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Characterization of promoter of a novel GTPase in T cell, TGTP Chang, Shiu Yuan (Martin) 2000

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CHARACTERIZATION OF PROMOTER OF A N O V E L GTPase IN T C E L L , TGTP By SHIU Y U A N (MARTIN) C H A N G B. Sc., McGILL University, 1995 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE F A C U L T Y OF G R A D U A T E STUDIES DEPARTMENT OF MICROBIOLOGY A N D I M M U N O L O G Y We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A August 2000 © Shiu Yuan (Martin) Chang In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Microbiology and Immunology The University of British Columbia Vancouver, Canada Date A u p „ g t i f 1 j 7000 DE-6 (2/88) 11 Abstract Our laboratory have previously described the cloning of a cDNA encoding a 50 kDa GTPase designated TGTP. TGTP s expression was induced rapidly after TCR cross-linking. However, subsequent studies showed that the induction was nevertheless mediated by IFN-y and that TGTP is inducible by IFN-y in cell lines of diverse lineages. More interestingly, studies in our laboratory showed that TGTP-transfected L cells displayed resistance to infection by the negative strand RNA virus, vesicular stomatitis virus. The main objective of my thesis project was to isolate, characterize and study the promoter of Tgtp, with the hope that this information may further elucidate the function of TGTP. The methologies employed in this study included: (1) identifying the genomic clones that contain the 5 end of the cDNA encoding TGTP; (2) mapping restriction sites and localizing the 5 end of cDNA in a genomic clone; (3) determining the orientation of the genomic DNA and, hence, the location of the promoter region; (4) assessing the promoter activity of a genomic DNA fragment that may carry the promoter region; (5) determining the sequence of the promoter region; and (6) analyzing the sequence of the promoter region to determine and locate different D N A motifs such as the GAS motif that is required for IFN-y induction. In this study I showed that the promoter of Tgtp contains a T A T A box, a GAS sequence (TTCCAGGAA), a Myb binding site, two CF-2 binding sites, three c-Myc/Max binding sites and a SP-1 binding site. In vitro functional assay of the Tgtp promoter with a luciferase reporter gene showed that the Tgtp promoter is optimally activated by low concentrations (5.5 U/ml) of IFN-y and, to a lesser extent, by high concentrations (5,500 U/ml) of IFN-a. A unique sequence of eleven repeats of T C T C C C C was also found in the Tgtp promoter. However, the potential role of these repeats in transcriptional regulation of TGTP remains to be determined. These repeats of high GC content (71%) also presented a special challenge to my sequencing efforts. The sequencing difficulty was finally resolved by using PCR templates synthesized in presence of 75% or 100% 7-deaza dGTP. iii T A B L E OF CONTENTS Abstract '. i i List of Tables iv List of Figures v Acknowledgement vii Introduction 1 Materials and Methods 4 Results 8 Discussion 16 References 21 LIST OF TABLES Table 1: Summary of D N A motifs present in the Tgtp promoter Table 2: D N A motifs or transcription complex-binding sites not found in the Tgtp promoter V LIST OF FIGURES Figure 1: A simplified restriction map of Tgtp cDNA 24 Figure 2: Size of the genomic D N A in the lambda phage clone 26 Figure 3: Restriction map and direction of the genomic D N A in the Sr 4-3 lambda phage clone 27 Figure 4: Orientation of the 1.7 kb Bam HI fragment in bluescript plasmid 28 Figure 5: Orientation of the 1.7 kb Bam HJ fragment 30 Figure 6: Graphic representation of the recombinant constructs 31 Figure 7: Preparation of the promoterless pGL-3 luciferase vector 32 Figure 8: Absence of contamination by pGL-3 vector (+SV40 promoter) in D N A sample of promoterless pGL-3 vector 33 Figure 9: Direction of the Tgtp promoter in the recombinant Tgtp promoter-luciferase reporter gene construct 34 Figure 10: Functional assay of the Tgtp promoter 35 Figure 11: Optimum induction of Tgtp promoter by titrated amount of IFN-y 36 Figure 12: Optimum induction of Tgtp promoter by titrated amount of IFN-ot 37 Figure 13: Time course of Tgtp promoter induction by IFN-y 38 Figure 14: Lack of Tgtp promoter induction by mouse IL-6 39 Figure 15: Sequence analysis of the Tgtp promoter 40 Figure 16: Locations of transcription-conmlex binding sites and D N A elements in the 1.5 kb Tgtp promoter region 41 Figure 17: Restriction map of the Tgtp promoter 42 Figure 18: Summary of sequencing strategies Figure 19: The amount of 100% vs 75% 7-deaza dGTP PCR products A C K N O W L E D G E M E N T Douglas Carlow kindly provided the Tgtp cDNA and the restriction map of the Tgtp cDNA. Dr. Nigel Killeen (UCSF) kindly provided the CD2 vector, pCDmCD2.1.1 am grateful to Christopher Ong for teaching me some essential techniques and for his assistance with the isolation of the Tgtp promoter. Soo-Jeet Teh, Douglas Carlow and Christopher Ong have provided very helpful and stimulating discussions. I also thank my thesis supervisor, Dr. Hung-Sia Teh, for his patience and understanding in the long time that it took to complete this thesis and the support that he has provided for this project. 1 Introduction Our laboratory has previously described the cloning of a cDNA encoding a 50 kDa GTPase designated TGTP (1). TGTP's expression was induced rapidly after TCR cross-linking and appeared to be restricted to T cells (1). However, the same cDNA was also cloned by Lafuse et al (2) from a library derived from IFN-y-treated macrophages. These authors designated their clone Mg21 and demonstrated that it was an immediate-early (IE) gene of IFN-y in macrophages. Our laboratory subsequently found that despite the rapid kinetics of TGTP RNA induction in T cells following TCR-linking, the induction of TGTP is nevertheless mediated by IFN-y. IFN-y is one of the most rapidly induced IE genes induced upon TCR stimulation and, as in macrophages, TGTP is an IE gene of IFN-y in T cells (3). TGTP/Mg21 had one relative in the sequence databases designated IRG-47 (4). IRG-47 was described as an IFN-y-inducible gene encoding a protein with a putative GTP-binding domain. IRG-47 is expressed predominantly in B cell lines and fibroblasts (4). Two other IFN-inducible relatives have been described in macrophages, including LRG-47 and IGTP. LRG-47 was shown to be induced by LPS and both type I IFN-a(3 and type II IFN-y (5). IGTP was induced by LPS and IFN-y and its GTPase activity was confirmed in vitro (6). Expression of TGTP/Mg21in macrophages and B cell lines was not induced by IL-2, 4, 10 and TNF-a (2). IL-1, -2, -4, -6, TNF-a , and G M -CSF was also ineffective in inducing LRG-47 (5). These observations reinforce the view that these genes function in response to IFN-y and therefore may contribute to the innate response to bacterial and/or viral infections. The importance of IFNs in host defense against viral and bacterial pathogens was demonstrated by the increased susceptibility to viral and bacterial challenge in mice lacking the capacity to respond to type I (a and |3) (7) and type II (y) (8, 9) IFNs, respectively. IFN-y is known to influence the expression of over 240 genes that fall into approximately 37 functional categories (10). It has been suggested that this extensive 2 response to IFN-y reflects the induction of four major genetic programs that collectively regulate immunity and promote the elimination of infectious agents (8-10). These programs include the regulation of cytokine secretion governing T and B cell differentiation, activation of phagocytic processes in macrophages and neutrophils, enhancement of antigen presentation, and direct anti-viral activity (10). IFN-y is also essential for increasing the activity of natural killer (NK) cells, macrophages and T cells; increasing M H C class I, class II and peptide transporter molecule expression of virally infected cells; and for increasing MHC-linked proteosome expression