Alternative Core Promoters Regulate Tissue-specific Transcription from the Autoimmune Diabetes-related ICA1 (ICA69) Gene Locus
2003; Elsevier BV; Volume: 278; Issue: 2 Linguagem: Inglês
10.1074/jbc.m210175200
ISSN1083-351X
AutoresRobert P. Friday, Susan L. Pietropaolo, Jennifer Profozich, Massimo Trucco, Massimo Pietropaolo,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoIslet cell autoantigen 69-kDa (ICA69), protein product of the human ICA1 gene, is one target of the immune processes defining the pathogenesis of Type 1 diabetes. We have characterized the genomic structure and functional promoters within the 5′-regulatory region of ICA1. 5′-RNA ligase-mediated rapid amplification of cDNA ends evaluation ofICA1 transcripts expressed in human islets, testis, heart, and cultured neuroblastoma cells reveals that three 5′-untranslated region exons are variably expressed from the ICA1 gene in a tissue-specific manner. Surrounding the transcription initiation sites are motifs characteristic of non-TATA, non-CAAT, GC-rich promoters, including consensus Sp1/GC boxes, an initiator element, cAMP-responsive element-binding protein (CREB) sites, and clusters of other putative transcription factor sites within a genomic CpG island. Luciferase reporter constructs demonstrate that the first two ICA1exon promoters reciprocally stimulate luciferase expression within islet- (RIN 1046-38 cells) and brain-derived (NMB7) cells in culture; the exon A promoter exhibits greater activity in islet cells, whereas the exon B promoter more efficiently activates transcription in neuronal cells. Mutation of a CREB site within the ICA1exon B promoter significantly enhances transcriptional activity in both cell lines. Our basic understanding of expression from the functional core promoter elements of ICA1 is an important advance that will not only add to our knowledge of the ICA69 autoantigen but will also facilitate a rational approach to discover the function of ICA69 and to identify relevant ICA1 promoter polymorphisms and their potential associations with disease. Islet cell autoantigen 69-kDa (ICA69), protein product of the human ICA1 gene, is one target of the immune processes defining the pathogenesis of Type 1 diabetes. We have characterized the genomic structure and functional promoters within the 5′-regulatory region of ICA1. 5′-RNA ligase-mediated rapid amplification of cDNA ends evaluation ofICA1 transcripts expressed in human islets, testis, heart, and cultured neuroblastoma cells reveals that three 5′-untranslated region exons are variably expressed from the ICA1 gene in a tissue-specific manner. Surrounding the transcription initiation sites are motifs characteristic of non-TATA, non-CAAT, GC-rich promoters, including consensus Sp1/GC boxes, an initiator element, cAMP-responsive element-binding protein (CREB) sites, and clusters of other putative transcription factor sites within a genomic CpG island. Luciferase reporter constructs demonstrate that the first two ICA1exon promoters reciprocally stimulate luciferase expression within islet- (RIN 1046-38 cells) and brain-derived (NMB7) cells in culture; the exon A promoter exhibits greater activity in islet cells, whereas the exon B promoter more efficiently activates transcription in neuronal cells. Mutation of a CREB site within the ICA1exon B promoter significantly enhances transcriptional activity in both cell lines. Our basic understanding of expression from the functional core promoter elements of ICA1 is an important advance that will not only add to our knowledge of the ICA69 autoantigen but will also facilitate a rational approach to discover the function of ICA69 and to identify relevant ICA1 promoter polymorphisms