (PLCgamma2) is a phospholipid-converting enzyme which, upon receptor stimulation, is activated within membrane-bound signalling complexes. In contrast to the highly ubiquitous PLCgamma1, PLCgamma2 is expressed predominantly in B-lymphocytes. Associated with antigen-coupling receptors it is activated by tyrosine phosphorylation after the triggering of B-cell surface immunoglobulin. We have cloned and sequenced the human PLCgamma2 promoter. Primer extension analysis reveals the existence of a major transcriptional start site. The TATA-less promoter contains G+C-rich stretches with a cluster of contiguous SP1 consensus sites, an NF1, and an AP2 site between bp -220 to -70. A construct containing the region from -189 to +78 confers full promoter activity, as shown by fusion to a luciferase reporter gene construct. The distal part of the promoter between bp -662 to -293 containing an SRE, EBF and CACCC box contributed negatively to promoter activity in the B-cell line Raji but not in three adherent cell lines. In Raji cells, PLCgamma2 mRNA is expressed at low levels with a half life greater than 4 h. After treatment with serum, TPA, retinoic acid, or with 5-azacytidine increased levels of PLCgamma2 mRNA were induced in B-cells. PMID: 8980110 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: Scand J Immunol. 1996 Dec;44(6):599-606. Genomic organization and promoter analysis of the gene ifngr2 encoding the second chain of the mouse interferon-gamma receptor. Ebensperger C, Rhee S, Muthukumaran G, Lembo D, Donnelly R, Pestka S, Dembic Z. F.Hoffmann-La Roche AG, Basel, Switzerland. A clone containing the gene ifngr2 for the second chain (IFN-gamma R2) of the mouse interferon gamma receptor complex was isolated from a cosmid library made of 129/Sv mouse genomic DNA. Sequence analysis revealed that the second chain is encoded by 7 exons. The complete gene spans about 17 kb of the genomic DNA. In the 5'-flanking region several transcription initiation sites between 27 and 136 nucleotides upstream from the translation initiation codon were mapped. This region has a high GC content, but no TATA or CAAT box. Potential binding sites were found for transcription factors Sp1, AP-2, NF1, EGR and NF kappa B. Promoter activity was assayed with a series of constructs with firefly luciferase as a reporter gene, under the control of the promoter fragments of various lengths. This region showed promoter activity in transiently transfected Chinese hamster ovary cells. PMID: 8972742 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: Nucleic Acids Res. 1996 Nov 1;24(21):4185-91. Characterization of the rat Class 3 aldehyde dehydrogenase gene promoter. Xie YQ, Takimoto K, Pitot HC, Miskimins WK, Lindahl R. Department of Biochemistry and Molecular Biology, University of South Dakota School of Medicine, Vermillion 57069, USA. The Class 3 aldehyde dehydrogenase gene (ALDH-3) is differentially expressed. Expression is either constitutive or xenobiotic inducible via an aromatic hydrocarbon (Ah) receptor-mediated pathway, depending upon the tissue. A series of studies were performed to examine the regulation of rat ALDH-3 basal expression. DNase I footprint analysis identified four DNA regions within the proximal 1 kb of the 5' flanking region of rat ALDH-3 which interact with regulatory proteins. Reporter gene and gel mobility shift assays indicate that Sp1-like proteins interact with two proximal DNase I footprinted sites to confer strong promoter activity. Two distal DNase I footprinted sites are found within a region that inhibits rat ALDH-3 promoter activity. This negative region is bound by NF1-like proteins and/or unique proteins. This 1 kb 5' flanking region of rat ALDH-3 may act constitutively in many cell types. In contrast with other Ah receptor regulated genes, no DNA elements or transcription factors acting within this region appear to be involved in regulating xenobiotic-inducible expression of rat ALDH-3. PMID: 8932370 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: Gene. 1996 Sep 16;173(2):155-61. The role of nuclear factor NF-Y/CP1 in the transcriptional regulation of the human aldehyde dehydrogenase 2-encoding gene. Stewart MJ, Dipple KM, Stewart TR, Crabb DW. Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202-5121, USA. Mitochondrial aldehyde dehydrogenase (ALDH2) activity is produced at low levels in many tissues, with highest production in liver. Transfection assays using the first 600 bp of upstream DNA provided evidence for both positive and negative regulatory elements in the proximal promoter. A region from -79 to -116 bp was protected in DNase I footprinting assays and bound in electrophoretic mobility shift assays (EMSA) by a nuclear factor found in all cell lines and tissues tested. This region, denoted FP160, contained the consensus recognition sites for Sp1 and AP2, and a CCAAT box. The CCAAT box was specifically protected by a nuclear factor in methylation interference assays. Mutagenesis of specific bp within the CCAAT box eliminated protein binding in vitro and decreased transcriptional activity from the ALDH2 promoter approximately 50% in reporter gene assays. Competition experiments showed that the nuclear factor binding to the FP160 oligodeoxyribonucleotide (oligo) was competed by oligos corresponding to an NY-Y/CP1-binding site to a greater extent than by those containing sites for CTF/NF1, C/EPB or CP2. The heat stability, resistance to proteinase K digestion, sensitivity to inhibition of DNA binding by o-phenanthroline, and immunological properties of the liver factor binding to FP160 were very similar to the corresponding properties of NF-Y/CP1. Thus, the proximal ALDH2 promoter was bound by NF-Y/CP1 and this transcription factor may be responsible for the basal expression of the gene observed in most tissues. The NFY-CP1 present in rat liver has similar properties to that previously characterized in M12 B-lymphoma cells and LMTK mouse fibroblasts. PMID: 8964492 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: J Cell Biochem. 1996 Sep 15;62(4):454-66. Developmental changes in transcription factors associated with the nuclear matrix of chicken erythrocytes. Sun JM, Chen HY, Litchfield DW, Davie JR. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada. The nuclear matrix has roles in organizing nuclear DNA and in controlling transcription. Transcription factors are associated with the nuclear matrix, with the spectra of transcription factors differing from one cell type to another. In this study we identified the transcription factors and enzymes functioning in the regulation of gene expression that were associated with nuclear matrix and nonmatrix nuclear fractions in erythrocytes isolated from chick embryos at different stages of development, anemic and normal adult birds. We found that the primitive erythroid nuclear matrix had the greatest histone deacetylase activity and highest levels of several transcription factors, including GATA-1, CACCC-binding proteins, and NF1. These transcription factors have key roles in erythroid-specific gene expression. The levels of these transcription factors were lower in the nonmatrix and matrix fractions isolated from definitive erythrocytes. For primitive and definitive erythrocytes, the level of CACCC-binding proteins in the nuclear matrix fraction was greater than that of Sp1. The relative levels of these transcription factors were reversed in the nonmatrix fraction. Casein kinase II was not found in erythroid nuclear matrices. The observed erythroid lineage specific alterations in erythroid nuclear matrix transcription factor composition and abundance may be involved in erythroid-specific gene expression. PMID: 8891891 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 55: J Biol Chem. 1996 Sep 6;271(36):22125-9. Characterization of the upstream sequence of the human CYP11A1 gene for cell type-specific expression. Chou SJ, Lai KN, Chung B. Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China. The CYP11A1 gene encodes the cholesterol side-chain cleavage enzyme P450scc, which catalyzes the synthesis of steroids from cholesterol. This gene is expressed only in steroidogenic organs such as the adrenal, gonad, placenta, and brain. We have characterized an upstream regulatory element of the human CYP11A1 gene, termed AdE, which contributed to its cell type-specific expression. The AdE sequence contains two protein binding regions, AdE1 and AdE2, which bind many proteins including NF1- and Sp1-like proteins as shown by electrophoretic mobility shift assay, footprinting, competition, antibody supershift, and mutagenesis of the binding sites. When cloned in front of the CYP11A1 promoter or the heterologous thymidine kinase promoter, AdE sequences enhanced expression of the reporter gene in steroidogenic cell lines of the adrenal, gonad, and placental origin but not in nonsteroidogenic cell lines such as COS-1 and Rat-1. The function of AdE1 and AdE2 was lower when present individually than together. The combined action of multiple transcription factors binding to the AdE sequence brings about the final activation of the CYP11A1 gene in a tissue-specific manner. PMID: 8703023 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: Endocrinology. 