in macrophages (11). Similarly, IFN-a has similar antiviral effects but does not increase surface M H C class II expression. IFN-a inhibits viral replication by inhibiting D N A replication and protein synthesis through activation of endoribonucleases that degrade viral RNA (11). Since TGTP is inducible by both type I and type IIIFN, the importance of this gene in host defense can not be overlooked. In fact, a recent report from our lab showed that forced expression of TGTP conferred an antiviral state for the negative-stranded RNA virus, vesicular stomatitis virus, but not to the D N A virus, herpes simplex virus (3). In this thesis I present the sequence analysis of the Tgtp promoter, the technical difficulties associated with the extensive secondary structure of this promoter, and the solution used to resolve this challenge. The Tgtp promoter was isolated by firstly screening a genomic library with a primer derived from the Tgtp cDNA. The genomic DNA containing the promoter region was then subcloned into a plasmid. A restriction map of the promoter region was determined and eventually sequenced to determine and locate putative binding sites of various transcription factors. It was found that the promoter of Tgtp contains a T A T A box, a GAS sequence (TTCCAGGAA) responsible for IFN-y induction, a Myb binding site, two CF-1 binding sites, three c-Myk/Max binding sites and a SP-1 binding site. The Tgtp promoter also contains eleven repeats of TCTCCCC. Surprisingly, the ISRE consensus sequence that is usually required for IFN-a induction is not found in the Tgtp promoter. 3 The function of the isolated Tgtp promoter was determined by assessing its ability to activate a luciferase reporter-gene construct. I showed that the isolated Tgtp promoter can be optimally activated by low concentrations (5.5 U/ml) of IFN-y and, to a lesser extent, by high concentrations (5500 U/ml) of IFN-a. 3 4 Materials and Methods Genomic library screening. In the primary and secondary genomic library screenings, lambda Fix II phage clones derived from 129 SvJ mouse genomic D N A were lifted from 2XYT plates with nitrocellulose membranes, denatured in 0.5 M NaOH solution followed by 1.5 M NaCl, washed with 3x SSC, dried, wiped to remove bacterial debris, washed in 2X SSC and dried again. The membranes were then hybridized overnight with a radioactive probe, an Eco RI/Hind III fragment (-400 bp) of cDNA generated by PCR from gene specific primers that were designed based on the TGTP cDNA sequence, at 45°C. Each membrane was then washed three times with 0.1X SSC, a 0.1% SDS blot wash at 50°C, and dried at 80°C for 2 hours. Finally, each membrane was exposed overnight to autoradiographic X-ray film. The same procedure was carried out for identifying clones containing the 5' flanking region of the TGTP cDNA, with the exception that a radioactive 70-mer probe corresponding to the 5' end of the 5' untranslated region of TGTP cDNA was used. The 70-mer probe was made by T4 D N A polymerase-extension of two 40-mer oligonucleotides that share 10 complementary bases (Fig. IB). One of the 40-mer oligonucleotides corresponds to nucleotides #1 to #40 of TGTP cDNA, while the other corresponds to nucleotides #70 to #30 of the complementary chain (Fig. IB, underlined). Subcloning of genomic DNA from a phage clone into bluescript plasmid. The Sr4-3 lambda phage clone was amplified in 2XYT plates, collected in SM buffer and spun. The supernatant was treated with RNase and DNase I (1 U/ml, 37°C, 30 minutes), bacteriophages were precipitated in a SM solution containing 20% (w/v) PEG and 2M NaCl at 10,000 g for 20 minutes at 4C, and washed once with SM. Phages were disrupted with 10% SDS (0.5% final concentration) and 0.5M E D T A (pH 8, 20mM final concentration) for 15 minutes at 68°C, followed by phenol chloroform extraction twice, 4 5 and DNA was precipitated with two volumes of 95% ethanol with 0.1 % NaCl. The lambda phage D N A was then digested with Not I restriction enzyme for 3 hours, and fragments separated in a 0.8% agarose gel. The genomic D N A band (-17.5 kb) was cut out, extracted with Gene-Clean Kit (Bio 101) and ligated to Bluescript KSII +/- plasmid that had been treated with Not I restriction enzyme, alkaline phosphatase and Gene-Clean Kit. Competent DH5a bacteria were transformed and screened on X G A L / I P T G plates. D N A was then collected from an overnight culture of blue transformants and digested with Not I enzyme for confirmation. DNA sequencing. Plasmid D N A from an overnight culture of a genomic recombinant DH5a transformant was digested with BamH I enzyme. This led to the isolation of a 1.7 kb Bam HI fragment from the gel by using the Gene-Clean Kit (Bio 101). The 1.7 kb fragment was ligated into the Bam HI site of the bluescript KSII +/- pretreated with Bam HI enzyme and alkaline phosphatase. Ligation was carried out overnight at 4°C. DH5ct bacteria were then transformed and screened on SBAL/IPTG plates. Among many positive transformants (Fig. 4 A ) , BI transformants' recombinant plasmid was isolated and purified by a modified mini-alkaline lysis/PEG precipitation procedure and sequenced using T3 and T7 primers at the NAPS Unit at the University of British Columbia (8 ul of terminator premix, 500 ng of dsDNA template, 3.2 pmol of each primer, and water to a final volume of 20 ul). The NAPS unit's standard sequencing conditions were: 96°C for 30 seconds, 50° C for 15 seconds, and 60° C for 4 minutes for 25 cycles. Three internal primers were also used to sequence the promoter region: MCT3, MCT7, and UNS2P2. The sequence of the internal primers are: A T T T C C A G A G T G G A T G T A for MCT7, and A C A G A A A T A G A A T A G A G T for MCT3. UNS2P2 primer was the reverse complement of MCT7 primer and was used to confirm the 5' sequence of the Tgtp promoter by subcloning the promoter into another vector, 5 6 pcmCD2. The sequence of standard T3 and T7 primers are: A T T A A C C C T C A C T A A A G G G A for T3 and T A A T A C G A C T C A C T A T A G G G for T7. Synthesis of PCR template in presence of 7-deaza dGTP for subsequent sequencing and the sequencing conditions. The 1.7 kb BamHI fragment in pBL plasmid (0.27 ug) was used as template with MCT3 and MCT7 internal primers ( 0.2 ug each) for amplification. The PCR product was -460 bp in length. Four sets of polymerization reactions were carried out; with 75% and 100% 7-deaza dGTP in the absence or presence of DMSO (5%). Other conditions included: 4mM MgCl 2 , 1.25 mM of each deoxyribonucleotide (with either 100% 7-deaza dGTP, or 75% 7-deaza dGTP and 25% dGTP) and 5 units of Taq D N A polymerase (lui). However, the Taq D N A polymerase was added following pre-heating of reaction mixture to 98°C for ten minutes and snap cooling on dry ice for five minutes. The volume of each set-up was 100 ul. Then, the polymerization mixture was pre-heated to 96° C for five minutes and cycled for 30 cycles with 96° C for 50 seconds, 55°C for 1 minute, 72° C for 6 minutes. The sequencing conditions were NAPS unit's standard conditions as stated previously. Construction of promoterless luciferase vector and TGTP promoter in promoterless luciferase vector. The endogenous SV40 promoter in the luciferase vector, pGL-3 (Promega), was removed by Bgl II and Hind III digests (Fig. 6), followed by blunt ending by T4 polymerase and self ligated to create promoterless pGL-3 vector. On the other hand, the TGTP promoter as a 1.7 kb Bam HI fragment was cloned into the Bam HI site of pUC 18 for amplification of the TGTP promoter (TGTPp/pUC 18, Fig. 6). The TGTPp/pUC 18 was then cleaved by Xba I and Sma I to clone the TGTP promoter into the promoterless pGL-3 vector digested with Nhe I and Sma I restriction enzymes to 6 7 create the TGTP promoter in the luciferase vector, which is designated TGTPpluc vector. The Nhe I and Xba I sites have compatible ends. Transient transfection and luciferase assay. L cells were seeded at l x l O 6 per well in 6-well plates in 4 ml of I media (37° C, 5% C0 2 ) . The next day two solutions were mixed together: one contained 0.5 ug of TGTPpLUc vector in 100 ul of Optimem medium (Gibco BRL), the other contained 4 ul of lipofectamine (Gibco BRL) per 100 ul of Optimem medium. After mixing these two solutions, the mixture (200 ul) was let sit for -45 minutes at room temperature to allow formation of a DNA-lipofectamine complex. Meanwhile, L cells that were cultured overnight were washed once with Optimem medium. After the DNA-lipofectamine complexes were formed, 0.8 ml of Optimem medium was added to the complex. The L cells were overlaid with this complex (1 ml). The cells were incubated for -5 hours for transfection to occur (day 0). 