and their potential associations with disease. untranslated region rapid amplification of cDNA ends reverse transcriptase RNA ligase-mediated the California Institute of Technology BAC Library the Centre d'Etude du Polymorphisme Humain initiator cAMP-responsive element-binding protein non-obese diabetic firefly relative light unit National Center for Biotechnology Information Human Genome Project gene-specific primers transcription factor Islet cell autoantigen 69 kDa (ICA69) is identified with a group of Type 1 diabetes-related islet autoantigens considered to be specific protein targets of the diabetogenic autoimmune response. By using sera from pre-diabetic individuals, Pietropaolo and co-workers (1Pietropaolo M. Castaño L. Babu S. Buelow R. Kuo Y.-L. Martin S. Martin A. Powers A.C. Prochazka M. Naggert J. Leiter E.H. Eisenbarth G.S. J. Clin. Invest. 1993; 92: 359-371Crossref PubMed Scopus (228) Google Scholar) first identified ICA69 through immunoscreening of a human islet cDNA expression library. The 1785-bp nucleotide sequence of the full-length clone and its deduced 483-amino acid protein coding region demonstrated no overt homology to known molecules at the time of its discovery, and nucleic acid and protein analyses revealed that the molecule is primarily expressed in neuroendocrine tissues (1Pietropaolo M. Castaño L. Babu S. Buelow R. Kuo Y.-L. Martin S. Martin A. Powers A.C. Prochazka M. Naggert J. Leiter E.H. Eisenbarth G.S. J. Clin. Invest. 1993; 92: 359-371Crossref PubMed Scopus (228) Google Scholar, 2Karges W. Pietropaolo M. Ackerley C. Dosch H.-M. Diabetes. 1996; 45: 513-521Crossref PubMed Scopus (35) Google Scholar). More recent subcellular fractionation studies of murine brain tissue have shown that the majority of ICA69 protein is cytosolic and soluble, although a subfraction appears to be membrane-bound and associates with synaptic-like microvesicles (3Pilon M. Peng X.R. Spence A.M. Plasterk R.H. Dosch H.-M. Mol. Biol. Cell. 2000; 11: 3277-3288Crossref PubMed Scopus (40) Google Scholar). Although of unknown significance, smaller isoforms of the protein may be expressed from at least three human transcript variants (1Pietropaolo M. Castaño L. Babu S. Buelow R. Kuo Y.-L. Martin S. Martin A. Powers A.C. Prochazka M. Naggert J. Leiter E.H. Eisenbarth G.S. J. Clin. Invest. 1993; 92: 359-371Crossref PubMed Scopus (228) Google Scholar, 4Gaedigk R. Karges W. Hui M.F. Scherer S.W. Dosch H.-M. Genomics. 1996; 38: 382-391Crossref PubMed Scopus (20) Google Scholar, 5Karges W. Gaedigk R. Hui M.F. Cheung R.K. Dosch H.-M. Biochim. Biophys. Acta. 1996; 1360: 97-101Crossref Scopus (16) Google Scholar, 6Miyazaki I. Gaedigk R. Hui M.F. Cheung R.K. Morkowski J. Rajotte R.V. Dosch H.-M. Biochim. Biophys. Acta. 1994; 1227: 101-104Crossref PubMed Scopus (29) Google Scholar), representing truncated cDNAs that arise from alternative splicing of coding region exons (4Gaedigk R. Karges W. Hui M.F. Scherer S.W. Dosch H.-M. Genomics. 1996; 38: 382-391Crossref PubMed Scopus (20) Google Scholar). ICA69 is encoded on human chromosome 7p22 by the ICA1 gene (7Gaedigk R. Duncan A.M.V. Miyazaki I. Robinson B.H. Dosch H.-M. Cytogenet. Cell Genet. 1994; 66: 274-276Crossref PubMed Scopus (16) Google Scholar), which is composed of 14 coding exons and three 5′-untranslated region (UTR)1 sequences, each of which splices with exon 1 in a mutually exclusive manner (4Gaedigk R. Karges W. Hui M.F. Scherer S.W. Dosch H.-M. Genomics. 1996; 38: 382-391Crossref PubMed Scopus (20) Google Scholar). In addition, multiple cDNA coding region splice variants from human, mouse, and rat have been identified within islet and brain bacteriophage λ cDNA libraries by immunoscreening with human serum or by DNA probe hybridization (1Pietropaolo M. Castaño L. Babu S. Buelow R. Kuo Y.