1996 Sep;137(9):3897-905. cis-acting elements and trans-acting proteins in the transcription of chorionic gonadotropin/luteinizing hormone receptor gene in human choriocarcinoma cells and placenta. Hu YL, Lei ZM, Rao CV. Department of Obstetrics and Gynecology, University of Louisville School of Medicine, Kentucky 40292, USA. We investigated the cis-acting elements and trans-acting proteins responsible for a higher basal rate of transcription of hCG/LH receptor gene in human choriocarcinoma JEG-3 cells compared with normal term pregnancy placenta. Sequential deletion of the 5'-flanking region of the gene revealed that there are three negative control regions (NCRs) designated NCR1 (-1457 to -1373 bp), NCR2 (-1051 to -835 bp), and NCR3 (-480 to -184 bp), and a promoter (-184 to -1 bp). NCR3 was more inhibitory than the other two; nearly 60-70% of the inhibitory activity resides in a sequence between -480 to -276 bp, and the rest resides in the sequence between -276 to -184 bp. Gel mobility shift assays showed that the nuclear extracts from JEG-3 cells contained proteins that form three complexes with NCR1, two with NCR2, and six with NCR3. Many of the proteins that form the complexes in NCR3 are shared with the other two NCRs. Most of the proteins that form these complexes are less abundant in nuclear extracts from JEG-3 cells than in those from placenta. The JEG-3 cell nuclear extracts also contained proteins that form three complexes with the proximal promoter of the hCG/LH receptor gene. These proteins were identified as Ap2, Ap2-like I, and Sp1 from the competition studies with synthetic excess unlabeled Ap2, Sp1, and CTF/NF1 consensus oligodeoxynucleotides and/or supershift in gel mobility assays with anti-Ap2 antibody. Although the JEG-3 cell nuclear extracts contained abundant Ap2-like protein I and low levels of Ap2 and Sp1 proteins, the placental nuclear extracts contained low levels of Ap2-like protein I and very low to nondetectable levels of Ap2 and Sp1 proteins. Deoxyribonuclease I footprinting revealed that the nuclear extracts from JEG-3 cells and placent protected the -116 to -93 bp and -65 to -45 bp regions in the proximal promoter of the hCG/LH receptor gene that contain Sp1 and Ap2 binding sites, respectively. However, the nuclear extracts from placenta only partially protected these regions, which is consistent with lower levels of proteins that bind to the proximal promoter of the gene. In summary, we conclude that the presence of low levels of proteins that bind to the NCRs and the high levels of proteins, especially Ap2-like I, that bind to the proximal promoter can potentially explain higher transcription of the hCG/LH receptor gene in JEG-3 cells compared with that in normal term pregnancy human placenta. PMID: 8756564 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: Biochem J. 1996 Jul 15;317 ( Pt 2):361-70. Effect of dietary protein restriction on liver transcription factors. Marten NW, Sladek FM, Straus DS. Biology Department, University of California, Riverside 92521-0121, USA. The transcription of several genes that are preferentially expressed in the liver, including the serum albumin, transthyretin and carbamyl phosphate synthetase-I genes, is specifically decreased in animals consuming inadequate amounts of dietary protein. The high level of transcription of these genes in the liver is directed in part by a number of liver-enriched transcription factors, including hepatocyte nuclear factors (HNF)-1, -3, and -4, and proteins of the CCAAT/enhancer-binding protein (C/EBP) family. In the present study, we investigated the possibility that the co-ordinate decrease in transcription of the nutritionally sensitive genes in protein-deprived rats results from altered activity of one or more of the liver-enriched transcription factors. For HNF-4, Western blots indicated no change in the level of nuclear HNF-4 protein in liver of protein-deprived animals, whereas we observed a 40% reduction in the DNA binding activity of HNF-4 as measured by electrophoretic mobility shift assay (EMSA). Furthermore, the binding affinity of HNF-4 for DNA was unaltered by dietary protein deprivation, while the number of HNF-4 molecules able to bind to DNA (Bmax) was reduced, as determined by Scatchard analysis. This indicates that in the protein-restricted rats a portion of the pool of HNF-4 protein is inactivated or otherwise prevented from binding to DNA. The overall DNA binding activity of C/EBP alpha and beta was increased in protein-restricted animals. This change occurred in the absence of a change in the amount of the full-length forms of these two proteins, quantified by Western blotting. Interestingly, dietary protein restriction specifically increased the level of a truncated form of C/EBP beta (liver-enriched transcriptional inhibitory protein, LIP), which is a protein dominant negative inhibitor of C/EBP function. Analysis of HNF-3 DNA-binding activity by EMSA revealed that HNF-3 alpha and beta DNA binding was increased and that HNF-3 gamma DNA-binding activity was unchanged in protein-restricted animals. We also detected two apparently novel shift complexes with the HNF-3 probe by EMSA, both of which were decreased in protein-restricted animals. HNF-1 DNA-binding activity was increased by dietary protein restriction. We also examined the effect of protein restriction on the DNA-binding activity of two ubiquitous transcription factors, NF1 and Sp1. The DNA binding activity of the major NF1 isoforms was unchanged whereas the binding activity of Sp1 was increased in the protein-restricted animals. In summary, restriction of dietary protein resulted in a number of specific changes in the DNA-binding activity of various transcription factors. Because transcriptional activation typically involves the synergistic action of more than one transcription factor, small changes in the amount/activity of several factors, could have a strong net effect on the transcription of many genes. PMID: 8713059 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: AIDS Res Hum Retroviruses. 1996 Jun 10;12(9):829-32. Genomic footprinting of HTLV type I and HIV type 1 in human T cell lines. Brown DA, Xu X, Nerenberg M. Department of Neuropharmacology, Scripps Research Institute, La Jolla, California 92037, USA. Genomic footprinting of integrated HTLV-I and HIV-1 confirmed many aspects of retroviral transcriptional regulation deduced from previous studies. However, many notable differences were seen. HTLV-I genomic protein-binding patterns corresponded more closely to elements defined by transient transfection expression studies than to those mapped by in vitro protein-binding studies. HIV-1 genomic footprinting showed activation-related binding to adjacent NF-KB/SP1 sites and a large (90 bp) region transversing the R/U5 boundary, but minimal protein binding to NFAT, NRE, LBP-1, and CTF/NF1 sites relative to previous in vitro footprinting studies. PMID: 8738435 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: DNA Cell Biol. 1996 Jun;15(6):461-73. Characterization of the rat catechol-O-methyltransferase gene proximal promoter: identification of a nuclear protein-DNA interaction that contributes to the tissue-specific regulation. Tenhunen J. Orion Corporation, Orion-Farmos, Research Center, Helsinki, Finland. The methylating enzyme catechol-O-methyltransferase (COMT) is an important inactivator of substrates containing catechol-structure, such as catechol neurotransmitters and hormones. In previous studies, the rat COMT gene has been cloned and characterized, and it has been shown that the two COMT polypeptides, S- and MB-COMT, are expressed from one gene by cooperation of two separate promoters. One promoter, P2, functions constitutively, whereas the other, the proximal P1 promoter, is regulated in a tissue-specific manner. In this report, a more detailed analysis of the rat P1 promoter is presented. By using reporter gene constructs, it is shown that upstream sequences of the P1 promoter contain several regions that modulate the expression either positively or negatively. These experiments also show that the region between the MB- and S-ATG translation initiation codons is indispensable for the activity of this promoter. Analysis of this region by DNase I footprinting and gel retardation assays identified the presence of several DNA elements with SP1 and NF1 recognition site homologies that bound both liver and brain nuclear proteins. However, one 11-nucleotide-long DNA region containing an overlapping consensus binding sequence for CREB and C/EBP-like factors reacted only with the liver nuclear lysate. Supershift experiments suggest that the transcription factor C/EBPalpha mediates the tissue-specific expression of the rat COMT P1 promoter. PMID: 8672242 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: Biochem Biophys Res Commun. 