4 ml of Optimem were added to the well and the mixutre incubated overnight. Next morning (day 1), 5 ml of Optimem medium were removed from the wells. 4 ml of fresh I medium were added per well and the cells were allowed to recover for 2-3 hours. The indicated concentration of cytokine was then added into each well of cells and incubate overnight (-17 hours). Next morning (day 2), luciferase activity was determined by the following procedure. The lx Cell Culture Lysis Buffer (Promega) and Luciferase Assay Reagent (Promega) were warmed to room temperature. Meanwhile, the cells were washed twice with PBS. Then, the cells were lysed with -150 ul of lx C C L B per well of a 6-well plate. The cells were scraped off with a rubber policeman and the lysate collected in a microcentrifuge tube, which was spun for ~5 seconds to pellet large debris. To assay luciferase activity, 20 ul of cell lysate were mixed with 100 ul of Luciferase Assay Reagent (Promega) and measured immediately with the luminometer (Dynex). 7 8 Results Genomic screening. A radioactive 5' fragment of the cDNA was used for preliminary screening of genomic library clones to identify those carrying D N A encoding 5' end of mRNA and possibly the promoter region of the Tgtp gene. The cDNA was 1611 bp in length with a 5' untranslated region (99 nucleotides) and a 3' untranslated region (264 nucleotides) (open boxes, Fig. 1 A). The 5' fragment of cDNA used for preliminary screening was an Eco RI/Hind III fragment (~413bp, Fig. 1A) that identified 19 positive clones out of 1.44 x 106 clones in the primary screening. Among these 19 clones, ten were probed again with the same probe in the secondary screening and all ten were positive. To further narrow down the number of clones to those carrying the sequence uspstream of the Tgtp cDNA , i.e. those containing the promoter region, a probe of 70 nucleotides (Fig. IB) derived from the 5'end of 5' untranslated region (left open box, Fig. 1 A) of the cDNA was used to screen the ten positive clones. Four out of ten clones were identified: Sr 1-3; Sr 4-3, Sr 6 and St 6 (Fig. 1C). Subcloning and characterization of genomic DNA. Mouse genomic D N A from the lambda phage clone Sr 4-3 was excised with Not I and subcloned into the Not I site of the Bluescript KS II +/- plasmid for amplification and subsequent construction of a restriction map. The genomic D N A from Sr 4-3 clone is approximately 17.5 kb in length (Fig. 2). The result of restriction digests of this genomic D N A with a variety of restriction endonucleases is shown in Fig. 3 and reveals that it does not contain restriction sites for Sma I, Kpn I and Sal I. The locations of three more Sac I sites and two more Hind III sites were not fully characterized. Hybridization of restriction fragments with the radioactive 70-mer probe, which is derived from the 5' end of the 5' untranslated region of the cDNA (the one used to identify this genomic clone), showed that the probe 8 9 hybridized with a 250 bp Eco RI/Bam HI fragment of the genomic D N A (Fig. 3, filled rectangle). The genomic D N A fragments adjacent to this Eco RI/Bam HI region are 4.8 kb (Not I to Eco RI) and 12.4 kb (BamHI to NotI) on the left and right side, respectively. Therefore, this implies that either the 4.8 kb or the 12.4 kb fragment contains the 5' upstream region of Tgtp cDNA. Identification of the 5' upstream region within the genomic clone. The identification of the 5' flanking region is based on the knowledge of cDNA sequence. By matching the sequence and the direction of the 5' end of cDNA with those of the 250 bp. Eco RI/Bam HI fragment (Fig. 3, filled rectangle) of the genomic DNA, one can determine the direction of the genomic D N A and, therefore, whether it is the 4.8 kb or the 12.4 kb portion of the genomic D N A that contains the 5' flanking region of the cDNA. For this purpose, a 1.7 kb Bam HI fragment of the genomic D N A was subcloned into the Bluescript plasmid for amplification and sequencing (Fig. 3, hatched box and filled box, where the filled box is the previously mentioned EcoRI/BamHI region). The Bam HI fragment containing the 70-mer probe binding region (filled box, or EcoRI/BamHI fragment, Fig. 3) was sublcloned into bluescript-plasmid as indicated in Fig. 4B, as revealed by enzymatic digests with EcoRI (1.45 kb and 3.25 kb fragments instead of 0.25 kb and 4.45 kb fragments, data not shown). Three transformant clones containing this Bam HI fragment (Fig. 4A, transformants B I , B5 and B8) were identified but only one (BI) was used for subsequent work. This construct was then sequenced using standard T3 and T7 primers. As indicated in the sequence data (Fig. 5), the Eco RI site at 5' end of Tgtp cDNA (Fig. 1 A) starts at nucleotide position 240 and the sequence of the 70-mer probe (5' end of cDNA) starts immediately upstream of the Eco RI site with perfect match (underlined in Fig. 5), ending with multiple cloning sites, such as BamHI site, close to the T7 primer binding site of the bluescript plasmid (Fig. 4B and 5). These results indicate that the 9 10 genomic DNA, or in this case the Bam HI fragment of the B l transformant clone, goes in the direction of Eco RI to Bam HI (filled box, Fig. 4B). This in turn implies that the 4.8 kb fragment on the left of the Eco RI/Bam HI fragment (Fig. 3) of the Sr 4-3 #4 genomic clone contains the 5' upstream region of the Tgtp cDNA. The Bam HI fragment (1.7 kb) of the genomic clone, which contains the Eco RI/Bam HI region, was then used to determine whether it actually carries the promoter activity. This was done by making a reporter-gene construct in which the SV40 promoter of a luciferase vector was replaced by the BamHI fragment. Construction of promoterless pGL-3 vector. The endogenous SV40 promoter of the luciferase vector, pGL-3, was removed by Bgl II and Hind III digests (Fig. 6), then blunt ended and ligated. The 210 bp band represents the cleaved SV40 promoter and the thick 5046 bp band represents the remainder of the pGL-3 vector (Fig.7). To exclude the possibility of the presence of the pGL-3 control vector in the sample of promoterless pGL-3 vector, enzymatic digests of the promoterless pGL-3vector were carried out. As shown in Fig. 8, the Hind III and Bgl II sites were absent from the sample of the promoterless pGL-3 vector (compare lanes 10 and 11 with 8), i.e. the SV40 promoter was absent in the promoterless pGL-3-3 vector. Furthermore, the possibility of partial digests was eliminated by the absence of D N A bands around the 5 kb region (lanes 10 and 11, Fig. 8). Construction of a luciferase vector under control of the Tgtp promoter. The Tgtp promoter as a 1.7 kb BamHI fragment was subcloned into the BamHI site of the pUC18 plasmid (TGTPp/pUC 18, Fig. 6). This was done to create compatible ends in order to facilitate subsequent cloning. The orientation of the promoter was confirmed by EcoRI digest (data not shown; the orientation is shown with the arrow in figure 6). The Tgtp promoter was then removed from TGTPp/pUC 18 construct by Xbal and Smal digests and 10 11 inserted into the promoterless pGL-3 vector treated with Nhe I and Sma I (Fig. 6). The Nhe I and Xba I sites produced compatible ends. The orientation of the Tgtp promoter was confirmed by Eco RI and Kpn I digests of the new construct. In the correct orientation, two fragments of 1.5 kb and 5.2 kb were expected and in fact observed (lane 4, Fig. 