-L. Martin S. Martin A. Powers A.C. Prochazka M. Naggert J. Leiter E.H. Eisenbarth G.S. J. Clin. Invest. 1993; 92: 359-371Crossref PubMed Scopus (228) Google Scholar, 4Gaedigk R. Karges W. Hui M.F. Scherer S.W. Dosch H.-M. Genomics. 1996; 38: 382-391Crossref PubMed Scopus (20) Google Scholar, 5Karges W. Gaedigk R. Hui M.F. Cheung R.K. Dosch H.-M. Biochim. Biophys. Acta. 1996; 1360: 97-101Crossref Scopus (16) Google Scholar, 6Miyazaki I. Gaedigk R. Hui M.F. Cheung R.K. Morkowski J. Rajotte R.V. Dosch H.-M. Biochim. Biophys. Acta. 1994; 1227: 101-104Crossref PubMed Scopus (29) Google Scholar). Intron-exon boundaries were established for the human and murine ICA1 genes using a combination of λ phage genomic DNA library screening and PCR experiments (4Gaedigk R. Karges W. Hui M.F. Scherer S.W. Dosch H.-M. Genomics. 1996; 38: 382-391Crossref PubMed Scopus (20) Google Scholar). Collectively, these data argue for a high level of evolutionary conservation of the ICA1 gene, not only upon comparison of the human protein to the rat (6Miyazaki I. Gaedigk R. Hui M.F. Cheung R.K. Morkowski J. Rajotte R.V. Dosch H.-M. Biochim. Biophys. Acta. 1994; 1227: 101-104Crossref PubMed Scopus (29) Google Scholar) and mouse (5Karges W. Gaedigk R. Hui M.F. Cheung R.K. Dosch H.-M. Biochim. Biophys. Acta. 1996; 1360: 97-101Crossref Scopus (16) Google Scholar) homologues but also in terms of exon/intron partitioning (4Gaedigk R. Karges W. Hui M.F. Scherer S.W. Dosch H.-M. Genomics. 1996; 38: 382-391Crossref PubMed Scopus (20) Google Scholar). Recently a protein from the nematode Caenorhabditis elegans, termed ric-19, was reported to exhibit amino acid homology with ICA69 (3Pilon M. Peng X.R. Spence A.M. Plasterk R.H. Dosch H.-M. Mol. Biol. Cell. 2000; 11: 3277-3288Crossref PubMed Scopus (40) Google Scholar). Based on functional studies of ric-19 in C. elegans, these authors have proposed that ICA69/ric-19 participates in the process of neuroendocrine secretion through an association with secretory vesicles. These data suggest that ICA69 may be involved in the insulin secretory pathway in islet β cells, as the molecule is known to be specifically expressed within islets (8Stassi G. Schloot N. Pietropaolo M. Diabetologia. 1997; 40: 120-122PubMed Google Scholar) and by insulin-producing cell lines maintained in culture (2Karges W. Pietropaolo M. Ackerley C. Dosch H.-M. Diabetes. 1996; 45: 513-521Crossref PubMed Scopus (35) Google Scholar). However, the true cellular function of ICA69 and its importance to normal mammalian pancreatic islet physiology remain unknown. Most basic and clinical research investigations concerning ICA69 have focused on the importance of the molecule as an autoimmune target in Type 1 diabetes. Three lines of evidence from at least four independent groups substantiate a role for ICA69 autoimmunity in diabetes. 1) First degree relatives of diabetic patients who developed the disease during follow-up have detectable serum levels of ICA69 autoantibodies (1Pietropaolo M. Castaño L. Babu S. Buelow R. Kuo Y.-L. Martin S. Martin A. Powers A.C. Prochazka M. Naggert J. Leiter E.H. Eisenbarth G.S. J. Clin. Invest. 1993; 92: 359-371Crossref PubMed Scopus (228) Google Scholar, 9Martin S. Kardorf J. Schulte B. Lampeter E.F. Gries F.A. Melchers I. Wagner R. Bertrams J. Roep B.O. Pfutzner A. Pietropaolo M. Kolb H. Diabetologia. 1995; 38: 351-355Crossref PubMed Scopus (1) Google Scholar). 2) T-cells isolated from newly diagnosed diabetic patients and from non-obese diabetic (NOD) mice demonstrate reactivity against the recombinant ICA69 molecule (10Roep B.O. Diabetes. 