1996 Mar 7;220(1):186-91. Identification of a 42-kDa nuclear factor (NF1-MUC5B) from HT-29 MTX cells that binds to the 3' region of human mucin gene MUC5B. Pigny P, Van Seuningen I, Desseyn JL, Nollet S, Porchet N, Laine A, Aubert JP. INSERM U 377, Lille, France. MUC5B gene is one of the four human mucin genes mapped to chromosome 11p15. The identification of three potential Sp1 binding sites located between the tandem repeat and the 3' end of MUC5B suggests a possible regulatory role for this region. In this report we show by electrophoretic mobility shift assay that only one potential Sp1 binding site (NAU62) leads to a specific interaction with a nuclear factor from HT-29 MTX cells which does not exist in parental HT-29 cells. By using mutated versions of NAU62, an 18 mer sequence within this later was shown to be directly involved in the interaction. The nuclear factor called NF1-MUC5B which binds to this element has a Mr of 42000 and is not Sp1. These results suggest that MUC5B contains a sequence in its 3' region that might act as a cis-element. This report opens the field of transcriptional regulation of human mucin genes encoding secreted mucins. PMID: 8602841 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: J Biol Chem. 1996 Mar 1;271(9):5131-42. Transcriptional regulation of murine beta1,4-galactosyltransferase in somatic cells. Analysis of a gene that serves both a housekeeping and a mammary gland-specific function. Rajput B, Shaper NL, Shaper JH. Cell Structure and Function Laboratory, Oncology Center Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21287-8937, USA. beta1,4-Galactosyltransferase (beta4-GT) is a constitutively expressed enzyme that synthesizes the beta4-N-acetyllactosamine structure in glycoconjugates. In mammals, beta4-GT has been recruited for a second biosynthetic function, the production of lactose which occurs exclusively in the lactating mammary gland. In somatic tissues, the murine beta4-GT gene specifies two mRNAs of 4. 1 and 3.9 kilobases (kb), as a consequence of initiation at two different start sites approximately 200 base pairs apart. We have proposed that the region upstream of the 4.1-kb start site functions as a housekeeping promoter, while the region adjacent to the 3.9-kb start site functions primarily as a mammary gland-specific promoter (Harduin-Lepers, A., Shaper, J. H., and Shaper, N. L. (1993) J. Biol. Chem. 268, 14348-14359). Using DNase I footprinting and electrophoretic mobility shift assays, we show that the region immediately upstream of the 4.1-kb start site is occupied mainly by the ubiquitous factor Sp1. In contrast, the region adjacent to the 3.9-kb start site is bound by multiple proteins which include the tissue-restricted factor AP2, a mammary gland-specific form of CTF/NF1, Sp1, as well as a candidate negative regulatory factor that represses transcription from the 3.9-kb start site. These data experimentally support our conclusion that the 3.9-kb start site has been introduced into the mammalian beta4-GT gene to accommodate the recruited role of beta4-GT in lactose biosynthesis. PMID: 8617793 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: DNA Cell Biol. 1996 Jan;15(1):65-74. Characterization of nuclear protein binding sites in the promoter of keratin K17 gene. Milisavljevic V, Freedberg IM, Blumenberg M. Ronald O. Perelman Department of Dermatology, New York University Medical Center, NY 10016, USA. Keratin K17, while not present in healthy skin, is expressed under various pathological conditions, including psoriasis and cutaneous allergic reactions. The regulatory circuits involved in transcription of the human keratin K17 gene are poorly understood. To begin an analysis of the molecular mechanisms that regulate K17 gene transcription, we have studied the interactions between the nuclear proteins and the promoter region of the human K17 gene. That promoter region comprised 450 bp upstream from the translation initiation site. For these studies, we used electrophoretic mobility-shift assays, computer analysis, site-directed mutagenesis, and DNA-mediated cell transfection. In addition to the previously characterized interferon-gamma-responsive elements, we identified eight protein binding sites in the promoter. Five of them bind the known transcription factors NF1, AP2, and Sp1 and three others bind still unidentified proteins. Using site-directed mutagenesis, we have demonstrated the importance of the protein binding sites for the promoter function involved in both constitutive and interferon-induced expression of the K17 keratin gene. PMID: 8561898 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 63: Biochem Biophys Res Commun. 1995 Dec 5;217(1):113-22. Regulatory elements in the promoter region of the renal kallikrein gene in normotensive vs hypertensive rats. Wang C, Chen YP, Chao L, Chao J. Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425, USA. The renal kallikrein-kinin system has been implicated in the pathogenesis of hypertension. The expression level of the renal kallikrein gene in the kidney is significantly lower in spontaneously hypertensive rats (SHR) as compared with that of normotensive (SD and WKY) rats. Deletion analysis showed that the fragment -356/-188 of the promoter contains a transcriptional silencer(s) and the GC rich region located between -77 and -187 is the minimal essential element for directing the expression of the CAT reporter gene in mouse L cells. In the kidney of normotensive vs hypertensive rats, the nuclear protein factors NF1/CTF and SP1 bind differently to the renal kallikrein promoter, but similarly in the salivary gland. The differential transcriptional regulation of the rat renal kallikrein gene in the kidney may be responsible for the genetic difference between normotensive and hypertensive rats. PMID: 8526898 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: J Biol Chem. 1995 Nov 10;270(45):26986-92. The mouse p44 mitogen-activated protein kinase (extracellular signal-regulated kinase 1) gene. Genomic organization and structure of the 5'-flanking regulatory region. Pages G, Stanley ER, Le Gall M, Brunet A, Pouyssegur J. Centre de Biochimie, CNRS UMR134, Nice, France. Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase are ubiquitous kinases conserved from fungi to mammals. Their activity is regulated by phosphorylation on both threonine and tyrosine, and they play a crucial role in the regulation of proliferation and differentiation. We report here the cloning of the murine p44 MAP kinase (extracellular signal-regulated kinase 1) gene, the determination of its intron/exon boundaries, and the characterization of its promoter. The gene spans approximately eight kilobases (kb) and can be divided into nine exons and eight introns, each coding region exon containing from one to three of the highly conserved protein kinase domains. Primer extension analysis reveals the existence of two major start sites of transcription located at -183 and -186 base pairs (bp) as well as four discrete start sites for transcription located at -178, -192, -273, and -292 bp of the initiation of translation. However, the start site region lacks TATA-like sequences but does contain initiator-like sequences proximal to the major start sites obtained by primer extension. 1 kb of the promoter region has been sequenced. It contains three putative TATA boxes far upstream of the main start sites region, one AP-1 box, one AP-2 box, one Malt box, one GAGA box, one half serum-responsive element, and putative binding sites for Sp1 (five), GC-rich binding factor (five), CTF-NF1 (one), Myb (one), p53 (two), Ets-1 (one), NF-IL6 (two), MyoD (two), Zeste (one), and hepatocyte nuclear factor-5 (one). To determine the sites critical for the function of the p44 MAPK promoter, we constructed a series of chimeric genes containing variable regions of the 5'-flanking sequence of p44 MAPK gene and the coding region for luciferase. Activity of the promoter, measured by its capacity to direct expression of a luciferase reporter gene, is strong, being comparable with the activity of the Rous sarcoma virus promoter. Progressive deletions of the approximately 1 kb (-1200/-78) promoter region allowed us to define a minimal region of 186 bp (-284/-78) that has maximal promoter activity. Within this context, deletion of the AP-2 binding site reduces by 30-40% the activity of the promoter. Further deletion of this minimal promoter that removes the major start sites (-167/-78) surprisingly preserves promoter activity. This result implicates a major role of this region that contains the Sp1 sites.(ABSTRACT TRUNCATED AT 400 WORDS) PMID: 7592946 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: Biochim Biophys Acta. 1995 Nov 7;1264(2):215-22. Molecular cloning and sequence analysis of the rat liver carnitine octanoyltransferase cDNA, its natural gene and the gene promoter. Choi SJ, Oh DH, Song CS, Roy AK, Chatterjee B. Bioproducts Research Center, Yonsei University, Seoul, South Korea. The full-length cDNA and the natural gene for rat peroxisomal carnitine octanoyltransferase (COT) have been isolated and sequenced. The 2681 bp long cDNA contains an open reading frame for 613 amino acids, resulting in a protein with a deduced molecular weight of 70,301, and a C-terminal peroxisomal targeting sequence (Ala-His-Leu). The isolated COT cDNA has 51 bp of the 5' untranslated region (UTR), 791 bp of 3' UTR, two putative polyadenylation sites, and a poly(A19-23) tail. Screening of a rat genomic DNA library in the lambda phage with the COT cDNA probe resulted in the isolation of seven overlapping clones, together containing the complete COT gene with seventeen exons. All of the exon-intron boundary sequences conform to the GT-AG rule. The COT gene appears to spread over 40 to 60 kbp region of the rat genome. The transcription initiation site of the COT gene was determined through primer extension, and the promoter sequence up to the position -1140 was established. The promoter lacks the canonical TATA box and a promoter-reporter construct containing the sequence encompassing -1140 to +84 base positions and the firefly luciferase reporter cDNA yielded about 100-fold increase in promoter activity in transfected hepatoma cells. Some of the consensus sequences for putative cis elements present in the promoter sequence are: the two CCAAT motifs for CTF/NF1/CBP binding (at -284 and -93), two GC boxes for Sp1 binding (at -160 and -68), two AP2 sites (at -359 and -25), a half site (TGACCT) for the peroxisome proliferator activated receptor (PPAR) binding at -737 within a partial palindromic sequence region. Potential regulatory elements, such as several palindromes and repeat motifs for five different sequence segments, are also identified. PMID: 7495866 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: Gene. 1995 Mar 10;154(2):219-23. Characterization of the 5' flanking region and gene encoding the mouse interferon-gamma receptor. Raval P, Obici S, Russell SW, Murphy WJ. Wilkinson Laboratory, University of Kansas Cancer Center, Kansas City 66160-7184, USA. The purpose of this investigation was to characterize the gene that encodes the receptor for mouse interferon-gamma (IFN-gamma R), including determination of its size, intronic boundaries and its transcription start points (tsp). The mouse IFN-gamma R gene is 22-kb long, with six introns that range in size from approx. 