Veterans Affairs Medical Center, Long Beach 90822; and University of California, Irvine, California 92697
Submitted 26 February 2003 ; accepted in final form 19 May 2003
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ABSTRACT |
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promoter analysis in vitro and in vivo; thiamin transport; transgenic mice
Because humans and other mammals cannot synthesize thiamin, they must obtain the vitamin from exogenous sources via intestinal absorption. Using a variety of human and animal intestinal preparations, studies have established the involvement of a specialized carrier-mediated mechanism for thiamin uptake (5, 14, 16, 17, 19). Several groups recently cloned a human thiamin transporter, SLC19A2, which specifically transports thiamin when expressed in a variety of cellular systems (4, 6, 7, 9). Subsequent studies from our laboratory have shown that this transporter is expressed in the human digestive system (13). In addition, we cloned the 5'-regulatory region of the human SLC19A2 gene, determined the minimal region required for basal promoter activity, and identified several putative cis-regulatory elements (13, 18). The putative cis-regulatory elements include nuclear factor-1 (NF-1), activator protein-1 (AP-1), Gut-enriched Krupple-like factor (GKLF), and stimulating protein-1 (SP-1) (13). Our aim in the present study was to further characterize the SLC19A2 promoter by investigating the role of the fore mentioned putative cis elements in regulating the activity of the minimal SLC19A2 promoter in vitro using Caco-2 cells and to confirm the activity of the SLC19A2 promoter in vivo using transgenic mice (mice have an ortholog of the human thiamin transporter SLC19A2 designated as Slc19a2, Ref. 12).
Our results from this study show that independent mutations of the GKLF/SP-1, NF-1, and SP-1 cis elements led to a decrease in SLC19A2 promoter activity. In addition, we established that the factors GKLF, NF-1, and SP-1 participate in forming DNA/protein complexes with the SLC19A2 promoter. Furthermore, SP-1 was found to specifically increase SLC19A2 promoter activity in vitro. We also confirmed the functionality of the full-length and the minimal SLC19A2 promoters in vivo using transgenic mice and established that the expression of the promoter-Luciferase transgene mimics the SLC19A2 RNA expression pattern reported in native human tissues.
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EXPERIMENTAL PROCEDURES |
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Cell culture, transfection, and Luciferase assay. Generation of the minimal promoter-Luciferase construct was described previously (13). The minimal promoter-Luciferase construct (4 µg) (356 to 36, see Fig. 3A) was transfected separately into human intestinal Caco-2 cells or human-derived liver HepG2 cells using the Lipofectamine reagent (Life Technologies) and the manufacturer's procedure. To normalize for transfection efficiency, the cells were cotransfected with 100 ng of pRL-TK (Promega, Madison, WI) plasmid along with the promoter-Luciferase construct. For SP-1 cotransfections in Schneider Drosophila SL2 cells (American Type Culture Collection, Rockville, MD), the identical transfection methods were used, except 2 µg of the minimal (356 to 36) or full-length (2,250 to 36) promoter-Luciferase constructs were used and either 1, 2, or 4 µg of an SP-1 construct. The SP-1 construct was generated by subcloning the open reading frame of the human SP-1 cDNA (a generous gift from Robert Tijan, University of California, Berkeley, Ref. 8) into the pPac expression vector (Clonetech). For all transfections, total cell lysate was prepared from cells 24 h posttransfection, and firefly Luciferase activity was assayed using the Dual Luciferase kit (Promega) and a Turner Design 20/20 Luminometer (Sunnyvale, CA). The activity was normalized to the Renilla Luciferase activity from pRL-TK in the same extract. Data presented are means ± SE of at least three independent experiments and given as fold expression over pGL3-basic expression set arbitrarily at one. Statistical analysis was performed using the Student's t-test.
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Semiquantitative PCR. A reverse transcription (RT) reaction was
performed on 5 µg of total RNA isolated from 2- to 5-day postconfluent
monolayers of Caco-2 or HepG2 cells using either an oligo-(dT) primer, a
random hexamer, or a gene-specific primer and an Invitrogen superscript kit
(Carlsbad, CA). A PCR with specific primers for SLC19A2 (forward
5'-GCTGCTGCGTGTATATCATG-3', reverse
5'-CACCAAATACTAGGGCATAG-3') was then performed on the firststrand
cDNAs. PCR conditions were 95°C denaturation for 5 min, followed by 25
cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, with a
final extension of 72°C for 10 min. PCR products were analyzed on a 2%
agarose gel and compared with identical -actin control reactions run
simultaneously. An Eagle Eye II system densitometer (Stratagene, La Jolla, CA)
was used to normalize the SLC19A2-specific reactions to the
-actin control.