1996; 45: 1147-1156Crossref PubMed Google Scholar, 11Roep B.O. Duinkerken G. Schreuder G.M. Th. Kolb H. DeVries R.R.P. Martin S. Eur. J. Immunol. 1996; 26: 1285-1289Crossref PubMed Scopus (68) Google Scholar, 12Miyazaki I. Cheung R.K. Gaedigk R. Hui M.F. Van der Meulen J. Rajotte R.V. Dosch H.-M. J. Immunol. 1995; 154: 1461-1469PubMed Google Scholar). 3) T-cells specific for the ICA69 peptide Tep-69 play a driving role in the acceleration of islet cell destruction in the NOD mouse model of Type 1 diabetes (13Winer S. Gunaratnam L. Astsatourov I. Cheung R.K. Kubiak V. Karges W. Hammond-McKibben D. Gaedigk R. Graziano D. Trucco M. Becker D.J. Dosch H.-M. J. Immunol. 2000; 165: 4086-4094Crossref PubMed Scopus (23) Google Scholar), whereas intraperitoneal injection of Tep-69 is associated with apparent immune toleration and decreased diabetes incidence in NOD mice (14Karges W. Hammond-McKibben D. Gaedigk R. Shibuya N. Cheung R. Dosch H.M. Diabetes. 1997; 46: 1548-1556Crossref PubMed Scopus (35) Google Scholar). It has been reported that a majority of patients with recent onset Type 1 diabetes shows evidence of autoreactive T-cells and/or autoantibodies with immune specificity for the ICA69 molecule (11Roep B.O. Duinkerken G. Schreuder G.M. Th. Kolb H. DeVries R.R.P. Martin S. Eur. J. Immunol. 1996; 26: 1285-1289Crossref PubMed Scopus (68) Google Scholar), but it must also be acknowledged that some investigators have questioned the significance of ICA69 autoantibodies based on their own studies (15Lampasona V. Ferrari M. Bosi E. Pastore M.R. Bingley P.J. Bonifacio E. J. Autoimmun. 1994; 7: 665-674Crossref PubMed Scopus (37) Google Scholar). Motivated by an interest in understanding how autoantigen expression in key body tissues relates to autoimmunity and as a prerequisite to searching for functional polymorphisms in the promoter region of the gene encoding ICA69, we have defined the basic structure and functional characteristics of the ICA1 promoter. Sequences adjacent to the multiple ICA1 transcription initiation sites contain motifs typical of a non-TATA, non-CAAT, GC-rich regulatory region, including consensus Sp1/GC box sites, Inr (initiator) elements, and CREB sites. The major alternative transcription initiation sites associate with independent 5′-UTR exons, and a detailed analysis ofICA1 transcripts from different tissues provides evidence for a tissue-specific utilization of the distinct initiation sites consistent with the observed 5′-UTR heterogeneity of mature protein-coding ICA69 transcripts. In vitro luciferase reporter gene assays of promoter function correlate with the observed preferential transcription initiation site usage within different tissues, whereas site-directed mutagenesis of promoter reporter constructs demonstrate the importance of an Sp1/GC box site and a CREB site to the regulation of expression from exons A and B, respectively. The significance and potential implications of the ICA1promoter structure and function are discussed in the context of understanding ICA69 biology and its role as a Type 1 diabetes autoantigen. Two adherent cell lines were maintained in culture in order to provide RNA for transcript analysis and for testing promoter activity of cloned ICA1 5′-flanking sequences in a firefly luciferase reporter assay. The human neuroblastoma cell line NMB7 was grown in RPMI containing 10% fetal bovine serum, supplemented with l-glutamine and penicillin/streptomycin. Rat insulinoma (RIN 1046-38) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum,l-glutamine, and penicillin/streptomycin. Cells were incubated at 37 °C in a 5% CO2 atmosphere. Passage of cells was conducted at 70–80% confluence as necessary. All cell culture reagents were obtained from