Regional variations in ABC transporter expression along the mouse intestinal tract.

The ATP-binding cassette (ABC) family of proteins comprise a group of membrane transporters involved in the transport of a wide variety of compounds, such as xenobiotics, vitamins, lipids, amino acids, and carbohydrates. Determining their regional expression patterns along the intestinal tract will further characterize their transport functions in the gut. The mRNA expression levels of murine ABC transporters in the duodenum, jejunum, ileum, and colon were examined using the Affymetrix MuU74v2 GeneChip set. Eight ABC transporters (Abcb2, Abcb3, Abcb9, Abcc3, Abcc6, Abcd1, Abcg5, and Abcg8) displayed significant differential gene expression along the intestinal tract, as determined by two statistical models (a global error assessment model and a classic ANOVA, both with a P < 0.01). Concordance with semiquantitative real-time PCR was high. Analyzing the promoters of the differentially expressed ABC transporters did not identify common transcriptional motifs between family members or with other genes; however, the expression profile for Abcb9 was highly correlated with fibulin-1, and both genes share a common complex promoter model involving the NFkappaB, zinc binding protein factor (ZBPF), GC-box factors SP1/GC (SP1F), and early growth response factor (EGRF) transcription binding motifs. The cellular location of another of the differentially expressed ABC transporters, Abcc3, was examined by immunohistochemistry. Staining revealed that the protein is consistently expressed in the basolateral compartment of enterocytes along the anterior-posterior axis of the intestine. Furthermore, the intensity of the staining pattern is concordant with the expression profile. This agrees with previous findings in which the mRNA, protein, and transport function of Abcc3 were increased in the rat distal intestine. These data reveal regional differences in gene expression profiles along the intestinal tract and demonstrate that a complete understanding of intestinal ABC transporter function can only be achieved by examining the physiologically distinct regions of the gut.

Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.

Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.

Struktur, Funktion und potentielle Anwendung der PKC- und SNZ,SNO-Promotoren aus Geodia cydonium und Suberites domuncula

Die Schwämme (Porifera) sind eine reiche Quelle bioaktiver Naturstoffe. Viele dieser Naturstoffe besitzen das Potential, als Pharmazeutika, molekulare Sonden usw. eingesetzt oder weiterentwickelt zu werden. Die Beschaffung dieser Naturstoffe in ausreichenden Mengen stellt jedoch eines der größten Probleme bei der Testung und Produktion vielversprechender Substanzen dar. Der Transfer von DNA in Schwammzellen bzw. in komplette Organismen wäre ein vielversprechender Ansatz, dieses Problem zu lösen. Das Ziel dieser Arbeit war es deshalb, die Funktion und Struktur homologer Promotoren zu untersuchen und eine Methode des Gentransfers in Schwammzellen auszuarbeiten. Zu diesem Zweck wurde zusätzlich zu der bereits vorhandenen 5'-flankierenden Region des conventional PKC-Gens aus Geodia cydonium eine genomische Bibliothek von Suberites domuncula konstruiert, um diese mit Hilfe des DNA-Homologiescreenings nach den 5'-flankierenden Regionen des cPKC- und des SNZ (SnooZe)-Gens (SD_SNZG) zu durchsuchen. Die Klonierung und Sequenzierung sowohl des 5'-Bereichs als auch die Charakterisierung der Exon-Intron Struktur beider Gene wurde erfolgreich durchgeführt. In der 5'-Region des SNZ-Gens konnte dabei ein weiteres Gen (SD_SNO; SNZ proximal Open Reading Frame) identifiziert werden, das in einer 'Kopf-an-Kopf' Anordnung zu SD_SNZG orientiert ist. Sowohl SD_SNZG als auch SD_SNO wurden hochkonservierten Genfamilien zugeordnet, deren Vorkommen in Metazoen hier erstmals beschrieben wird.Funktionelle Studien mit Hilfe der Reportergene Luciferase und Enhanced Green Fluorescent Protein (EGFP) im heterologen System der NIH 3T3 Zellen wiesen sowohl dem cPKC-Promotor aus G. cydonium als auch dem SNZ-Promotor aus S. domuncula eine starke Promotoraktivität im Verhältnis zum SV40-Promotor nach. Die Aktivität des cPKC-Promotors aus S. domuncula dagegen war relativ schwach. Darüber hinaus konnte geklärt werden, daß die 5'-flankierende Region des SNZ-Gens bidirektionale Promotoraktivität aufweist und daß der G. cydonium cPKC-Promotor keine TATA-Box besitzt, sondern eine GC-Box für die basale Funktion benötigt.Als geeignete Methode zur Transfektion von Zellen des Schwamms S. domuncula erwies sich der ballistische Gentransfer mit Hilfe der Gene Gun. Homologe Promotoren konnten die sichtbare Expression des Reportergens EGFP jedoch nicht bewirken. Nur der virale CMV-Promotor erwies sich als hierfür geeignet.