9). Transformant #2 was confirmed to contain the Tgtp promoter upstream of the luciferase reporter-gene in the correct orientation. In the wrong orientation, two fragments of 0.2 kb and 6.5 kb would be expected (Fig. 6, Tgtp promoter in luciferase vector, or symbolized as TGTPpLUc). Once the recombinant reporter construct was made, tests of its inducibility by IFN-y and - a were required to determine whether this Bam HI fragment of the genomic clone actually possesses the promoter activity. This Bam HI fragment contains the first 80 nucleotides of the cDNA (underlined and dash-lined sequence in Fig. 5), 114 nucleotides of the first intron (nucleotide #154 to #40 of Fig. 5) and approximately 1.5 kb of sequence upstream of the 5'end of cDNA. Subsequent induction experiments with IFN-y, IFN-a and other cytokines were carried out with this construct. Induction of Tgtp promoter is IFN-y specific. Transfection of L cells with an irrelevant plasmid (CD2 vector) does not induce expression of luciferase (lane 1, Fig. 10). This eliminates the possibility of cross-reactivity of endogenous enzymes of L cells with the luciferase assay reagents. The promoterless pGL-3 vector (lane 3, Fig. 10) had a background luciferase level of 854.59 R L U . Nevertheless, the possibility of contamination by pGL-3 vector, which has SV40 promoter, was eliminated as discussed previously (lanes 8 and 11 vs. 10, Fig. 8). IL-2 and IL-4 did not induce luciferase expression beyond the basal level (843.96 R L U ; lanes 5, 14 and 15 of Fig. 10). Activation of the Tgtp promoter by IFN-y as indicated by induction of luciferase expression was achieved with 10 U/ml of IFN-y (lanes 6 vs. 5, Fig. 10) and inhibited in the presence of anti-IFN-y antibodies (lanes 6 to 10, Fig. 10), and with 5,000 U/ml of 11 12 IFN-a (lane 13, Fig. 10). Lack of response of Tgtp promoter to IL-2 and - 4 indicates the specificity of this promoter. A more detailed analysis of Luciferase upregulation by varying concentrations of IFN-y and IFN-a is shown in Fig. 11 and Fig. 12. Luciferase was up-regulated 4.7 fold by the Tgtp promoter in presence of 0.5 U/ml of IFN-y (2768 R L U vs. 590 R L U , Fig. 11), and 6.3 fold with 100 U/ml IFN-y (3715 R L U vs. 590 R L U , Fig. 11). By contrast, luciferase was up-regulated 1.7 fold by 55 U/ml IFN-a (1001 R L U vs. 590 R L U , Fig. 12), and 4 fold with 5500 U/ml of IFN-a (2297 R L U vs. 590 R L U , Fig. 12). A higher concentration of IFN-a (10000 U/ml, Fig. 12) did not further up-regulate expression of luciferase. Time course of Tgtp induction by IFN-y. Assays were carried out to determine the time required for activation of Tgtp promoter by 5.5 U/ml IFN-y. The amount of luciferase induced by IFN-y was determined at 0.5, 1, 2, 4, 6, and 24 hours after the addition of IFN-y. As shown in Fig. 13, a minimum incubation of 6 hours was required to double the luciferase expression (770.53 R L U vs. 366.93 R L U , lane 9 vs. 4) but five-fold induction was achieved only with overnight (24 h) incubation of IFN-y (1855 R L U vs. 367 RLU). Lack of induction of Tgtp promoter by mIL-6. Previous studies have shown that different transcription complexes are recruited by IFN-y and IL-6 but they bind to the same D N A element, namely the GAS (gamma-interferon activated sequence) (12). As the Tgtp promoter contains the GAS element (see below), test of induction by mIL-6 was assessed in vitro. Concentrations of mIL-6 used included 0.55, 1, 5.5, 10, 55, 100, 550, 1000, 5500, and 10000 U/ml. None was effective in activating the Tgtp promoter (Fig. 14). 12 13 DNA elements present in the Tgtp promoter region. Having shown that the 1.7 kb Bam HI fragment of the genomic D N A contains the functional Tgtp promoter by its specific induction by IFN-y and -oc, I proceeded to determine the complete sequence of this Bam HI fragment. These sequence data were essential for determining not only the locations of IFN-y and IFN-a response elements, but also whether DNA-response-elements of other cytokines were present, or whether other D N A elements of interest were present that may provide additional hints as to the function of TGTP. The length of the genomic D N A fragment upstream of Tgtp cDNA that was isolated from the genomic clone Sr 4-3 is approximately 4.8 kb. This 4.8 kb fragment contains the 1.7 kb BamH I fragment (striped bar and filled rectangle, Fig. 3) used in the transfection experiments. The complete sequence of this 1.7 kb BamHI fragment is shown in Fig. 15. The strategies that were used to determine this complete sequence are outlined in the next section. Here I will highlight the main features of this 1.7 kb promoter region. The D N A elements present in the Tgtp promoter region are summarized in Table 1, and their locations are indicated in Fig. 15 and illustrated in Fig. 16. These D N A elements include: a T A T A box ( T A T A A A A , nucleotides 1212-1218), a GAS sequence (TTA/CNNNT/GAA, nucleotides 1258-1266), two CF-1 binding sites (ANATGG, nucleotides 8-13, 22-27) , three c-Myk/Max binding sites (CAT/CGTG, nucleotides 145-150,446-451,1037-1042) , a SP-1 binding site (T/GRGGCT/GRRT/G, nucleotides 191-199), two human NF IL-6 binding sites (A/CTTNCNNA/CA, nucleotides 296-304 , 613 -621), a Myb binding site ( Y A A C G / T G , nucleotides 1402-1407), two HI-boxes ( A A A C A C A , nucleotides 1157-1163, 1216-1222), an IBP-lb site ( A A G T G A , nucleotides 1591-1595), s ix LBP-1 sites (WCTGG, nucleotides 189-193, 3 8 8 - 3 9 2 , 4 0 6 -410 , 476-480, 764-768 , 1072-1076) , and two N F - E 1 sites ( W G A T A M M , nucleotides 1314-1320 ,1514-1520) and two T G G C A elements (nucleotides 11-15, 1335-1339). The first exon extends from nucleotides 1508 to 1587 (boxed, F i g . 15). N o I S R E ( I F N - a stimulated response element) that is usually needed for I F N - a induction was found. In 13 14 addition, no binding sites were found for the following factors that are associated either with the host defense mechanisms or with the overall transcription: heat shock factor, N F - K B , URF (UV-response factor) and LEF-1 (lymphoid enhancer binding factor) and others (Table 2) (13). The restriction map of the Tgtp promoter was determined using D N A Strider 1.1 software and unique restriction sites for this region are illustrated in Fig. 17. Strategies used for sequencing the Tgtp promoter. The 1.7 kb Bam HI fragment was cloned into pBL plasmid and sequenced with standard T3 and T7 primers. The T3 primer revealed approximately 500 nucleotides of the 5' end of 1.7 kb Bam HI fragment (MC/T3, Fig. 18A); whereas the T7 primer revealed about 600 nucleotides at 3' end of the Bam HI fragment that included the T A T A box, first exon, part of first intron, and 400 nucleotides of upstream sequence with one Hind i site (MCT77 of Fig. 19A, D and H). The sequence at the 5' end was confirmed by using an internal primer (UNS2P2) with thel.7 kb Bam HI fragment in a CD2 vector, pCDmCD2.1 (1.7BH1 2P2 of Fig. 18C). The sequence also revealed presence of two Sea I sites at 5' region. Subsequently, an internal Scal/HincII fragment was isolated from the BamHI fragment (~0.7 kb, Fig. 16) and subcloned into the pBL plasmid and sequenced with T7 and T3 primers. The T7 primer revealed about an additional 150 nucleotides (on the 5' end region) (MCT7, Fig. 18A). The combined sequence of 5' region of the Bam HI fragment is shown in Fig. 18B, revealing about 650 nucleotides of the 5' region. The sequence of the 3' region of the BamHI fragment (-935 nucleotides) was revealed by four, sequencing reactions with 1.7 kb BamHI fragment in pBL with T7 primer, the internal Scal/HincII fragment from BamHI fragment in pBL with T3 primer, ds PCR template with MCT3 primer, and BamHI fragment in CD2 vector with MCT3 primer (Fig. 19). Difficulty was encountered in determining the sequence of the most 14 15 internal 100 nucleotides and of its adjacent region, probably due to high degree of secondary structure and/or the presence of a GC rich region. Sequencing the most internal 100 bp of the Tgtp's promoter region was