Invitrogen and lot-certified in-house. NMB7 cells were provided by Dr. Ira Bergman and Judi Griffin (Children's Hospital of Pittsburgh, Pittsburgh). Human total genomic DNA was purified from whole blood using the Qiagen Genomic DNA Extraction Kit (Qiagen, Inc., Valencia, CA). Briefly, 10 ml of heparinized blood was successively subjected to cellular lysis, nuclear isolation, nuclear lysis, and anion-exchange chromatography using the buffers and prepared columns supplied with the Qiagen kit. Typical yields ranged from 150 to 350 μg of total genomic DNA per 10 ml of whole blood. Bacterial plasmid DNA was isolated by one of two methods, depending upon the quantity and concentration desired. For DNA sequencing and restriction enzyme analysis of subcloned DNA, the QIAprep Spin Miniprep Kit (Qiagen) was used to isolate 10–20 μg of plasmid DNA from 3 to 5 ml of an overnight bacterial culture. Alternatively, plasmid DNA used for transfection of cultured cells was prepared from 100 to 200 ml of overnight bacterial culture using the Qiagen Maxiprep DNA Isolation Kit. Typical DNA yields ranged from 150 to 450 μg. For BAC clone DNA, a Qiagen Maxiprep protocol modified for use with BACs was followed (protocol available from manufacturer). Major modifications to the basic protocol included the use of a larger culture volume (500 ml) and elution of BAC clone DNA from the column with buffer warmed to 50 °C. Yields of BAC clone DNA ranged from 100 to 150 μg. YAC clone DNA was co-purified along with yeast genomic DNA according to a standard protocol (16Chaplin D.D. Brownstein B.H. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York1992Google Scholar) with some modifications. Briefly, following 2000 × g centrifugation of 100 ml of fresh yeast cell culture at room temperature for 10 min, the cell pellet was resuspended in SCE buffer (0.9 m sorbitol, 0.1 m sodium citrate, 0.06 m EDTA) with freshly added 0.3 mβ-mercaptoethanol and lyticase enzyme (Sigma). Formation of yeast spheroplasts was allowed to proceed for 1–2 h at 37 °C with gentle shaking. Spheroplasted cells were then pelleted by centrifugation at 1000 × g for 10 min. Yeast cell lysis was achieved by suspension of spheroplasts in lysis buffer (0.5 m Tris-Cl, pH 8.0, 3% N-lauroylsarcosine, 0.2 m EDTA, 1 mg/ml proteinase K) buffer and incubation for 30–45 min at 65 °C in a water bath. Overnight treatment of cell lysate with RNase PLUS (5 Prime → 3 Prime, Inc., Boulder, CO) at 37 °C adequately removed contaminating yeast RNA from the sample. Isolation of the DNA fraction was achieved through two successive organic extractions with an equal volume of 50:50 phenol/chloroform, followed by 3–4 extractions of the aqueous phase with chloroform only. YAC and yeast DNA were co-precipitated from the aqueous phase with 0.1 volumes of 3m NaCl and 2.5 volumes of ice-cold 100% ethanol. After gentle spooling, the precipitated DNA was washed in 70% ethanol and air-dried. Purified DNA was resuspended in TE buffer, pH 8.0, with a typical preparation yielding 300–700 mg of nucleic acid as measured byA 260 measurement. Due to the nature of the sequences being amplified, the PCR technology used in an experiment was adapted to each DNA target and template. Reaction components were used in amounts and concentrations as recommended by the manufacturer unless otherwise noted. Suggested annealing temperatures for each PCR kit were adjusted based on the sequence identity of the amplification primers and the sequence composition of the amplification target with the assistance of Oligo 4.0 (Molecular Biology Insights, Inc., Cascade, CO) primer design software. Reactions were cycled 30–35 times unless otherwise specified. For PCR amplification