1 to 7 kb. The first six exons encode the extracellular and transmembrane (TM) domains of the protein, while the last exon of about 1 kb encodes most of the intracellular domain. No canonical TATA box can be found in the 5' flanking sequence of the gene, and primer extension analysis indicates multiple tsp. In addition, the gene's 5' promoter region was sequenced to identify candidate responsive elements that might regulate expression of the gene. Among the putative regulatory motifs identified by computer-assisted analysis are multiple SP1 and AP-2 sites, an NF1 and CCAAT box, as well as a potential cyclic AMP-responsive element (CRE). PMID: 7890167 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: J Biol Chem. 1994 Sep 30;269(39):24321-7. Activation of nuclear factor kappa B and oncogene expression by 12(R)-hydroxyeicosatrienoic acid, an angiogenic factor in microvessel endothelial cells. Laniado-Schwartzman M, Lavrovsky Y, Stoltz RA, Conners MS, Falck JR, Chauhan K, Abraham NG. Department of Pharmacology, New York Medical College, Valhalla 10595. 12(R)-Hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid (12(R)-HETrE) is an arachidonic acid metabolite formed by the corneal epithelium of several species, porcine leukocytes, and human and rat epidermal cells. It is a potent, stereospecific proinflammatory and angiogenic factor and its synthesis is increased manyfold in inflamed tissues, e.g. cornea and skin. It is possible that the angiogenic activity of 12(R)-HETrE is due to a direct mitogenic effect on microvessel endothelial cells via yet to be elucidated cellular and molecular mechanisms. In the present study, we demonstrated the ability of 12(R)-HETrE to stimulate the growth of quiescent endothelial cells in a time- and concentration-dependent manner with a maximal effect at 0.1 nM. This effect was highly stereospecific since its enantiomer, 12(S)-HETrE, had no effect within the same concentration range. Northern blot analysis and transient transfection experiments with chloramphenicol acetyltransferase constructs of oncogene promoter regions demonstrated significant increases over control (0.5% fetal calf serum) in c-myc-, c-jun, and c-fos mRNA levels and expression in cells treated with 0.1 nM 12(R)-HETrE. Electrophoretic mobility shift assay of nuclear protein extracts from cells treated with 12(R)-HETrE with specific radiolabeled oligonucleotides corresponding to known transcriptional binding sites, including AP-1, AP-2, SP1, TRE, NF kappa B, TFIID, OKT1, CREB, CTF/NF1, and GRE demonstrated a markedly rapid and specific increase in the binding activity of NF kappa B and to a lesser extent, AP-1. No significant increase was observed in the binding of other transcription factors assayed as compared to control (untreated) cells. Since the protooncogenes (c-fos, c-jun, and c-myc) are immediate early response genes that are implicated in the process of cell proliferation and differentiation, and activation of certain transcription factors, in particular NF kappa B, is associated with the immediate response of the cell to an injury, we propose that 12(R)HETrE's mitogenic and angiogenic activities are mediated, in part, via the activation of NF kappa B and expression of these protooncogenes. PMID: 7523372 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: Biochem Biophys Res Commun. 1994 Jun 30;201(3):1526-33. Study of 5'-flanking region of human Cu/Zn superoxide dismutase. Kim HT, Kim YH, Nam JW, Lee HJ, Rho HM, Jung G. Department of Biology Education, Seoul National University, Korea. The 5'-flanking region of human Cu/Zn superoxide dismutase (SOD1) was cloned from human genomic library for the study of regulation of human SOD1 gene. We determined 3678 nucleotide sequences of 5'-flanking region of human SOD1. The putative binding sites of transcriptional factors such as NF1, Sp1, AP1, AP2, GRE, HSE and NF kappa B were found. The upstream region of this gene was analyzed by deletion and measuring the linked chloramphenicol acetyltransferase (CAT) activities. Several deletion analyses of promoter activity indicated that there were positive and negative regulatory regions. The region from -1325 bp to -1040 bp was found to have a heat shock response element. PMID: 8024598 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 69: Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5987-91. Identification of binding sites for transcription factors NF-kappa B and AP-2 in the promoter region of the human heme oxygenase 1 gene. Lavrovsky Y, Schwartzman ML, Levere RD, Kappas A, Abraham NG. Rockefeller University Hospital, New York, NY 10021. Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and its activity is induced by many agents, including its substrate heme, heavy metals, UV radiation, and other injurious oxidant conditions. We examined the presence of several regulatory elements in the promoter region of the human HO-1 gene which could possibly account for its induction in response to diverse agents or influences. Heme treatment increased both HO activity and HO-1 mRNA in the human erythroleukemic cell line K562. Electrophoretic mobility-shift assays of nuclear protein extracts from heme-treated and control cells with specific oligonucleotide probes containing binding sites for known transcription factors, including AP-1, AP-2, Sp1, NF-kappa B, CTF/NF1, TFIID, OKT1, and CREB, and oligonucleotides containing serum-, metal-, and glucocorticoid-responsive elements demonstrated a specific and marked increase in the NF-kappa B and AP-2 transcription factors and, to a lesser extent, an increase in AP-1. No significant increase in other transcription factors over the control, untreated cells was observed. DNase I footprint assays using purified transcription factors revealed the presence of NF-kappa B and AP-2 binding sites in the proximal part of the promoter region of the human HO-1 gene. Moreover, nucleotide sequence analysis of the HO-1 promoter region showed that the protected regions encompassed NF-kappa B and AP-2 consensus binding sites. The presence of regulatory sequences for the binding of transcription factors such as NF-kappa B and AP-2, whose activation is associated with the immediate response of the cell to an injury, may be an indication of the important role which HO-1 may play in defense mechanisms against tissue injury. PMID: 8016102 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 70: Biochem Biophys Res Commun. 1994 Jun 15;201(2):871-7. Role of zinc-coordination and of the glutathione redox couple in the redox susceptibility of human transcription factor Sp1. Knoepfel L, Steinkuhler C, Carri MT, Rotilio G. Department of Biology, University of Rome, Tor Vergata, Rome, Italy. We show that thiol-groups confer redox-susceptibility to the zinc-finger transcription factor Sp1 and that this redox-susceptibility is prevented by DNA-binding and depends on zinc-coordination of the protein. Apo-Sp1 contained in metal depleted nuclear extracts of human K562 cells exhibited a markedly increased susceptibility towards oxidizing and alkylating agents, as compared to holo-Sp 1. Moreover, DNA binding of apo-Sp1, but not of the holo-protein, was dramatically decreased in the presence of GSH/GSSG ratios within the physiological range. We compared these results with the redox behaviour of two other transcription factors, OTF-1 and NF1, which was found to be different in several aspects from that of Sp1. PMID: 8003025 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: J Cell Biochem. 1994 Jun;55(2):252-63. Nuclear factor 1 is a component of the nuclear matrix. Sun JM, Chen HY, Davie JR. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada. Chicken histone H5 is an H1-like linker histone that is expressed only in nucleated erythrocytes. The histone H5 promoter has binding sites for Sp1 (a high affinity site) and UPE-binding protein, while the 3' erythroid-specific enhancer has binding sites for Sp1 (one moderate and three weak affinity), GATA-1, and NF1. In this study we investigated whether trans-acting factors that bind to the chicken histone H5 promoter or enhancer are associated with adult chicken immature and mature erythrocyte nuclear matrices. We show that NF1, but not Sp1, GATA-1, or UPE-binding protein, is associated with the internal nuclear matrices of these erythroid cells. Further, we found that a subset of the NF1 family of proteins is bound to the mature erythrocyte nuclear matrix. These results suggest that in chicken erythrocytes NF1 may mediate an interaction between the histone H5 enhancer and the erythroid internal nuclear matrix. NF1 was also present in the internal nuclear matrices of chicken liver and trout liver. The observations of this study provide evidence that NF1 may have a role in a variety of cell types in targeting specific DNA sequences to the nuclear matrix. PMID: 8089200 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: Radiat Res. 1994 Apr;138(1 Suppl):S47-51. Alterations in transcription factor binding in radioresistant human melanoma cells after ionizing radiation. Sahijdak WM, Yang CR, Zuckerman JS, Meyers M, Boothman DA. Department of Human Oncology (K4/626), University of Wisconsin-Madison 53792. We analyzed alterations in transcription factor binding to specific, known promoter DNA consensus sequences between irradiated and unirradiated radioresistant human melanoma (U1-Mel) cells. The goal of this study was to begin to investigate which transcription factors and DNA-binding sites are responsible for the induction of specific transcripts and proteins after ionizing radiation (Boothman et al., Proc. Natl. Acad. Sci. USA 90, 7200, 1993). Transcription factor binding was observed using DNA band-shift assays and oligonucleotide competition analyses. Confluence-arrested U1-Mel cells were irradiated (4.5 Gy) and harvested at 4 h. Double-stranded oligonucleotides containing known DNA-binding consensus sites for specific transcription factors were used. Increased DNA-binding activity after ionizing radiation was noted with oligonucleotides containing the CREB, NF-kappa B and Sp1 consensus sites. Increased DNA binding activity after ionizing radiation was noted with oligonucleotides containing the CREB, NF-kappa B and Sp1 consensus sites. No changes in protein binding to AP-1, AP-2, AP-3 or CTF/NF1, GRE or Oct-1 consensus sequences were noted. X-ray activation of select transcription factors, which bind certain consensus sites in promoters, may cause specific induction or repression of gene transcription. PMID: 8146325 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: EMBO J. 1994 Feb 1;13(3):641-5. Functional differences between mammalian transcription activation domains at the yeast GAL1 promoter. Kunzler M, Braus GH, Georgiev O, Seipel K, Schaffner W. Mikrobiologisches Institut, Eidgenossische Technische Hochschule (ETH), ETH-Zentrum, Zurich, Switzerland. We have fused representatives of three structurally and functionally distinct classes of mammalian transcription activation domains for RNA polymerase II to the yeast GAL4 DNA binding domain. All fusion proteins were stable when expressed in yeast and were tested for their ability to activate transcription from various positions in the yeast GAL1 promoter. Activation domains functional from remote as well as TATA-proximal positions in mammalian cells, e.g. the acidic-type domain of VP16, also stimulate transcription in yeast from various promoter positions. Proline-rich domains, as e.g. in AP-2 and CTF/NF1, with considerable promoter activity and low enhancer activity in mammalian cells stimulate transcription in yeast only from a position close to the TATA box. The glutamine-rich domains of Oct1, Oct2 and Sp1, which activate transcription in mammalian cells from close to the TATA box in response to a remote enhancer, are inactive in the yeast GAL1 promoter. This finding might reflect some basic difference between the organization of yeast and mammalian promoters. PMID: 8313909 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: J Virol. 1993 Nov;67(11):6476-86. Transcriptional control of human papillomavirus (HPV) oncogene expression: composition of the HPV type 18 upstream regulatory region. Butz K, Hoppe-Seyler F. Projektgruppe Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany. The malignant transformation potential of high-risk human papillomaviruses (HPVs) is closely linked to the expression of the viral E6 and E7 genes. To elucidate the molecular mechanisms resulting in HPV oncogene expression, a systematic analysis of the cis-regulatory elements within the HPV type 18 (HPV18) upstream regulatory region (URR) which regulate the activity of the E6/E7 promoter was performed. As the functional behavior of a given cis-regulatory element can be strongly influenced by the overall composition of a transcriptional control region, individual elements were inactivated by site-directed mutagenesis in the physiological context of the complete HPV18 URR. Subsequently, the effects of these mutations on the activity of the E6/E7 promoter were assessed by transient transfection assays. We found that the transcriptional stimulation of the E6/E7 promoter largely depends on the integrity of cis-regulatory elements bound by AP1, SP1, and in certain epithelial cells, KRF-1. In contrast to previous reports by implying a key role for NF1 and Oct-1 recognition motifs in the stimulation of papillomavirus oncogene expression, the inactivation of these elements in the context of the HPV18 URR did not strongly affect the transcriptional activity of the E6/E7 promoter. Mutation of a promoter-proximal glucocorticoid response element completely abolished dexamethasone inducibility of the HPV18 E6/E7 promoter and resulted in an increase of its basal activity. Functional dissection of the HPV18 constitutive enhancer region indicates that its transcriptional activity is largely generated by functional synergism between a centrally located AP1 module and thus far undetected cis-active elements present in the 5' flank of the enhancer. Furthermore, comparative analyses using homologous and heterologous promoters show that the transcriptional activity of HPV18 enhancer elements is influenced by the nature of the test promoter in a cell-type-specific manner. PMID: 8411351 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 75: Infect Immun. 1993 Oct;61(10):4427-33. Shigella flexneri invasion of HeLa cells induces NF-kappa B DNA-binding activity. Dyer RB, Collaco CR, Niesel DW, Herzog NK. Department of Microbiology, University of Texas Medical Branch, Galveston 77555-0605. Although information about the genetic basis and mechanisms of Shigella flexneri cellular invasion is accumulating, little is known about changes in cell signaling and their consequences following bacterium-host cell interactions. A general result of signal transduction is alterations in the levels and/or activities of transcription factors. Alterations in transcription factor binding activities were observed following challenge with S. flexneri. Changes in the DNA-binding activities of cellular transcription factors to AP1, AP2, cyclic AMP response element, CTF1/NF1, NF-kappa B/Rel, OCT1, and SP1 DNA-binding sites were investigated by electrophoretic mobility shift assays. NF-kappa B/Rel DNA-binding activity was enhanced more than 11-fold by cellular invasion; noninvasive S. flexneri strains induced low levels of kappa B DNA binding. Both subunits of the NF-kappa B transcription factor, p50 and p65, but not c-Rel (p85), are components of the kappa B DNA-binding activity. These data suggest that changes in cellular transcription factor binding activity are a consequence of S. flexneri invasion, and these changes could play a role in the initial host response or in the pathogenesis of the disease. PMID: 8406833 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 76: FEBS Lett. 1993 Sep 27;331(1-2):141-4. Repression of histone H5 gene expression in chicken mature erythrocytes is correlated with reduced DNA-binding activities of transcription factors Sp1 and GATA-1. Sun JM, Penner CG, Davie JR. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada. During the final stages of erythroid maturation, the expression of the chicken histone H5 gene ceases. The histone H5 promoter has binding sites for Sp1 and UPE-binding protein. The 3' histone H5 enhancer has binding sites for Sp1, GATA-1 and NF1. Here, we show that the DNA-binding activities of transcription factors Sp1 and GATA-1 is reduced 5- to 10-fold in mature cells, while the activities of UPE-binding protein and NF1 remain the same in mature and immature erythrocytes. The reduced activities of Sp1 and GATA-1 may contribute to the inactivation of the histone H5 gene in mature erythrocytes. PMID: 8405392 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: Mol Cell Biol. 1993 Aug;13(8):4632-9. Mechanism of developmental regulation of alpha pi, the chicken embryonic alpha-globin gene. Knezetic JA, Felsenfeld G. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892. The chicken alpha pi-globin gene is expressed during development only in the primitive erythrocyte lineage and not in the definitive lineage. We show that stage-specific expression is maintained when plasmids containing the alpha pi promoter are transfected into primitive and definitive lineage primary erythroid cells and that the information contained in the promoter is sufficient to confer this specificity. Detailed analysis of binding sites in the promoter for trans-acting factors, together with studies of the effects of mutagenesis on expression, reveals that the factors critical to stage-specific expression are all present in both primitive and definitive lineages, but at various concentrations. We identify three proteins, an NF1 family member, a Y-box factor, and an Sp1-like factor, which interact to stimulate or inhibit transcription. We propose that the concentration-dependent action of these factors, together with the general erythroid factor GATA-1, is responsible for the stage-specific expression of the alpha pi-globin gene. PMID: 8336706 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: J Biol Chem. 1993 Mar 15;268(8):5694-702. Human casein kinase II. The subunit alpha protein activates transcription of the subunit beta gene. Robitzki A, Bodenbach L, Voss H, Pyerin W. German Cancer Research Center, Heidelberg. Casein kinase II (CKII), an ubiquitous serine/threonine protein kinase in control of a variety of crucial cellular functions, is composed of catalytic subunits (alpha and alpha') and regulatory subunits (beta). The adjusted activity of CKII is determined by the actual conformational state of CKII beta and the stoichiometry of the CKII subunits. Thus, the expression control of CKII beta is of particular concern. Carrying out gel shifts and footprints with affinity-purified proteins and cellular extracts in combination with mutational analysis we find that aside NF1 and Sp1, two out of the many factors predicted to bind to the upstream promoter region of the human CKII beta gene (Voss, H., Wirkner, U., Jakobi, R., Hewitt, N. A., Schwager, C., Zimmermann, J., Ansorge, W., and Pyerin, W. (1991) J. Biol. Chem. 266, 13706-13711), CKII alpha protein is able to complex with the CKII beta gene promoter. The complex of CKII beta-DNA/CKII alpha-protein is shown to occur within the 170-239-base pair (bp) segment upstream of the first transcription start site of the gene. The DNA motif contains, in a distance of 44 bp, two GC-rich boxes, 5'-GGGGCCC and 5'-CCCCTGGGC, and represents a novel cis-acting element; the binding of the CKII alpha protein activates the CKII beta gene promoter. This is manifested by driving the expression of the indicator gene luciferase or of CKII alpha-cDNA in HeLa cells. The binding of the CKII alpha protein is inhibited due to CKII beta protein addition or by mimicking the corresponding situation in vivo by overexpression of the CKII subunits. The data suggest that cells may maintain a certain CKII subunit stoichiometry via transcriptional control; excess of nuclear CKII alpha protein could activate the CKII beta gene transcription causing CKII beta protein to increase which, in turn, could feed back to abolish the action of CKII alpha at the CKII beta gene promoter. PMID: 8449932 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: Nucleic Acids Res. 1992 Dec 11;20(23):6385-92. Analysis of erythroid nuclear proteins binding to the promoter and enhancer elements of the chicken histone H5 gene. Sun JM, Penner CG, Davie JR. Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada. The chicken erythroid proteins binding to the histone H5 5' promoter and 3' erythroid-specific enhancer regions were identified. In DNase I footprinting and gel mobility shift experiments with immature adult erythrocyte nuclear extracts, we have demonstrated the binding of proteins to the GC-box, a high affinity Sp1 binding site, and to the upstream promoter element. We have previously demonstrated that a multisubunit complex containing the transcription factor GATA-1 was associated with the enhancer. Here, we show that the enhancer region also has four Sp1 binding sites (one medium and three weak affinity, one of which may also bind the CACCC factor), a potential NF-E4 binding site, and a binding site for a NF1-like factor. The results of gel mobility-shift and competition experiments provide evidence that the Sp1 binding sites are associated with a high molecular mass (greater than 450 kDa), Sp1 containing protein complex. We propose that Sp1 multimers bound at the promoter and enhancer interact to mediate the juxta-positioning of the enhancer and promoter elements, bringing the GATA-1 multisubunit complex next to the initiation site. The GATA-1 complex may contribute to the protein-protein interactions between the enhancer and promoter. PMID: 1475200 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: EMBO J. 1992 Dec;11(13):4961-8. Different activation domains stimulate transcription from remote ('enhancer') and proximal ('promoter') positions. Seipel K, Georgiev O, Schaffner W. Institut fur Molekularbiologie II, Universitat Zurich, Switzerland. We reported previously that the lymphocyte-derived octamer transcription factor 2A (Oct-2A or OTF-2A) activated both natural immunoglobulin promoters and synthetic promoters which contain the 'octamer' site, but was unable by itself to stimulate transcription from a remote enhancer position. Here we examine a larger set of transcription factors with respect to their proximal versus remote activation. Since a transcription factor may contain more than one activation domain, we have chosen to study the potential of individual activation domains in the context of fusion proteins that contain the DNA binding domain of GALA. We have identified at least two distinct functional classes of transcriptional activation domains. 'Proximal' activation domains, exemplified by glutamine-rich domains of Oct-1, Oct-2A and Sp1, stimulate transcription only from a position close to the TATA box, usually in response to a remote enhancer. 'General' activation domains, derived from VP16, GAL4, p65 (NF-chi B), TFE3, ITF-1 and ITF-2, can activate transcription from remote as well as proximal positions. These domains contain many acidic amino acids and/or other features such as clusters of serine and threonine. The proline-rich activation domains of AP-2 and CTF/NF1 may represent a third class with considerable promoter activity and low but significant enhancer activity. Furthermore, activation domains of both the acidic and glutamine-rich types seem to have a modular structure, since duplicated subdomains can substitute for the entire domain. PMID: 1464321 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: Pharmacogenetics. 1992 Oct;2(5):185-96. Regulation of human alcohol dehydrogenase genes. Edenberg HJ, Brown CJ. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122. This review focuses upon the regulation of the three human class I alcohol dehydrogenase genes, ADH1, ADH2 and ADH3. These closely related genes are expressed at high levels in liver, and at different levels in other tissues. Multiple cis-acting sequences to which nuclear proteins bind have been mapped, and transcription factors that can bind to these sequences have been identified; these include C/EBP alpha, Sp1, USF, HNF1, CTF/NF1, the glucocorticoid receptor, and RAR alpha. There are interesting but often subtle differences in the binding to these three closely related genes, that presumably account for the differences in patterns of their expression. Publication Types: Review PMID: 1339084 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: J Biol Chem. 1992 Sep 5;267(25):17944-8. Sp1 DNA binding efficiency is highly reduced in nuclear extracts from aged rat tissues. Ammendola R, Mesuraca M, Russo T, Cimino F. Department of Biochemistry and Medical Biotechnology, University of Naples Federico II, Italy. To explore the role of transcriptional factors in the genesis of the senescent phenotype, nuclear extracts from 4- and 30-month-old rat brains were analyzed for the presence of DNA-binding proteins able to interact with double-stranded oligonucleotides containing recognition sites for sequence-specific DNA-binding factors. Gel shift assays revealed that the DNA-binding efficiency of Sp1 is significantly reduced in aged animals compared to young ones, whereas CTF/NF1 and AP1 from young and old rat nuclear extracts bind their DNA targets with the same efficiency. The quantitative analysis of Sp1 by immunoblotting indicated that equivalent quantities and degrees of heterogeneity of Sp1 protein are present in both nuclear extracts, suggesting that the observed difference is not due to a different expression of this transcriptional factor. DNase I footprinting of the heavy chain ferritin gene promoter, which contains a Sp1 binding site, demonstrated that the nuclear extract from 30-month-old rat brain does not protect the region involved in the regulation of the H ferritin gene by Sp1. This results in a reduction of about 50% of the expression of the H ferritin mRNA in aged rat brains. Furthermore, the Sp1 binding sites present in the SV40 early promoter are not protected in a DNase I footprinting assay where a nuclear extract from 30-month-old rat brain was used as a source of DNA binding proteins. Liver nuclear extracts prepared from young and aged rats demonstrated that a decrease of Sp1 binding efficiency is similarly present in this tissue. PMID: 1381357 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 83: Mol Cell Biol. 1992 Sep;12(9):3991-7. Transcriptional regulation by triiodothyronine requires synergistic action of the thyroid receptor with another trans-acting factor. Voz ML, Peers B, Wiedig MJ, Jacquemin P, Belayew A, Martial JA. Laboratoire de Biologie Moleculaire et de Genie Genetique, Institut de Chimie B6, Universite de Liege, Sart-Tilman, Belgium. Human placental lactogen B (hCS-B) promoter activity