Genomische Struktur und Promotorcharakterisierung des humanen ADAM10-Gens

Das ADAM10-Gen kodiert für eine membrangebundene Disintegrin-Metalloproteinase, die das Amyloidvorläuferprotein spaltet. Im Mausmodell konnte bewiesen werden, dass die Überexpression von ADAM10 die Plaquebildung vermindern und das Langzeitgedächtnis verbessert. Aus diesem Grund ist es für einen möglichen Therapieansatz für die Alzheimer’sche Erkrankung erforderlich, die Organisation des humanen ADAM10-Gens und seines Promotors aufzuklären. Beim Vergleich der genomischen Sequenzen von humanem und murinem ADAM10 zeigte sich eine hohe Übereinstimmung. Beide Gene umfassen 160 kbp und bestehen aus 16 Exons. Die ersten 500 bp stromaufwärts vom Translationsstartpunkt zwischen dem Menschen, der Maus und der Ratte sind hoch konserviert. Diese Region beinhaltet spezifische regulatorische Elemente, die die ADAM10-Transkription modulieren. In den ersten 2179 bp stromaufwärts vom humanen ADAM10-Translationsstartpunkt fanden sich einige potentiellen Transkriptionsfaktor-bindungsstellen (Brn-2, SREBP, Oct-1, Creb1/cJun, USF, Maz, MZF-1, NFkB und CDPCR3HD). Es wurde eine charakteristische GC-Box und eine CAAT-Box, aber keine TATA-Box identifiziert. Nach Klonierung dieser 2179 bp großen Region wurde eine starke Promotoraktivität, insbesondere in neuronalen Zelllinien, gefunden. Bei der Analyse von Deletionskonstrukten wurde die Region zwischen -508 und -300 als essentiell für die Transkriptionsaktivierung bestimmt. Die Promotoraktivität wird zudem streng herunterreguliert, wenn in die Region 317 bp stromaufwärts vom Startpunkt der Translation eine Punktmutation eingeführt wird. Diese per Computeranalyse als USF-Bindungsstelle deklarierte Region spielt eine zentrale Rolle bei der ADAM10-Transkription. Im EMSA wurde eine Protein-DNA-Interaktion für diese Region gezeigt. Durch transienten Transfektionen in Schneider Drosophila Insektenzellen konnte nachgewiesen werden, dass die Überexpression von Sp1 und USp3 für die ADAM10-Promotoraktivität entscheidend ist. In EMSA-Studien bestätigte sich eine Protein-DNA-Interaktion für die Region -366 bp stromaufwärts vom Translationsstartpunkt. Die Punktmutation in der CAAT-Box veränderte die die Promotoraktivität nicht. Da weiterhin für diese potentielle Bindungsstelle kein Bindungsfaktor vorausgesagt wurde, scheint die CAAT-Box keine Bedeutung bei der Promotorregulation zu spielen. Schließlich fand sich im EMSA eine Protein-DNA-Interaktion für die Bindungsstelle 203 bp stromaufwärts vom Translationsstartpunkt. Diese in Computeranalysen als RXR-Bindungsstelle identifizierte Region ist ebenfalls von Bedeutung in der Promotorregulation. Auf der Suche nach Substanzen, die die ADAM10-Promotoraktivität beeinflussen, wurde ein negativer Effekt durch die apoptoseauslösende Substanz Camptothecin und ein positiver Effekt durch die zelldifferenzierungsauslösende Substanz all-trans Retinsäure festgestellt. Mit dieser Arbeit wurde die genomische Organisation des ADAM10-Gens zusammen mit dem zugehörigen Promotor aufgeklärt und ein neuer Regulationsmechanismus für die Hochregulation der Expression der alpha-Sekretase ADAM10 gefunden. Im Weiteren sollen nun die genauen Mechanismen bei der Hochregulation der alpha-Sekretase ADAM10 durch Retinsäure untersucht und durch Mikroarray-Analysen an RNA-Proben transgener Mäuse, welche ADAM10 überexpremieren, neue therapeutische Ansätze zur Behandlung der Alzheimer´schen Erkrankung identifiziert werden.