of simple target DNAs (i.e. 5 kb), or when other PCR methods failed, the eLONGase long PCR enzyme mix (Invitrogen) was employed. Oligonucleotides were synthesized in the DNA Sequencing and Synthesis Core Facilities of the Diabetes Institute, Children's Hospital of Pittsburgh. PCR primers designed from theICA1 exon 2 intron-exon boundary sequences were used to screen the Centre d'Etude du Polymorphisme Humain (CEPH) Mega-YAC Human DNA Library by systematic amplification of YAC clone DNA pools (primer sequences: 5′-CCTGGGACTTACAGGATCGA-3′ and 5′-GACAGCAATAAAGAGCTCAC-3′, annealing temperature 55 °C, 178-bp PCR product). The California Institute of Technology BAC Library (CITB Release IV, Research Genetics, Inc., Pasadena, CA) was similarly screened using a PCR approach. The PCR amplimer used for BAC library screening was a microsatellite (CA repeat) centered 1830 bp upstream of the ICA1 translation initiation codon (primer sequences: 5′-TATGAAACAGTGTTATTCTGGACCT-3′ and 5′-GTACAGTATAGTAGTGCTAACA-3′, annealing temperature 55 °C, 540-bp PCR product). Stab vials or frozen aliquots of each PCR-positive YAC and BAC library clone identified through screening were obtained, and purified DNA extracted from their respective cultures was retested under PCR conditions similar to those used for library screening to verify that the targetICA1 sequences were present. If necessary, amplified products from GenomeWalker-PCR, RT-PCR, and 5′-RACE experiments were gel-purified using the Qiagen Gel Extraction Kit (Qiagen) before subcloning, or they were subcloned by direct ligation of an aliquot of the PCR. The gel-purified or neat PCRs were subcloned into the pCR 2.1 vector (Original TA Cloning Kit, Invitrogen). When PCR primers were designed to include restriction sites, they were digested with the appropriate restriction enzyme(s) and ligated into an overhang-compatible aliquot of the pGL3 basic luciferase reporter vector. All ligation reac-competent Escherichia coli(Invitrogen) and plated on 100-mm LB agar plates containing 50 μg/ml ampicillin or kanamycin and 50 μg/ml 5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside (X-gal) for blue-white color selection of transformants (if applicable). In situations where T/A PCR product ligation proved to be inefficient because of 3′→ 5′-exonuclease activity from the PCR enzyme or enzyme mix used for amplification, the PCR product(s) were “tailed” with dA-overhangs prior to ligation. Briefly, 1 unit of AmpliTaqDNA polymerase was added to the completely cycled PCRs, incubated for 15 min at 37 °C, and immediately extracted with an equal volume of phenol/chloroform. The tailed products were then ethanol-precipitated, resuspended in a small volume (∼½ of original reaction volume), and directly used in the T/A ligation step. The FirstChoice RLM-RACE Kit (Ambion, Austin, TX) was used for RNA ligase-mediated (RLM) RACE analysis of ICA1 transcripts, because it permits selective amplification of capped RNA molecules from non-poly(A)-selected RNA. Briefly, total cellular RNA is treated with calf intestinal alkaline phosphatase to remove the 5′-phosphate group from uncapped mRNA precursors, tRNA, rRNA, and small nuclear RNA molecules, followed by phenol/chloroform extraction and recovery of the dephosphorylated RNA by ethanol precipitation. Dephosphorylated RNA is then incubated with tobacco acid pyrophosphatase to remove m7Gpp from the cap structure of the 5′ end of capped RNAs, leaving a single 5′-terminal phosphate group. Ligation of a synthetic RNA adapter of known sequence to the calf intestinal alkaline phosphatase- and tobacco acid pyrophosphatase-treated RNA proceeds in the presence of E. coli RNA ligase. Adapter-ligated RNA is