problematic. Attempts with 98°C denaturation temperature (in the absence of DMSO), high annealing and elongation temperatures, 5-10% DMSO, internal primers, linearized vector, single and double stranded PCR templates, and sequencing in presence of betaine (up to 2.5 M) were all nonproductive. Another attempt involved amplification of the most internal sequence by PCR (-450 bp in size) and then trying to find an endonuclease that would cut somewhere near the middle of the fragment. Twenty-four different endonucleases were tested but none could cleave the PCR product. The previously stated Sac I and Hinc II sites lie external to this PCR product. The sequencing of the most internal 100 bp was eventually resolved by synthesizing double stranded PCR template in the presence of 100%7-deaza-dGTP. The double-stranded 7-deaza dGTP-containing PCR template (-460 bp) was synthesized with internal primers MCT3 and MCT7. A better yield was obtained when the reaction contained 75% 7-deaza dGTP instead of 100% dGTP, in the absence of DMSO (Fig. 20). However, when the sequence of the PCR template was analysed with the same primers, the PCR template with 100% 7-deaza dGTP yielded better sequencing results (Fig. 21). The resolution of the sequence of the middle region of the Tgtp promoter allowed the identification of overlaps that led to the determination of the complete sequence of this 1.7 kb fragment (Fig. 22). 15 16 Discussion Recent studies have shown that for many IFN-y inducible genes, the promoter region contains an IFN-y inducible element located within 1 kilobase upstream of the transcription initiation site (14-17). In the Tgtp promoter, the T A T A box and the GAS sequence lie within 300 bp upstream of Tgtp's first exon. The presence of a T A T A box in the promoter region is variable among many IFN-y or -a induced genes (16, 17). The T A T A box is located 297 bp upstream of the cDNA 5' end. Previous studies have shown that the GAS (gamma-interferon activated sequence) element is required for IFN-y induction of many IFN-y inducible genes (ICSBP, IRF-1, GBP, FcyRl and MIG), and its consensus sequence is a palindrome, usually T T A / C C N N G / T A A with small variations. In fact, there is a perfectly matched GAS element located 47 bp downstream of the T A T A box. This is of particular interest as most IFN-y inducible promoters have the GAS element upstream of the T A T A box. In addition, a thymidine residue immediately upstream of the GAS element may be important, as its mutation in ICSBP makes its GAS less active than the wild type GAS in inducing reporter gene expression (18). A thymidine residue is also present upstream of the GAS in the Tgtp promoter. The importance of this thymidine residue in the function of Tgtp's GAS could be determined by mutation or deletion constructs in future experiments. Another DNA element one expects to find in the promoter region of Tgtp gene is the interferon stimulated response element for IFN-a (ISRE, (GAAAGT) x 2) (19). However, there is no ISRE in the promoter region of Tgtp. The absence of consensus ISRE sequence in the Tgtp promoter may explain the weak induction by IFN-a. However, a previous report has shown that the GAS motif can also serve as an IFN-a-responsive element in vivo (20). Other reports also show that there is no absolute correlation between IFN-y responsiveness with GAS and IFN-a responsiveness with ISRE (14,15,17,21). Therefore, the activation of the TGTP promoter by IFN-a may 16 17 also be mediated via the GAS motif. Another noteworthy observation is that in the presence of 10 U/ml IFN-y, thebasal level of luciferase from the promoterless pGL-3 was reduced by -70% compared to that in absence of IFN-y (lanes 4 and 3, Fig. 10). This is probably due to the anti-viral effect of IFN-y. In contrast, 5.5 U/ml IFN-y did not have such an inhibitory effect (lanes 3 and 4, Fig. 13). The specificity of the Tgtp promoter for IFN-y and -a is further evidenced by the finding that it was not induced by cytokines such as IL-2 (100 U/ml), IL-4 (100 U/ml) and IL-6 (10,000 U/ml), and that the strong induction by IFN-y is specifically blocked by anti-IFN-y antibodies. These observations confirm previous immunobloting experiments that TGTP was induced by stimulating L cells with IFN-y or IFN-a (3). The lack of induction by IL-6 is noteworthy since two human NF IL-6 sites are present in the Tgtp promoter. Furthermore, other studies have shown that one single D N A element can confer responsiveness to both IFN-y and IL-6 (12). However, I did not observe activation of the Tgtp promoter by either human or mouse IL-6 (up to 10,000 U/ml). One potential explanation for the lack of induction by IL-6 is that this D N A motif may not be accessible because of secondary structures arising from the eleven repeats of TCTCCCC that are in the vicinity of this motif. Although the promoterless luciferase vector expresses approximately 50 - 65% luciferase compared to that expressed by the positive control (pGL-3 control vector, with SV40 promoter), Fig. 8 shows digests of the promoterless vector with various restriction enzymes and confirms the absence of the SV40 promoter in the promoterless vector. The pGL-3 control vector linearized by Hind III is 5.3 kb in length (lane 8, Fig. 8). The absence of pGL-3 control vector in the sample of promoterless pGL-3 vector is evidenced by the absence of the 5.3 kb bands in lanes 10 and 11 (Fig. 8), due to the absence of Hind III and Bgl II sites as a result of the removal of the SV40 promoter and blunt-ending by T4 D N A polymerase. It is likely that the high luciferase background of the promoterless pGL-3 vector in transfection assays is due to the presence of the S V40 enhancer in the 17 18 promoterless vector and/or the cell type (L cell) used (personal communication with technical staff of luciferase-assay reagents, Promega). Interestingly, it seems the SV40 enhancer activity can be inhibited by 10 U/ml IFN-y (lanes 3 and 4, Fig. 10), probably through the anti-viral effect of IFN-y. The Tgtp promoter has a unique sequence: eleven repeats of TCTCCCC (nucleotides 642-718, Fig. 15). These repeats lie 616 bp upstream of the GAS (interferon-gamma activated sequence). Each repeat is 71% GC content and contributed to the difficulty in sequencing the most internal 100 bp fragment of the promoter. The possible function of these repeats is not known yet, but a search in the gene bank has revealed that some genes have as many as four repeats at most. None, with the exception of the Tgtp promoter, has eleven repeats. Whether these repeats play a role in transcription could be determined by gradual deletion of these repeats and then assessment of the inducibility by IFN-y and - a , as well as the resistance of cells in vitro to VSV. Similarly, the function of the Tgtp promoter region could be further tested by 5' or 3' deletional constructs or by introducing mutations to the GAS consensus motif. The difficulty in sequencing was also caused by strong hairpin structures formed in this region, and these include stem length of 10, 9, 8, 7 and 6 nucleotides with 2, 2, 1,0 and 1 mismatch(es), respectively. Another noteworthy observation is that the internal 500 bp region has restriction sites only at its ends, leaving its middle portion deprived of any restriction site (Fig. 17). This confirms the absence of restriction sites in the 450 bp PCR product, which was synthesized to test for the presence of restriction sites in an attempt to resolve secondary structures in the template to facilitate sequencing. The difficulty in sequencing the most internal 100 bp region was finally resolved by synthesizing PCR template in presence of 100% 7-deaza dGTP. In addition, it was observed that the presence of betaine, which has been reported to facilitate sequencing of stretches of DNA with high GC content, in fact reduced the efficiency of sequencing by FS Taq DNA polymerase with dye-terminator technique (ABI, model 377) 18 19 The Tgtp promoter also contains binding sites for CF-1, SP-1 and Myb. The CF-1 