Transcriptional regulation of the human Fas (CD95) promoter

The coordination of the apoptotic program necessitates the timely expression of sensor, effector, and mediator molecules. Fas/CD95, a transmembrane receptor which tethers the cell-death machinery, triggers apoptosis to maintain immune homeostasis, tolerance, and surveillance. Dysregulation in Fas-mediated apoptosis, either from disproportionate expression or disruptions in the downstream signaling pathway, manifests in autoimmune disorders and certain malignant progression. ^ In this project, the transcriptional requirements underlying two modulators of Fas expression were investigated. In T-lymphocytes, activation results in potent Fas upregulation followed by an acquisition of sensitivity towards FasL-mediated apoptosis. Human fas promoter cloning and analysis have identified a cis-element critical for inducible Fas expression. EMSA studies using this region demonstrated a constitutive association with the transcription factor Sp1 and inducible NF-κB binding in response to activation. These interactions were mutually exclusive, as the rB/Sp1 element bound with recombinant Sp1 was readily displaced by increasing amounts of NF-κB p50. Thus, Fas upregulation by T-cell activation stimuli is dependent upon NF-κB binding at the fas promoter. ^ The capacity of Sp1 to direct basal Fas expression was examined through mutagenesis of several GC-rich regions within the core fas promoter. Reporter analysis of single or combinatorial mutant GC-box constructs revealed usage of a particular GC-element in moderating over 50% of basal fas transcription. Inducible expression was Sp1-independent, however, since activated Jurkat cells containing fas Sp1-mutant constructs retained equivalent reporter induction. Overall, a dual-level of transcriptional control exists in fas, where constitutive activity is monitored through Sp1 binding, whereas T-cell activation obligates NF κB transactivation. ^ In response to genotoxic damage, p53 modulates Fas levels partly by a transcription-dependent mechanism. Reconstitution of wild-type p53 in the hepatoma cell line Hep3B readily induced Fas transcription. Furthermore, fas promoter analysis identified an undescribed p53 responsive element which, when deleted, ablated p53-mediated reporter activity. Therefore, the pro-apoptotic function mediated by p53 is driven partially through the enhancement of Fas expression. ^ Altogether, events elicting Fas transcription may invoke single or overlapping mechanisms that converge at the level of promoter activity. Agents that enhance or attenuate these pathways may be therapeutically beneficial in modulating the expression and sensitivity towards Fas-dependent apoptosis. ^

The effects of DNA cytosine methylation on H -ras promoter structure and function

The effect of DNA cytosine methylation on H-ras promoter activity was assessed using a transient expression system employing the plasmid H-rasCAT (NaeI H-ras promoter linked to the chloramphenicol acetyltransferase (CAT) gene). This 551 bp promoter is 80% GC rich, enriched with 168 CpG dinucleotides, and contains six functional GC box elements which represent major DNA methylation target sites. Prokaryotic methyltransferases HhaI (CGm$\sp5$CG) and HpaII (Cm$\sp5$CGG) alone or in combination with a human placental methyltransferase (HP MTase) were used to introduce methyl groups at different CpG sites within the promoter. To test for functional promoter activity, the methylated plasmids were introduced into CV-1 cells and CAT activity assessed 48 h post-transfection. Methylation at specific HhaI and HpaII sites reduced CAT expression by 70%, whereas more extensive methylation at generalized CpG sites with HP MTase inactivated the promoter $>$95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in nonpromoter regions. We also observed that DNA cytosine methylation of a 360 bp promoter fragment by HP MTase induced a local change in DNA conformation. Using three independent methodologies (nitrocellulose filter binding assays, gel mobility shifts, and Southwestern blots), we determined that this change in promoter conformation affected the interaction of nuclear proteins with cis-regulatory sequences residing in the promoter region. The results provide evidence to suggest that DNA methylation may regulate gene expression by inducing changes in local promoter conformation which in turn alters the interactions between DNA and protein factors required for transcription. The results provide supportive evidence for the hypothesis of Cedar and Riggs, who postulated that DNA methylation may regulate gene expression by altering the binding affinities of proteins for DNA. ^

Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene

Twenty-four base pairs of the human antioxidant response element (hARE) are required for high basal transcription of the NAD(P)H:quinone oxidoreductase1 (NQO1) gene and its induction in response to xenobiotics and antioxidants. hARE is a unique cis-element that contains one perfect and one imperfect AP1 element arranged as inverse repeats separated by 3 bp, followed by a “GC” box. We report here that Jun, Fos, Fra, and Nrf nuclear transcription factors bind to the hARE. Overexpression of cDNA derived combinations of the nuclear proteins Jun and Fos or Jun and Fra1 repressed hARE-mediated chloramphenicol acetyltransferase (CAT) gene expression in transfected human hepatoblastoma (Hep-G2) cells. Further experiments suggested that this repression was due to overexpression of c-Fos and Fra1, but not due to Jun proteins. The Jun (c-Jun, Jun-B, and Jun-D) proteins in all the possible combinations were more or less ineffective in repression or upregulation of hARE-mediated gene expression. Interestingly, overexpression of Nrf1 and Nrf2 individually in Hep-G2 and monkey kidney (COS1) cells significantly increased CAT gene expression from reporter plasmid hARE-thymidine kinase-CAT in transfected cells that were inducible by β-naphthoflavone and tert-butyl hydroquinone. These results indicated that hARE-mediated expression of the NQO1 gene and its induction by xenobiotics and antioxidants are mediated by Nrf1 and Nrf2. The hARE-mediated basal expression, however, is repressed by overexpression of c-Fos and Fra1.

The rat Na+-sulfate cotransporter rNaS2: functional characterization, tissue distribution, and gene (slc13a4) structure

Inorganic sulfate is essential for numerous functions in mammalian physiology. In the present study, we characterized the functional properties of the rat Na+-sulfate cotransporter NaS2 (rNaS2), determined its tissue distribution, and identified its gene (slc13a4) structure. Expression of rNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by phosphate, thiosulfate, tungstate, selenate, oxalate, and molybdate, but not by citrate, succinate, or DIDS. Transport kinetics of rNaS2 determined a K-M for sulfate of 1.26 mM. Na+ kinetics determined a Hill coefficient of n=3.0 +/- 0.7, suggesting a Na+:SO42- stoichiometry of 3:1. rNaS2 mRNA was highly expressed in placenta, with lower levels found in the brain and liver. slc13a4 maps to rat chromosome 4 and contains 17 exons, spanning over 46 kb in length. This gene produces two alternatively spliced transcripts, of which the transcript lacking exon 2 is the most abundant form. Its 5' flanking region contains CAAT- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, SP1, and AP-2 consensus sequences. This is the first study to characterize rNaS2 transport kinetics, define its tissue distribution, and resolve its gene (slc13a4) structure and 5' flanking region.

Functional characterization and genomic organization of the human Na+-sulfate cotransporter hNaS2 gene (SLC13A4)

Sulfate plays an essential role in human growth and development. Here, we characterized the functional properties of the human Na+-sulfate cotransporter (hNaS2), determined its tissue distribution, and identified its gene (SLC13A4) structure. Expression of hNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by thiosulfate, phosphate, molybdate. selenate and tungstate, but not by oxalate, citrate, succinate, phenol red or DIDS. Transport kinetics of hNaS2 determined a K, for sulfate of 0.38 mM, suggestive of a high affinity sulfate transporter. Na+ kinetics determined a Hill coefficient of 1.6 +/- 0.6, suggesting a Na: SO42- stoichiometry of 2:1. hNaS2 mRNA was highly expressed in placenta and testis, with intermediate levels in brain and lower levels found in the heart, thymus, and liver. The SLC13A4 gene contains 16 exons, spanning over 47 kb in length. Its 5'-flanking region contains CAAT- and GC-box motifs, and a number of putative transcription factor binding sites, including GATA-1, AP-1, and AP-2 consensus sequences. This is the first study to characterize hNaS2 transport kinetics, define its tissue distribution, and resolve its gene (SLC13A4) structure and 5' flanking region. (C) 2004 Elsevier Inc. All rights reserved.