is strongly stimulated by triiodothyronine (T3) in pituitary GC cells through interaction between the thyroid receptor and a thyroid receptor-binding element (TBE) spanning coordinates -67 to -41. This TBE is adjacent to the binding site for pituitary factor GHF1 (-95 to -68) which seems necessary for T3 stimulation of hCS-B promoter activity (M. L. Voz, B. Peers, A. Belayew, and J. A. Martial, J. Biol. Chem. 266:13397-13404, 1991). We here demonstrate actual synergy between the thyroid receptor and GHF1. Indeed, in placental JEG-3 cells devoid of factor GHF1, hCS promoter activity is barely stimulated by T3, while a strong response is observed in pituitary GC cells. In the latter, furthermore, neither the TBE nor the GHF1-binding site alone is sufficient to render the thymidine kinase promoter responsive to T3, while in combination they promote strong T3 stimulation. Close proximity between these sites is required for optimal synergy: T3 stimulation globally decreases with increased spacing. Furthermore, synergy occurs not only with a GHF1-binding site but also with all other factor recognition sequences tested (Sp1, NF1, CP1, Oct1, and CACCC boxes) and even with two other copies of the TBE. Nor is it specific to hCS TBE, since the palindromic sequence TCAGGTCA TGACCTGA (TREpal) also exhibits cooperativity. PMID: 1324411 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 84: Biochem J. 1992 Aug 1;285 ( Pt 3):925-8. Cloning, sequencing and characterization of the human alpha glutathione S-transferase gene corresponding to the cDNA clone pGTH2. Klone A, Hussnatter R, Sies H. Institut fur Physiologische Chemie I, Heinrich-Heine-Universitat Dusseldorf, Germany. The human Alpha glutathione S-transferase gene corresponding to the human liver cDNA clone pGTH2 was isolated from a cosmid genome library. The gene, represented by the clone cosGTH2, spans nearly 12 kb and contains seven exons. The intron/exon borders conform to the standard rules, and an open reading frame is present, starting at position 67 in exon 2, the double-stop codon being at position 733 in exon 7. Exons 1, 2 and 7 differ in length from the known rat gene coding for the Ya enzyme. A 209 bp 5'-upstream region contains TATA and CAT boxes and, in addition, motifs for Sp1-, NF1- and HNFI-binding factors. Clone cosGTH2 represents the less basic subunit, alpha y, of two Alpha glutathione S-transferase subunits (alpha x and alpha y) expressed in liver, which is identical with the kidney subunit alpha 2. PMID: 1497629 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 85: Mol Cell Biol. 1992 Mar;12(3):1352-6. The core promoter region of the tumor necrosis factor alpha gene confers phorbol ester responsiveness to upstream transcriptional activators. Leitman DC, Mackow ER, Williams T, Baxter JD, West BL. Metabolic Research Unit, University of California, San Francisco 94143. Activators of protein kinase C, such as 12-O-tetradecanoylphorbol 13-acetate (TPA), are known to regulate the expression of many genes, including the tumor necrosis factor alpha (TNF) gene, by affecting the level or activity of upstream transcription factors. To investigate the mechanism whereby TPA activates the TNF promoter, a series of 5'-deletion mutants of the human TNF promoter linked to chloramphenicol acetyltransferase was transfected into U937 human promonocytic cells. TPA produced a 7- to 11-fold activation of all TNF promoters tested, even those promoters truncated to contain only the core promoter with no upstream enhancer elements. The proximal TNF promoter containing only 28 nucleotides upstream and 10 nucleotides downstream of the RNA start site confers TPA activation to a variety of unrelated upstream enhancer elements and transcription factors, including Sp1, CTF/NF1, cyclic AMP-response element, GAL-E1a, and GAL-VP16. The level of activation by TPA depends on the TATA box structure, since the TPA response is greater in promoters containing the sequence TATAAA than in those containing TATTAA or TATTTA. These findings suggest that the core promoter region is a target for gene regulation by second-messenger pathways. PMID: 1545816 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 86: Oncogene. 1991 Nov;6(11):2067-75. Regulatory domains within the P0 promoter of human c-myc. Lang JC, Wilkie NM, Clark AM, Chudleigh A, Talbot S, Whitelaw B, Frame MC. Beatson Institute for Cancer Research, Glasgow, UK. Expression of P0 RNA in some Burkitt lymphoma cell lines varies independently of levels of RNA derived from P1 and P2. These data suggest the possibility that expression of P0 RNA may be capable of independent regulation. In order to investigate this possibility we have isolated putative regulatory domains flanking P0 RNA starts within the human c-myc gene and analysed both their ability to direct expression of control reporter genes and their ability to interact with specific transcription factors. Regulatory regions necessary for expression of P0 RNA have been located within 131 bp 5' of the first major P0 RNA start. DNAase 1 footprint analysis and gel retardation assays demonstrate binding of transcription factors Sp1, NF1 and CBP to this region. NF1 binds specifically to two consensus sequences. The more distal site overlaps with the binding site for CBP, and it is likely that concomitant binding of NF1 and CBP within the distal region of the P0 promoter is not possible. Previous work from our laboratory has described a negative regulatory domain within the 5' flanking region of c-myc. The P0 promoter resides within this domain and therefore may contain a negative regulator of c-myc gene expression. PMID: 1945411 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 87: Bioessays. 1991 Oct;13(10):499-503. The interactions of transcription factors and their adaptors, coactivators and accessory proteins. Martin KJ. Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605. Consistent with the complexity of the temporally regulated processes that must occur for growth and development of higher eukaryotes, it is now apparent that transcription is regulated by the formation of multicomponent complexes that assemble on the promoters of genes. These complexes can include (in addition to the five or more general transcription factors and RNA polymerase II) DNA-binding proteins, transcriptional activators, coactivators, adaptors and various accessory proteins. The best studied example of a complex that includes a transcriptional adaptor, accessory proteins and a DNA-binding protein is that involving the herpes simplex virus VP16 protein. Evidence suggests that the adenovirus E1a protein and the cellular Sp1 and CTF/NF1 transcription factors also function through adaptors or coactivators. Each additional component of the transcription complex provides the cell with another point at which to exert control of gene expression. Publication Types: Review Review, Tutorial PMID: 1661580 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 88: Genes Dev. 1990 Aug;4(8):1277-87. In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Pfeifer GP, Tanguay RL, Steigerwald SD, Riggs AD. Beckman Research Institute of the City of Hope, Department of Biology, Duarte, California 91010. The promoter region of the X-linked human phosphoglycerate kinase-1 (PGK-1) gene is a CpG island, similar to those often found near autosomal genes. We used ligation-mediated polymerase chain reaction (PCR) for a genomic sequencing study in which 450 bp of the human PGK-1 promoter region was analyzed for the presence of in vivo protein footprints and cytosine methylation at all CpG sites. A technique was devised to selectively visualize the DNA of the inactive X chromosome (Xi), even in the presence of the active X chromosome (Xa). We found that the human Xa in both normal male lymphocytes and hamster-human hybrids is completely unmethylated at all 120 CpG sites. In contrast, 118 of the CpG sites are methylated on the human Xi in hamster-human hybrids. The Xi in normal female lymphocytes is also highly methylated, but some GCG or CGC trinucleotides partially escape methylation; all other CpGs are fully methylated. In vivo footprinting studies with dimethylsulfate (DMS) revealed eight regions of apparent protein-DNA contacts on the Xa. Four of the footprints contained the consensus sequence of the binding site for transcription factor Sp1. The other regions include potential binding sites for transcription factors ATF, NF1, and a CCAAT-binding protein. The Xi did not show any specifically protected sequences, and with the exception of four hyperreactive sites, the in vivo DMS reactivity profile of Xi DNA was very similar to that of purified, linear Xi DNA. The implications of these findings with regard to the maintenance of methylation-free islands, X chromosome inactivation, and the chromatin structure of facultative heterochromatin are discussed. PMID: 2227409 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 89: Biochem Biophys Res Commun. 