is a transcription factor that is ubiquitously expressed in murine tissues. The SP-1 transcription complex has been shown to increase RNA synthesis by five to eight fold in vitro. Other motifs are also present in the Tgtp promoter but their functions in the transcription regulation of Tgtp are unclear (13). These include: LBP-1, which binds to the 5' untranslated leader of HIV-1 and HIV-2 and functions as a cellular activation of transcription initiation; NF-E1, a transcription factor found in megakaryocyte erythroid and mast cell lineages; and HI-box, which protects only the upstream region of the coding strand of rat albumin gene. Recent work has indicated that several IFN-responsive genes may share a capacity to bind a common transcription factor (p91 or STAT1) that is rapidly activated by tyrosine phosphorylation through JAK1 and JAK2 (22). In some systems, p91 appears to bind directly to the D N A element (GAS element of GBP and IFP-53 genes). In other systems, p91 appears to be a component of a complex, such as ISGF-3 complex that binds to the ISRE site of the GBP gene (20). Alternatively, p91 may also be a component of the FcyRl and FcyR2 complexes that utilize an associated protein (p43) to bind to the GRR site of the FcyRI gene. Whether the GAS element of the Tgtp promoter interacts with the various transcription factors or complexes mentioned needs to be studied in the future. However, at present, it appears that a pattern is emerging for a set of immediate response genes induced by IFN-y; these genes utilize cis-active elements that share a palindrome and an ability to bind p91, either alone or in combination with other factors such as p43. The importance of defining specific DNA-protein interactions in distinct genes is to target critical sequences to rationally disrupt or enhance the immune response. IFN-y is a pleiotropic cytokine produced primarily by activated T lymphocytes and natural killer cells to modulate macrophage superoxide production, macrophage migration, T cell proliferation as well as lymphokine production and function. The selective inducibility of Tgtp by IFN-y and - a in cells of lymphocyte/macrophage lineage 19 20 suggests that Tgtp may play a regulatory role in cells of the immune system. The importance of TGTP in host defense is underscored by the observation that the forced expression of TGTP conferred an antiviral state for the negative-stranded RN A virus, V S V , but not to the D N A virus, HSV (3). 20 21 References 1. Carlow, D.A., J.D. Marth, I. Clark-Lewis, and H.-S. Teh. 1995. Isolation of a gene encoding a developmentally regulated T cell specific protein with a GTP-binding motif. J. Immunol. 154:1724-1734. 2. Lafiise, W.P., D. Brown, L. Castle, and B.S. Zwilling. 1995. Cloning and characterization of a novel cDNA that is IFN-y-induced in mouse peritoneal macrophages and encodes a putative GTP-binding protein. J. Leukocyte Biol. 51:411-483. 3. Carlow, D.A., S.-J. Teh, and H.-S. Teh. 1998. Specific antiviral activity demonstrated by TGTP, a member of a new family of interferon-induced GTPases. J. Immunol. 161:2348-2355. 4. Gilly, M . , and R. Wall. 1992. The IRG-47 gene is IFN-y induced in B cells and encodes a protein with GTP-binding motifs. J. Immunol. 148:3275-3281. 5. Sorace, J .M., R.J. Johnson, D.L. Howard, and B.E. Drysdale. 1995. Identification of an endotoxin and IFN-inducible cDNA: possible identification of a novel gene family. J. Leukocyte Biol. 58:477-484. 6. Taylor, G.A., M . Jeffers, D.A. Largaespada, N .A. Jenkins, N.G. Copeland, and G.F.V. Woude. 1996. Identification of a novel GTPase, the inducibly expressed GTPase that accumulates in response to interferon gamma. J. Biol. Chem. 271:20399-20405. 7. Muller, U . , U . Steinhoff, L.F. Reis, S. Hemmi, J. Pavlovic, R. Zinkernagel, and M . Aguet. 1994. Functional role of type I and type II interferons in antiviral defense. Science 264:1918-1921. 8. Huang, S., W. Hendriks, A . Althage, S. Hemmi, H . Bluethmann, R. Kamijo, J. Vilcek, R. Zinkernagel, and M. Aguet. 1993. Immune response in mice that lack the interferon-y receptor. Science 259:1742-1745. 21 22 9. Dalton, D.K., S. Pitts-Meek, S. Keshav, I.S. Figari, A. Bradley, and T.A. Stewart. 1993. Multiple defects of immune cell function in mice with disrupted interferon y genes. Science 259:1739-1742. 10. Boehm, U. , T. Klamp, M . Groot, and J.C. Howard. 1997. Cellular responses to interferon y. Annu. Rev. Immunol. 15:749-795. 11. Abbas, A .K . , A . H . Lichtman, and J.S. Pober. 1997. Cellular and Molecular Immunology. 3rd ed. Saunders, pp. 494 12. Yuan, J., U . M . Wegenka, C. Lutticken, J. Buschmann, T. Decker, C. Schindler, P.C. Heinrich, and F. Horn. 1994. The signaling pathways of interleukin-6 and gamma interferon converge by the activation of different transcription factors which bind to common responsive D N A elements. Mol. Cell. Biol. 14:1657-1668. 13. Boulikas, T. 1994. A compilation and classification of D N A binding sites for protein transcription factors from vertebrates. Crit. Rev. Euk. Gene Exp. 4:117-321. 14. Chon, S.Y., H.H. Hassanain, R. Pine, and S.L. Gupta. 1995. Involvement of two regulatory elements in interferon-gamma-regulated expression of human indoleamine 2,3-dioxygenase gene. J. Interferon Cyt. Res. 15:517-526. 15. Chon, S.Y., H.H. Hassanain, and S.L. Gupta. 1996. Cooperative role of interferon regulatory factor 1 and p91 (STATI) response elements in interferon-gamma-inducible expression of human indoleamine 2,3-dioxygenase gene. J. Biol. Chem. 271:17247-17252. 16. Nicolet, C M . , and D . M . Paulnock. 1994. Promoter analysis of an interferon-inducible gene associated with macrophage activation. J. Immunol. 152:153-162. 17. Paquette, R.L., M.R. Minosa, M.C. Verma, S.D. Nimer, and H.P. Koeffler. 1995. An interferon-gamma activation sequence mediates the transcriptional regulation of the IgG Fc receptor type IC gene by interferon-gamma. Mol. Immunol. 32:841-851. 18. Kanno, Y . , C A . Kozak, C. Schindler, P.H. Driggers, D.L. Ennist, S.L. Gleason, J.E. Darnell, and K . Ozato. 1993. The genomic structure of the murine ICSBP gene 22 23 reveals the presence of the gamma interferon-responsive element, to which an ISGF3-a subunit (or similar) molecule binds. Mol. Cell. Biol. 13:3951-3963. 19. Naf, D., S.E. Hardin, and C. Weissmann. 1991. Multimerization of A A G T G A and G A A A G T generates sequences that mediate virus inducibility by mimicking an interferon promoter element. Proc. Natl. Acad. Sci. USA 88:1369-1373. 20. Decker, T., D.J. Lew, and J.E. Darnell Jr. 1991. Two distinct alpha-interferon-dependent signal transduction pathways may contribute to activation of transcription of the granulocyte-binding protein. Mol. Cell. Biol. 11:5147-5153. 21. Ohmori, Y . , and T.A. Hamilton. 1993. Cooperative interaction between interferon (IFN) stimulus-response element and kappa-B sequence motifs controls IFN-y-stimulated and lipopolysaccharide-stimulated transcription from the murine IP-10 promoter. J. Biol. Chem. 268:6677-6688. 22. Shuai, K. , G.R. Stark, I.M. Kerr, and J.E. Darnell Jr. 1993. A single phosphotyrosine residue of Stat91 required for gene activation by interferon-gamma. Science 261:1744-1746. 23 24 1 A EcoRI (0) Rsa I (243) Hind 111(413) Rsa I (1339) EcoRV (970) Sspl (685) Rsa I (1086) A T G Hind III (1347) EcoRI (161 1) T G A A A T A A A 1 B 5' ACT TTT AGA TAA AG A CGT TCC CTA AGA GGA AAG CCA TCA CAG AAG TGT TAT • TCA AAA TCT ATT T C T G C A A G G G A T TCT CCT TTC GGT AGT GTC TTC ACA ATA -TGC CAC CAG ATC AAG GTC A 3' ACGGTGGTC TAG TTC CAGT 25 Figure 1: (A) A simplified restriction map of Tgtp c D N A . The open boxes at the two ends indicate 5' (left) and 3' (right) untranslated regions; (B) Sequence of the 70-mer probe, derived from 5' end of 5' untranslated region of c D N A , that was used to identify phage clones carrying genomic D N A with the upstream sequence of the Tgtp cDNA. The 5' end of the cDNA is shown. The underlined sequence represent the two 40-mer oligonucleotides with 10 complementary bases that were used to generate this 70-mer probe; (C) Autoradiograph showing that four lambda phage clones were identified to carry geneomic D N A with sequence upstream of the cDNA: Sr 1-3, Sr 4-3, Sr 6 and St 6. 