reverse-transcribed into cDNA using Moloney murine leukemia virus-reverse transcriptase enzyme in the presence of random decamer primers. The resultant single-stranded cDNA then serves as template in nested PCRs using adapter sequence-specific primers (provided with the RLM-RACE kit) and gene-specific primers (GSP1 and GSP2) designed from ICA1 exons 1 and 2. The sequences of these latter primers are as follows: GSP1 (antisense exon 2), 5′-TGCATCTTATTTACAACTGACTTATCTTGA G-3′ and GSP2 (antisense exon 1/2 boundary), 5′-TGTAAGTCCCAGGGATAACTGCATTTGTGT CCTGA-3′. The Advantage GC cDNA enzyme was used in all nested RLM-RACE PCRs. To clone segments of the ICA1 5′-flanking region and UTR exons, a 1028-bp genomic segment spanning the entire region was amplified from CITB-503D2 DNA via PCR, followed by T/A ligation of the product into the pCR2.1 vector (amplification primers, 5′-TAGGAAGCAGCTATGCCAACACT-3′ and 5′-CAGAGAAGGCAGCTCCTACCA-3′). Excision of various segments of the cloned PCR product using pairs of restriction endonucleases recognizing sites found in the pCR2.1 vector arms, internal restriction sites of the insert, or a combination of the two allowed for directional cloning of defined ICA1 sequences into a pGL3 Basic vector having compatible overhangs. A second strategy for cloning ICA1sequences into pGL3 Basic involved the design ofICA1-specific primers with restriction endonuclease sites added at the 5′ end. After spin column chromatography purification of a PCR product amplified with these primers, the product was digested with one or more restriction enzymes to create overhangs compatible with those generated on an aliquot of the pGL3 Basic vector. Heat inactivation and gel purification or spin column purification of the digested PCR product was then followed by ligation into pGL3 Basic. Two pGL3 promoter reporter constructs, ExA −957 and ExB −440, were modified using the QuikChange Site-directed Mutagenesis Kit (Stratagene, Cedar Creek, TX) to introduce mutations at suspected key sites within these promoters. Sequence mutations were introduced to the Sp1/GC box, Inr, (Sp1/GC box + Inr), and CREB sites within ExA −957 using the following oligonucleotides, respectively (mutant name and bases mutated are in boldface and in parentheses): 5′-CCTGCCGGAGAGCAGGGtattGGTCACTCTGGGCGGCG (ExA-GC, −564 to −561), 5′-CGGAGAGCAGGGGCGGGGTggaggTGGGCGGCGGATCCG (ExA-Inr, −557 to −553), 5′-CCTGCCGGAGAGCAGGGtattGGTggaggTGGGCGGCGGATCCGAGC (ExA-GC/Inr, mutations of −564 to −561 and −557 to −553), and 5′-CCTGTCCGCCAGGTCATcggcACGCAAACGCTATGGCCACGTGG (ExA-CREB, −612 to −609). For the ExB −440 construct, the Sp1/GC box and CREB sites were modified with the following oligonucleotides, respectively: 5′-CCGGTTCCTGCGCTCCCCaataCCCTTTCCCTCGCCTTCG (ExB-GC, −196 to −193) and 5′-CCCTTTCCCTCGCCTTCGatccACGCTGACGTCGGATGAGTG (ExB-CREB, −174 to −171). The mutation strategy of the QuikChange protocol was adhered to for all site-directed mutation reactions, using the above oligonucleotides in combination with a reverse complement sequence primer in each PCR-based mutagenesis reaction. After digestion of the reactions with DpnI to remove non-mutated, methylated DNA, each mutated plasmid reaction was used to transform XL1-Blue supercompetent cells. Resultant colonies were then miniprepped and screened via automated fluorescent sequencing for successful mutation incorporation. For luciferase transfection experiments, NMB7 and RIN 1046-38 cells were plated at a density of 0.8 × 105 cells/well of a 12-well plate the day before transfection. Growth in the appropriate complete medium for 20–24 h generally resulted in 50–70% cellular confluence in each well at the time of transfection. Transient transfection of