1990 Jul 16;170(1):140-6. Transcription factor levels in medullary thyroid carcinoma cells differentiated by Harvey ras oncogene: c-jun is increased. Nelkin BD, Borges M, Mabry M, Baylin SB. Oncology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231. In the TT cell line of human medullary thyroid carcinoma, the viral Harvey ras (v-rasH) oncogene induces differentiation, marked by morphological changes, diminution of growth, and increased expression of the calcitonin gene. Here, we show that the transcriptional factor c-jun is increased during v-rasH induced differentiation of TT cells both at the mRNA and functional protein levels. In contrast, nuclear proteins with binding activities related to AP2, AP3, NF1/CTF, and Sp1 were unchanged in v-rasH differentiated TT cells. PMID: 2115330 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 90: Exp Eye Res. 1990 Jun;50(6):759-70. Analysis of the human ornithine aminotransferase gene family. Zintz CB, Inana G. Molecular Pathology Section, National Eye Institute, National Institutes of Health, Bethesda, MD 20892. Ornithine aminotransferase is a mitochondrial matrix enzyme that is deficient in patients with gyrate atrophy, an autosomal recessive disease of the eye. Southern blots of human DNA probed with a previously characterized OAT cDNA showed a complex pattern of gene fragments, suggesting a gene family. Hybridization of these blots with 5' and 3' OAT cDNA probes indicated that there are at least three to four copies of the OAT (approximately 22 kbp) and OAT-related gene sequence(s). We have isolated and partially characterized human OAT gene clones from total genomic and X-chromosome DNA libraries. Sequence analysis confirmed the following previously reported findings on the functional OAT gene: 11 exons, ten introns, an atypical TATA box (TTTAA), two CCAAT boxes, several GC-rich binding sites, 5' sequence homologous to SV40 enhancer core sequence (GTGGA/GA/GA/GG) and promoter region of three urea cycle enzymes (GTATCCTGCCCTC). In addition, we extended the OAT gene sequence in both the 5' and 3' directions and found its promoter region also contained a sequence homologous to the progesterone receptor (TGTTCA/TCC/T), several of the glucocorticoid responsive element (AGAACA), a cyclic AMP-responsive element (TGACGTCG), and recognition motifs for transcription factors AP-2, NF1 and Sp1. Partial sequence analyses of X-chromosome clones demonstrated an intron-less pseudogene with 77% identity to the functional OAT gene. These results demonstrate that the OAT gene is a gene family that contains both functional and related OAT gene sequence(s). PMID: 2373169 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 91: Cell. 1990 May 4;61(3):467-74. Extinction of an immunoglobulin kappa promoter in cell hybrids is mediated by the octamer motif and correlates with suppression of Oct-2 expression. Junker S, Pedersen S, Schreiber E, Matthias P. Institute of Human Genetics University of Aarhus, Denmark. When immunoglobulin-expressing B cells are fused with fibroblasts, immunoglobulin expression is rapidly and selectively suppressed. here we demonstrate that the conserved octamer motif of a kappa light chain gene promoter plays a crucial role in mediating this "extinction" phenomenon. Replacement of this octamer site by an Sp1 or NF1 binding site is sufficient to bypass extinction. Furthermore, in early cell hybrids, immunoglobulin suppression is correlated with absence of the cell-specific transcription factor Oct-2 and its transcripts. Such hybrids cannot support transcription of a transiently introduced reporter plasmid, driven by an octamer-containing promoter, unless an expression vector encoding Oct-2 is cotransfected. Transfection of the same Oct-2 expression vector into hybrid cells is also sufficient to "reactivate" an integrated kappa promoter construct. Thus, our data further establish the role of Oct-2 for immunoglobulin transcription and show that in B cell x fibroblast hybrids, the lack of a necessary cell-specific transcription factor is involved in the extinction of immunoglobulin expression. PMID: 2110507 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 92: Arch Biochem Biophys. 1989 Jun;271(2):479-87. Structural characterization of the bovine CYP17 (17 alpha-hydroxylase) gene. Bhasker CR, Adler BS, Dee A, John ME, Kagimoto M, Zuber MX, Ahlgren R, Wang XD, Simpson ER, Waterman MR. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235. The complete exonic and partial intronic sequence of the bovine CYP17 (P45017 alpha) gene has been determined. The gene contains eight exons with exon/intron boundaries which are identical to those determined previously for the human CYP17 gene. The site of initiation of transcription of this gene is located within a 6-base sequence 52 bp from the initiation of translation. Considerable sequence homology (58.7%) is found when approximately 500 bp of the 5'-flanking sequences of the bovine and human CYP17 genes are compared. A computer-based search of this region of bovine CYP17 for consensus sequences associated with binding of transcription factors (i.e., GR, PR, CREB/ATF, AP1, AP2, AP3, AP4, AP5, OTF, CTF/NF1, SP1) shows only the consensus CREB/ATF sequence TGACGT which is also found to be at approximately the same position in the human CYP17 gene. In bovine adrenal cortex, transcription of the CYP17 gene is regulated by the peptide hormone adrenocorticotropin via cAMP. Whether the consensus CREB/ATF sequence is associated with the cAMP-mediated transcription of the CYP17 gene remains to be elucidated. PMID: 2543297 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 93: J Virol. 1989 Apr;63(4):1514-24. An element of the BK virus enhancer required for DNA replication. Del Vecchio AM, Steinman RA, Ricciardi RP. Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104-4268. The human papovavirus BK virus contains three 68-base-pair (bp) repeats that act as transcriptional enhancers. An analysis of plasmids containing the BK virus origin revealed that sequences within the 68-bp enhancer are required for DNA replication as well as transcription of the early promoter in COS-1 cells. Origins with a single 68-bp repeat replicated as efficiently as did those with three repeats when transfected into COS-1 cells. Replication did not occur in the absence of enhancer sequences and could not be restored by distal placement of enhancers to enhancerless origins. However, as with simian virus 40, replication in vitro was not dependent on the presence of any enhancer sequences. Deletion analysis showed that replication of BK virus origins was dependent on the presence of the first 21 bp of the enhancer contiguous with the A-T-rich stretch of the origin. This 21-bp element is referred to as the rep element. Although in combination with rep the remaining 47 bp of the enhancer appear to increase replication by two- to fivefold, they alone are not sufficient to support replication. Deletions or insertions in the enhancer which did not alter the rep element had no major effect on replication. Site-directed mutagenesis of the Sp1-like site within the rep element, the NF1 site present in the enhancer, or the NF1 site in adjacent late-side sequences each reduced transcription by two- to fivefold, but had no effect on replication, suggesting that replication and transcription can be uncoupled. PMID: 2538642 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 94: Virology. 1989 Mar;169(1):172-81. Unidirectional deletion and linker scan analysis of the late promoter of the human papovavirus BK. Cassill JA, Deyerle KL, Subramani S. Department of Biology, University of California, San Diego, La Jolla 92093. We have previously shown that the late promoter of the human papovavirus BK (prototype) is contained within the three 68-bp repeats and a 66-bp region to the late side of the repeats which together constitute the early promoter enhancer. We have now carried out unidirectional deletion and linker scan analyses of these sequences to identify the major elements of the late promoter in human and monkey cells. Several important sequence motifs involved in late promoter function are found throughout this region. The most active ones correspond to previously defined binding sites for the transcription factors NF1 and Sp1 and a GC-rich region known to be important for early promoter function. The NF1 sequences may also be involved in negative regulation in some situations. PMID: 2538030 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 95: Nucleic Acids Res. 1988 Aug 11;16(15):7527-44. Multiple nuclear factors interact with promoter sequences of the urokinase-type plasminogen activator gene. von der Ahe D, Pearson D, Nakagawa J, Rajput B, Nagamine Y. Friedrich Miescher-Institut, Basel, Switzerland. To characterize proteins that bind to the cyclic AMP inducible promoter of the urokinase-type plasminogen activator gene, we performed a DNAase I footprinting analysis. Within 500 nucleotides upstream of the transcription start site we found eight protected regions due to at least four different binding proteins. Among these is a single binding site for the transcription factor CTF/NF1, which is flanked on each side by two conserved binding sites for the transcription factor Sp1. A region at -380, which shares a similarity with sequences observed in the corresponding regions of other cyclic AMP regulated genes, was protected. This binding site contains a sequence of ten nucleotides which is repeated further upstream at -480 and also protected against DNAase I digestion. Comparisons of extracts from four different cell lines revealed that all DNA binding factors are present in nuclei of uPA expressing and nonexpressing cells. Mechanism underlying hormonal regulation of the gene is discussed. PMID: 3412894 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------