26 Chromosomal D N A 15 kb Genomic D N A (-17.5 kb) Figure 2: Size of the genomic D N A in the Sr 4-3 lambda phage cloe is approximately 17.5 kb as revealed by Not I digestion and gel electrophoresis. The molecular weight indicators on the left ae those of 1 kb ladder. 27 L i n e a r r e s t r i c t i o n - e n z y m e map o f S r 4-3 #4 (17500 bp) Sma 1 Spe 1 EcoR 1 Bam HI Xho 1 ' > / ' ' > EcoR V J_ Xho I EcoR V EcoR V Hind III , / N o t I Bam HI EcoR 1 Hind III Not I Sac I BamH 1 EcoR V BamH 1 4.8 kb. 12.4 kb Figure 3: Restriction map and orientation of the genomic D N A in the Sr 4-3 lambda phage clone. The filled rectangle (Eco RI/Bam HI fragment) represents where the 70-mer probe hybridized, hence, it represents the 5 end of cDNA. The length of the genomic D N A to the left of this Eco RI/Bam HI fragment is about 4.8 kb, whereas that to the right is approximately 12.4 kb. A 1.7 kb Bam HI fragment (hatched plus filled rectangles) was subsequently subcloned into the bluescript plasmid for sequencing and determination of orientation of the genomic DNA. Figure 4 shows that the orientation of the genomic D N A is as shown by the arrow in this figure. This also indicated that the 4.8 kb fragment is the upstream sequence of the cDNA where one may find the promoter. 28 4 A . 3 kb —> 2 kb —> 1.5 kb —> 29 4 B Figure 4: Orientation of the 1.7 kb Bam HI fragment in bluescript plasmid. (A) Bam HI digest of plasmid DNA from plausible DH5a transformants. First lane represents the 1 kb molecular weight marker. Three transformants (BI, B5 and B8 in lanes 4, 8 and 11, respectively) were confirmed to carry the 1.7 kb Bam HI fragment. The 3 kb band represents the linearized bluescript plasmid; (B) Orientation of the 1.7 kb Bam HI fragment in the BI transformant. The orientation of the genomic DNA is as shown and confirmed by Eco RI digest. 30 A Modal Varalon 2.1.0 AB1 PRISM" MC/T7 Martin Chang Signal G:102A:121 T:77C:43 DT6%Ac(A Sat-AnyPrlnwr) 1995matit< PolnU886to7258 B a w l : 888 Pag* 1 o( 2 Fri, Mar 1,1896 4:40 AM Thu, Fab 29,1998 4:04 PM Spacing: 9.91 ABISO eCCT 0 » .T . « . »CCT«»aCTCTCCTT , C CTOOTOTC»Oa«C»«C»CTCC.C«IO i CI»TC»C»e«TC C A T C T T c j a A T T A T C A A A C A A O C T O A A d A C A C A O C C C T T O C T C T C T A C A C T T T T C T O C C A Q O A T C T O A O C C C T A T T T A I C TA A A C C A C C I T C C . 'J Figure 5: DNA sequence revealing the orientation of the 1.7 kb Bam HI fragment. The sequence of the 1.7 kb Bam HI fragment revealed by T7 primer showed the 5 end of the cDNA started at nucleotide 234 and proceeded toward the multiple cloning sites. The Eco RI site at 5 end of cDNA corresponded to nucleotide 240 to 235. The seqence shown was actually complementary to the cDNA. The boxes represent Sac I (4), Sac II (3), Not I (2) and Bam HI/ Spe I (1) sites downstream of the T7 primer binding-site. The complementary sequence of the first 80 nucleotides of the cDNA is underlined (nucleotide #234 to #155). 31 Figure 6: Representation of the recombinant vector constructs that were made for functional assay of the Tgtp promoter. 2kb 1.5 kb 1 kb 0.5 kb 0.4 kb Fig. 7: Preparation of the promoterless pGL-3 luciferase vector. The pGL-3 vector was treated with Hind III and Bgl II restriction enzymes and fragments separated by gel electrophoresis. These two endonucleases removed the SV40 promoter (210 bp) from the vector and the linearized promoterless vector was isolated and purified from the gel. 1 2 3 4 5 6 7 8 9 10 11 12 <— 5.3 kb Fig. 8. Absence of contamination by pGL-3 vector (with SV40 promoter) in DNA sample of promoterless pGL-3 vector. Lane 8: pGL-3 vector linearized by Hind III (5.3 kb), lane 9: circular pGL-3 vector, lane 10: promoterless pGL-3 vector treated with Hind III, lane 11: promoterless pGL-3 digested with Bgl II, lane 12: promoterless pGL-3 vector linearized with Xho I endonuclease. Note the absence of Hind III and Bgl II sites in the promoterless pGL-3 vector (lanes 10 and 11), confirming the absence of SV40 promoter in the promoterless recombinant vector. Also note the absence of pGL-3 vector (5.3 kb) in lanes 10 and 11. 34 5.2 kb — 1.5 kb Fig. 9. Direction of the Tgtp promoter in the recombinant Tgtp prommoter-luciferase reporter gene construct (TGTPpLUC). When treated with Eco RI and Kpn I, the transformant with correct orientation of the Tgtp promoter would reveal two fragments of 5.2 kb and 1.5 kb in length. Lanel: transformant #1 digested with EcoR I, lane 2: transformant #1 treated with EcoR I and Kpn I, lanes 3: transformant #2 digested with EcoR I, lane 4: transformant #2 treated with EcoR I and Kpn I, lane 5: transformant #3 treated with EcoR I, lane 6: transformant #3 treated with EcoR I and Kpn I, lane 7: transformant #4 treated with EcoR I, lane 8: transformant #4 treated with EcoR I and Kpn I, lane 9: transformant #5 digested with EcoR I, lane 10: transformant #5 treated with EcoR I and Kpn I. Transformant #2, lane 4, clearly shows the presence of the Tgtp promoter in correct orientation in the recombinant Tgtp promoter4uciferase reporter gene construct. 35 TGTPp + 100 U/ml IL-4 TGTPp+ 100 U/ml IL-2 TGTPp + 5,000 U/ml IFN-a TGTPp + 2,000 U/ml IFN-a TGTPp + 100 U/ml IFN-a •TGTPp + 60 ul a-IFNg *TGTPp + 26 ul a-IFNg *TGTPp + 5.1 ul a-IFNg *TGTPp + 2.6 ul a-IFNg TGTPp + 10 U/ml IFN-g TGTPp Prless pGL-3 + 10 U/ml IFN-g Prless pGL-3 (-SV40p) pGL-3 (+SV40p) Irrelevant vector (CD 2) 1 h 0 H 2,334 1,910 1,322 h -1,000 + 2,000 RLU H 3,238 h 3,735 H 3,000 4,000 Figure 10: Functional assay of the Tgtp promoter using the Tgtp promoter-luciferase recombinant vector (TGTPpLuc). The activation of the Tgtp promoter by IFN-y (lane 6) and its subsequent down-regulation by anti-IFNy antibodies (lanes 7 to 10) were tested. Also tested were Tgtp promoter induction by IFN-a (lanes 11,12,13) and other cytokines (lanes 14,15). +/- SV40p: with/without SV40 promoter, Prless: promoterless, i.e. without SV40 promoter, IFN-g: IFN-y, TGTPp: Tgtp promoter-luciferase recombinant vector, T G T P p : TGTPp + 10 U/ml IFN-y, a-IFNg: anti-IFNy, IFN-a: IFN-a. 36 TGTPp + 0.1 U/ml IFN-g TGTPp + 0.5 U/ml IFN-g TGTPp + 1 U/ml IFN-g TGTPp+ 5.5 U/ml IFN-g TGTPp + 10 U/ml IFN-g TGTPp + 55 U/ml IFN-g TGTPp + 100 U/ml IFN-g TGTPp Prless pGL-3 pGL-3 ctl 2,765 0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 R L U Figure 11: Determination of the optimum induction of Tgtp promoter (TGTPp) by titrated amount of IFN-y. The activation of the TGTPp was tested in various concentrations of IFN-y: 0.1, 0.5,1, 5.5, 10, 55, and 100 U/ml. Ctl: control, Prless: promoterless, TGTPp: Tgtp promoter-luciferase recombinant vector, IFN-g: IFN-y. 37 TGTPp + 100 U/ml TGTPp + 100 U/ml TGTPp + 10,000 U/ml TGTPp + 5,500 U/ml TGTPp + 1,000 U/ml TGTPp + 550 U/ml I TGTPp + 100 U/ml I TGTPp + 55 U/ml I TGTPp + 10 U/ml I TGTP Prless pGL pGL-3 c 0 500 1,000 1,500 2,000 2,500 RL Figure 12: Determination of the optimum induction of Tgtp promoter (TGTPp) by titrated amount of IFN-a. The activation of the U2p was tested in various concentrations of IFN-a: 10000, 5500,1000, 550,100, 55 and 10 U/ml. IL-2 and -4 were used as specificity controls. Ctl: control, Prless: promoterless, TGTPp: Tgtp promoter-luciferase recombinant vector, IFN-a: IFN-a. TGTPp + 100 U/ml mIL-2 (O/N) • T f T D n 4- < \" I I /ml I F M n lf\fM\ H i MrntWh 430 l u l r p + 3.J U/ml lrIN-g(U/INJ TGTPp + 5.5 U/ml IFN-g(6 hr) • TGTPp + 5.5 U/ml IFN-g (4 hr) • < 488 TGTPp + 5.5 U/ml IFN-g (2 hr) • HI-, 239 TGTPp + 5.5 U/ml IFN-g (1 hr) • | H 144 TGTPp + 5.5 U/ml IFN-g (30 min.) • | H 148 TGTPp (O/N) ! • • • H 3 6 7 Prless pGL-3 + 5.5 U/ml IFN-g (O/N) • ' 550 Prless pGL-3 (O/N) • nnr i M\ mm 632 1 i i i i i 0 l l i 500 1000 1500 RLU l 1 2000 2500 Figure 13: Time course of Tgtp promoter induction by IFN-y. Time lapse of IFN-y presence that is required for luciferase reporter gene expression was tested with transfected L cells incubated with 5.5 U/ml IFN-y for: 30 minutes, 1,2,4, 6 hours and overnight (O/N; 16 hr). Mouse IL-2 (mIL-2) was used as a specificity control. Ctl: control, Prless: promoterless, TGTPp: Tgtp promoter-luciferase recombinant vector, IFN-g: IFN-y. 3 9 TGTPp+l 0,000 U/ml IL-6 TGTPp+5,500 U/ml IL-6 TGTPp+1,000 U/ml IL-6 TGTPp+550 U/ml IL-6 TGTPp+100 U/ml IL-6 TGTPp+55 U/ml IL-6 TGTPp+10 U/ml IL-6 TGTPp+5.5 U/ml IL-6 TGTPp+1 U/ml IL-6 TGTPp+0.55 U/ml IL-6 TGTPp+5.5 U/ml IFN-g TGTPp Prless pGL3+100 U/ml IL-6 Prless pGL3 pGL-3 ctl Figure 14: Lack of Tgtp promoter induction by mouse IL-6. To test whether mIL-6 would cross-activate Tgtp promoter, L cells transfected with Tgtp promoter-Luciferase recombinant reporter vector (TGTPpLuc vector of figure 6, abbreviated as TGTPp in this figure) was incubated with 1, 10,100, 1000, and 10000 U/ml of mIL-6. 