luciferase constructs and mutants thereof into the various cultured cell lines using Effectene Transfection Reagent (Qiagen) was followed by incubation of the transfected cells at 37 °C and cellular lysis 35–45 h after transfection. Luminescence assays of cellular lysates allowed for a semi-quantitative measure of luciferase production driven by each cloned segment of the ICA1 5′-flanking region. Within a given assay, plate wells were set up in triplicate for each transfected construct or control vector. The amount of DNA transfected was held constant for each construct and cell line, with a total amount 0.3 μg/well of a 12-well plate. Each Effectene reagent was used in the amount recommended by the manufacturer's protocol in proportion to the amount of DNA applied to each well. The strength of the promoting activity for each construct was assessed by comparison to basal luciferase expression from the promoterless pGL3 Basic vector transfected into triplicate samples of the same cell type within the same assay. To allow for normalization of firefly luciferase values based on transfection efficiency, a co-reporter vector expressingRenilla luciferase from the thymidine kinase promoter (pRL-TK) was included at a ratio of 1:10 of co-reporter plasmid to experimental promoter construct (or control vector) in the transfection mixture. Careful optimization of transfection conditions to maximize transfection efficiency provided an assay system yielding consistent results from repeated experiments. Transfected cells were lysed by adding 100 μl of Passive Lysis Buffer (Promega, Madison, WI) to each well of a 12-well plate, followed by vigorous pipetting of the detached cells. Cell lysates were subjected to two freeze-thaw cycles (liquid N2 and 20 °C H2O) and either immediately assayed for luciferase activity or stored at −70 °C for analysis the following day. Firefly andRenilla luciferase activities of each lysate were measured sequentially via manual reagent injection in a Monolight 2010 luminometer using the Dual-Luciferase Reporter Assay System (Promega). In order to compare inter-construct firefly (FF) luciferase activity values, the raw data relative light unit (RLU) readings were corrected by normalizing each sample according to transfection efficiency. OneRenilla luciferase RLU (R-RLU) measurement from the pGL3 Basic control transfectant samples in a given experiment was selected and used to normalize each measured FF luciferase value as follows: ((normalizing R-RLU) ÷ (sample R-RLU)) × (sample FF-RLU) = (Nml sample FF activity). The normalized (Nml) triplicate values for each construct were then averaged to arrive at a relative measure of luciferase activity for that ICA1promoter reporter construct. Fold increases in promoter activity over the pGL3 Basic vector were calculated from the following formula: (Avg Nml sample FF activity) ÷ (Avg Nml pGL3 Basic control) = (sample fold increased activity over pGL3 Basic); where Avg is average. These calculations were performed independently for each transfection experiment data set (n = 3–5), and the average of all results obtained for a given ICA1 promoter reporter construct was used as a measure of relative promoter strength. Where indicated, statistical analysis of luciferase reporter data was performed using the Mann-Whitney U test. All oligonucleotides used as primers in the various PCR-based methods were synthesized on an ABI 394 DNA Synthesizer (Applied Biosystems, Inc.) using solid phase synthesis and phosphoramidite nucleoside chemistry, unless a primer was provided with a particular molecular biology kit. Automated fluorescent sequencing of plasmid DNA or p
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