Human KLF17 is a new member of the Sp/KLF family of transcription factors

The Sp/KLF transcription factors perform a variety of biological functions, but are related in that they bind GC-box and CACCC-box sequences in DNA via a highly conserved DNA-binding domain. A database homology search, using the zinc finger DNA-binding domain characteristic of the family, has identified human KLF17 as a new family member that is most closely related to KLFs 1-8 and 12. KLF17 appears to be the human orthologue of the previously reported mouse gene, zinc finger protein 393 (Zfp393), although it has diverged significantly. The DNA-binding domain is the most conserved region, suggesting that both the murine and the human forms recognize the same binding sites in DNA and may retain similar functions. We show that human KLF17 can bind G/C-rich sites via its zinc fingers and is able to activate transcription from CACCC-box elements. This is the first report of the DNA-binding characteristics and transactivation activity of human KLF17, which, together with the homology it displays to other KLF proteins, put it in the Sp/KLF family. (c) 2006 Elsevier Inc. All rights reserved.

Molecular cloning and characterization of the mouse Na+ sulfate cotransporter gene (Slc13a4): structure and expression

Sulfate is an essential ion required for numerous functions in mammalian physiology. Due to its hydrophilic nature, cells require sulfate transporters on their plasma membranes to allow entry of sulfate into cells. In this study, we identified a new mouse Na+-sulfate cotransporter (mNaS2), characterized its tissue distribution and determined its cDNA and gene (Slc13a4) structures. mNaS2 mRNA was expressed in placenta, brain, lung, eye, heart, testis, thymus and liver. The mouse NaS2 cDNA spans 3384 nucleotides and its open frame encodes a protein of 624 amino acids. Slc13a4 maps to mouse chromosome 6131 and contains 16 exons, spanning over 40 kb in length. Its 5'-flanking region contains CART- and GC-box motifs and a number of putative transcription factor binding sites, including GATA-1, MTF-1, STAT6 and HNF4 consensus sequences. This is the first study to define the tissue distribution of mNaS2 and resolve its cDNA and gene structures, which will allow us to investigate mNaS2 gene expression in vivo and determine its role in mammalian physiology.

Computational analysis of base composition pattern and promoter elements in the putative promoter regions in relation to expression profiles of 682 human genes on chromosome 22

The base composition pattern (BCP) in the putative promoter region (PPRs) up to 5 Kb lengths of 682 human genes on Chromosome 22 (Chr22) was examined. Two-dimensional (2D) and three-dimensional (3D) functions were designed to delineate the DNA base composition, with four major patterns identified. It is found that 17.6% genes include TATA box, 28.0% GC box, 18.9% CAAT box and 38.4% CpG islands, and approximately 10% genes have one of four putative initiator (Inr) motifs. The occurrence of the promoter elements is tightly associated with the base composition features in the promoter regions, and the associations of the base composition features with occurrence of the promoter elements in the promoter regions mediate tissue-wide expression of the genes in human. The occurrence of two or more promoter elements in the promoter regions is required for the medium- and wide-range expression profiles of the human genes on Chr22. Thus, the reported data shed light on the characteristics of the PPRs of the human genes on Chr22, which may improve our understanding of regulatory roles of the PPRs with occurrence of the promoter elements in gene expression.

Esi(+)-ms And Gc-ms Study Of The Hydrolysis Of N-azobenzyl Derivatives Of Chitosan.

New N-p-chloro-, N-p-bromo-, and N-p-nitrophenylazobenzylchitosan derivatives, as well as the corresponding azophenyl and azophenyl-p-sulfonic acids, were synthesized by coupling N-benzylvchitosan with aryl diazonium salts. The synthesized molecules were analyzed by UV-Vis, FT-IR, 1H-NMR and 15N-NMR spectroscopy. The capacity of copper chelation by these materials was studied by AAS. Chitosan and the derivatives were subjected to hydrolysis and the products were analyzed by ESI(+)-MS and GC-MS, confirming the formation of N-benzyl chitosan. Furthermore, the MS results indicate that a nucleophilic aromatic substitution (SnAr) reaction occurs under hydrolysis conditions, yielding chloroaniline from N-p-bromo-, and N-p-nitrophenylazo-benzylchitosan as well as bromoaniline from N-p-chloro-, and N-p-nitrophenylazobenzyl-chitosan.