5.5 U/ml of IFN-y was used as a positive activation control. Ctl: control, Prless: promoterless, IFN-g: IFN-y. 40 10 20 30 40 50 1234567890 1234567890 1234567890 1234567890 1234567890 'ftirrirriVTft rrrrlrrtTTr'Tr ifefaTTTtirr Aiccmiur CTCAQCTCCA 50 AAcnrcrcT c ron^c icc r m x i u y u r GATTCTTICT A M T M ? ^ G A 100 AQGAQCAAAG TCirXACACT TI03ICMCG TU.'I'K.TIjft (jl'I'll 'ATT7TTJ 150 TmucfflraG TCBCCTIAT AICTCACTRT ACTAAcrnfir ffrrrrroATk 200 TrrAATrrATr AGIGAGTACA TncATiTCA GTicirrrGr GAnooGm 250 CCICACICAG GAIGATOCCC TOCAG3TCCA ACCATTIQCC T A C Y J A K T T T H 300 lATA^ATICAT 'ILTl'l'l'lAAT GGCIGfiGTAG TftCTOCATIG TCIPAATGTA 350 OCACMmTT TIGTAICCAT TCCICIGTIG AQ3ftQCA^LgfenCTTTC 400 CAGCTEES} CEATEAIAAA CAAG3CIGCT AIGAACATAG TAfWt-ATTTrl 450 SlCCTTCTEA. C0QGTIG3GA CAICltood A3KTATOCCC AGGAGAGGTA 500 TIGCQGflSTC CICOGGTAGT ACTA3X3lSI^ITICTGAG GAACCACCAG 550 ACIGA3TICC AGAGIG3ATC TACAA03CTT GCAATCCCAC CAACAATOGA 600 QGAAGIGTIC c:it' i ' i 'u ' ' irr^ATrr^rrrr CAOCATCAGC ATCTOCCCTC 650 T033C^CICC CCTCIOCCCT CTOCCXTICIC CCCTCICCCC TCTCCCCICT 700 CCCCICICCC CTCTCOCCIC TCCICATICC TCICICCCCT CICCICICAT 750 cicccricTC oocbnrisfic CCKXTIOCGT GCICCTCCIG CATCTCCCTT 800 GCTCCICICA TCTCCCTTCT OTO0CCTOC TCCCC'l'llUC GGITICATOC 850 TOCTCICICC TATCTICTCT CCIL 'iCIGlT CTCTCCICGT GTtXTCICTC 900 CTmuSCTT CTCCTCTCTC CTI'ICICCCC TITClCCrTr c n c c c c m c 950 TAQCCCmCA AICTAOOQCC CTATICACIC 'lCl'l'lUl'I'IC CCCCTAQCCC 1000 CXriGITOCCT TICATCICTT OOCAACEAOC CXrnrxfcAEZ^DOCIAAAT 1050 AGAACICACT ATGTICAIAT i f ^ ^ I G A C TAGTOOCTIC CXXTO3AGAA 1100 AAGIQCCTCA ACCAQQ3GOC CAACAGAGGC TAICCTICAT CTAOCACCAA 1150 OCCCCCAAAC ACA^CAOCA TACIQCACCT TCEA.TCAAAC ATATICCTO3 1200 TmmCTIT TtTATAAA^ CA CftATKEftGIT GACATTTGAT TCCTACAAGC 1250 TOrrrATtrrr rarraarrjGA AACCGAAACC TACICTCATT ACAQCCCCCC BOO TCITIQ3IGG TTlt&fiATAA^ TAG3X3CAG A l C c E i r i b AAAAGTGTAG 1350 AGAGCAAGQ3 CTOIGTGTO3 ACCACTAAGG ACAAAGAAAC TCAGGGAGAT 1400 rbAAnrrnrr ATCCCACGGT CTCCTOCTTT CTATCIGCAG TTCTICCIGG 1450 GACACCAGAA CIOCACAGCT OgGTOCTCT CTICATAAAC AICCTCAAIC 1500 TCAATITKTFTTTW^ATAA?! GACiJi'ia-rr A A G A T T ^ A A A G opATTArAnAl 1550 lAffrnrrATir, rrArrArwrr AvrrrrArrA r rorr r jhrTr AAGIGAAGGG 1600 OGTCISGdT CAQCTIGTIT GATAAIOCAA GATCIGTGAT AGCATCAGGA 1650 GIGCTCirXT GCACCAGGA AAGGAGAQCT CAG3TTTCIA CTCAGGGAGG 1700 A 1701 Figure 15: Complete sequence analysis of the 7g#> promoter region. The identity and location of various DNA elements or transcription complex-btmiing sites are summarized in Table 1. 41 Max/c-Myc (-1363) Max/c-Myc (-1062) ,SP-1 (-1317) CF-1 (-1486)\ \ h N F - I L e ^ L ^ " 8 9 5 ) (-1212) \ \ ( T C T C C C C ) x l l (-866)| CF-1 (-1500)' 7 BamHI Max/c-Myc (-471) T A T A (-296) Sea I (-1173) V Sea I (-984) Myb (-106) AS (-250)/ c D N A ( l ) t Hinc II (-274) BamHI Eco RI Figure 16: Locations of transcription-complex binding sites and D N A elements relative to the transcription initiation site in the 1.5 kb Tgtp promoter region. The promoter extends from the Eco RI site to the Bam HI on the left margin. Not all binding sites or D N A elements are shown. 42 19 Xbal J 316 Msel 289 Sty I 289 Avril 460 Cfr10 I 460 Age I 445 Nsp7524l 445 Nsp I 1267 Taq 1249 Nhe I 1117 Hae III 1116 Bsp120l 1116 Apal 1115 PssI 1115 EcoO109l 1079 SpeI 966 Aci I 1228 Hinc 11 1676 Sac I 1599 BsaH I 1522 Mae II 1414 Dsal 1676 Ecl136 1502 EcoR I J U I L 1 7 0 1 base pairs Unique Sites Figure 17: Restriction map of the Tgtp promoter region. The sequence of the Tgtp promoter region was analyzed by D N A Strider 1.1 software and unique restriction sites are shown. C O j_ Q. I-o o UJ o Q. C O C L co h-o o c C L C O H o C L < M 0_ C \ J CD o T3 O CC o 0_ CO o 00 CO o co s t CO 1 C L h-T C L h-O 5 0 E 5 co CO 0 9 - E o c a) co E! O) JD cc E co ca ° - TD . f § O Q _ -2 O •° ^ cu J Z Q . ~ * CO .E t-E d co i— 0 TD J Z c *- co TD co c < Z Q o E o c 0 c 0 CO 0 1 CL 0 X — 0 _ -F. CO CO 0 •4—' 0 .E cz +2 CO Q . 0 (1) E ~ 0 TD .E o ~ CO 1 co ° 0 " O) co 0 . -2 0 * i— c c o g c o 0 J Z 3 CO % g CO 2 CO — Q _ C O o I— c -—• X £ — 0 co E O c CO a. 0 F £ >» c c CO Q CO c c ^ ® o-C L M _ 0 o CO 0 c •- .2 -4—' - O " ~ 0 O .!= CO TD CL CL J D £ g C M " CO ^ Z 3 3 O 1 « CO CO a. co H- CO O c 2 ® . CO Q _ CO P | o 5 T D M _ 11 TD CJ) i - c F 0 C L t= \— -C D C TD ° " co co 0 N CO 0 C L 0 •a I h-" O 0 c J Z o 0 t: £ c « S co ^ 0 5 0 0 J Z _ Q 5 0 - o 2 5 0 5 E w C TD ° - 0 TD J Z CO r-1 2 5 0 w E 0 c C0 CL CO 1^  C H 0) -a c c 5 7 3 0 0 J Z 1 18 C L " O E 0 ~ H D C 0 2 01 0 TO E c c N C L co CL 0 1^  -c I— c O =^  ^ CO TD C J D TD 0 CO 0 > 0 0 CO | CL o-eo H o — CO CO iz co O Q) co a o ^ s co E .2 1^ c7) 2 T3 CL 0 C O C r — .CO 0 CO •= 5 1- ? ^ E CL O 2 | S 0 a h D « TD c; co _ TD ^ E « CO r -CO C 5 0 o •£ J Z J Z * 1 ° TD 0 0 o o c c 0 0 ZJ ZJ cr cr 0 0 co co co ZJ o i co > J D TD 0 C J D O 0 O cz 0 Z) cr 0 CO I £ CO CD C L C O I— G O 0 k_ ZI C D 44 Figure 19: The amounts of 100% (lane 2) vs 75% (lane 4) 7-deaza dGTP PCR products (-460 bp) synthesized in the presence of 100% and 75 % 7-deaza dGTP, respectively. 45 Table 1: Surnmary of D N A motifs present in the Tgtp promoter. Name Sequence Location T A T A box T A T A A A A 1212-1218 GAS T T A / C N N N T / G A A 1258-1266 (TCTCCCC)x l l TCTCCCC 642-718 CF-1 A N A T G G 8-13, 22-27 cMyk/Max CAT/CGTG 145-150,446-451, 1037-1042 SP-1 T/GRGGCT/GRRT/G 191-199 hNF-IL6 A / C T T N C N N A / C A 296-304, 613-621 Myb Y A A C G / T G 1402-1407 H - l box A A A C A C A 1157-1163, 1216-1222 IBP-lb A A G T G A 1591-1595 LBP-1 W C T G G 189-193,388-392, 406-410, 476-480, 764-768, 1072-1076 NF-E1 W G A T A M M 1314-1320 T G G C A T G G C A 11-15, 1335-1339 First exon AGTTTT C T G C T G A 1508-1587 First intron G G T A A G G G A G G A 1588-1701 46 Table 2: D N A motifs or transcription complex-binding sites not found in the Tgtp promoter. D N A elements or transcription factors Name Symbol Sequence Activating protein 2 AP-2 CCCA/CN(GGG/CCC) cAMP response element binding protein ATF, CRE-BP1 T G A C G Y C / A R ; or Y G / T R C G T C A C/EBP C/EBP GTGG(TTT/AAA)G; or C(AAA/TTT)CCAC CAT box transcription factor CTF C C A A T ; or A T T G G Fos Fos G C T G A C T C A ; or T G A G T C A G C H-2 M C H class I genes transcription factor H2TF1, KBF1 , MBP2 A G A T " G G G G A A T C C C C " A G C C ; or G G C T " G G G G A T T C C C C " A TCT Hox 1.3 Hox 1.3 C Y Y N ATT A T / G Y Heat shock factor HSF N G A A N N T T C N N G A A N ; or N T T C N N G A A N N T T C N ISGF-3 ISGF-3 GCTTCAGTTT; or A A A C T G A A G C Interferon-a stimulated response element ISRE G G G A A A N 2 G A A A C ; or GTTTCN 2 TTTCCC 47 Table 2: Continued. Lymphoid enhancer binding factor LEF-1 CCTTTGAA; or T T C A A A G G N F - K B N F - K B G G G A A / C T N Y C C ; or GGRNAT/GTCCC NF-1 NF-1 T G G C / A N 5 G C C A A ; or TTGGCN5G /TCCA Nucelar factor of activated T cells NF-AT G G A G G A A A A A C T G T T T C A T ; or A T G A A A C A G T T T T T C C T CC Octamer factor Octl TNATTTGCAT; or A T G C A A A T N A Tumor suppressor gene product P53 RRRC(AT/TA)GTTT RAG-1 RAG-1 C A C A G T G ; or C A C T G T G RAG-2 RAG-2 A C A A A A A C C ; or GGTTTTTGT Sis-inducible factor SIF CCCGTC/A; or G/TACGGG UV-response element U R E T G A C A A C A ; or TGTTGTCA 

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