Mutational analysis. Mutations were introduced into the SLC19A2 minimal promoter-Luciferase construct individually at the sites indicated in Fig. 3A using a Stratagene site-directed mutagenisis kit and the manufacturer's protocols. The sequences were verified using the Largen DNA sequencing facility (Los Angeles, CA). The mutated constructs were then transiently transfected into Caco-2 cells using the identical method described above.
EMSA. Nuclear extracts were prepared from 2- to 5-day
postconfluent monolayers of Caco-2 cells using standard published methods
(1). Nuclear extract
(510 µg) was incubated for 20 min at room temperature with
-[32P]-ATP end-labeled DNA fragments in a binding buffer
consisting of 20 mM Tris · HCl (pH 7.5), 2 mM MgCl2, 1 mM
EDTA, 50 mM NaCl, 0.1% Triton X, 6 µg bovine serum albumin, 2 µg
poly(dI-dC), 0.5 mM dithiothreitol, and 10% glycerol. Competition analysis was
performed with 50- or 500-fold molar excess of commercial competitor consensus
oligonucleotides (Santa Cruz Biotech, Santa Cruz, CA) or with 100-fold molar
excess of unlabeled SLC19A2 DNA fragment (397 to 121).
For supershift assays, the nuclear extract was pretreated with nonspecific
IgG-, GKLF-, NF-1-, or SP-1-specific antibodies (Santa Cruz Biotech) for 15
min in binding buffer before the addition of labeled DNA. DNA/protein
complexes were separated on 6% nondenaturing polyacrylamide gels in 1x
Tris-acetate-EDTA (TAE; pH 8.5) at 3035 mA. The gels were dried and
exposed to film for autoradiography.
Generation of transgenic mice. We utilized the expertise of the transgenic mouse facility at the University of California Irvine (UCI-TMF) to provide us with founders carrying human SLC19A2 promoter-Luciferase constructs. The procedure utilizes the method of pronuclear DNA injection and is described briefly below. Two constructs, the 2,210-base pair full-length or the 320-base pair minimal 5'-regulatory regions of the SLC19A2 gene fused to the Luciferase reporter gene, described previously (13), were excised with the enzymes KpnI and BamHI, gel purified, and provided to the UCI-TMF. Eggs from superovulating females were injected and implanted into foster mothers. Eleven pups were born from the full-length construct-implanted mothers and 49 pups from the minimal construct-implanted mothers 1920 days after implantation, and tail tips were cut about 14 days after birth. Genomic DNA was extracted from the tail tip and sent to us. We genotyped the pups by performing a PCR with specific primers for the human SLC19A2 promoter (forward 5'-GTGGAGTTCCACATGTCC-3') and Luciferase gene (reverse 5'-ATGCGAGAATCTCACGCAG-3') that would yield a 366-base pair product. We reconfirmed these results by performing another PCR with the same forward primer designed from the promoter sequence and a different reverse primer from the Luciferase gene sequence. Identical results were obtained (data not shown). Southern blots were performed and the copy number of the inserted transgene was determined as previously described (25). The transgenic mice were paired with wild-type CB6F2 litter mates to ensure the transgene was passed to the F1 progeny and to establish transgenic human SLC19A2 promoter-Luciferase mouse colonies. The Institutional Animal Care and Use Committee (IACUC) at the Long Beach VA, as well as the IACUC at UCI, approved the experimental procedures used for mice in this study.
Tissue isolation, RNA, and Luciferase analysis for transgenic mice. Mice were euthanized, and specific tissues were immediately removed and split into either ice-cold Trizol (Invitrogen) for RNA isolation or ice-cold passive lysis buffer (Promega) for Luciferase assays. The tissue was immediately lysed on ice using a PowerGen125 (Fisher, Pittsburgh, PA) hand blender and frozen at 80°C. RNA was isolated using the manufacturers procedure (Invitrogen), and semiquantitaive PCR was performed as described above using the SLC19A2-specific primer 5'-AGGGAGAAGGCGTCACTC-3' and the Luciferase-specific primer 5'-ATGCGAGAATCTCACGCAG-3'. Luciferase assays were performed using the manufacturer's procedures and a Turner Design TD-20/20 luminometer. Luciferase assays were normalized to total protein for each sample measured using the DC protein assay kit (Bio-Rad, Hercules, CA).
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RESULTS |
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Role of the putative cis-regulatory elements on the activity of the SLC19A2 minimal promoter: mutational analysis. Previous studies from our laboratory with Caco-2 cells identified the minimal promoter region and potential cis-regulatory elements for SLC19A2 (13). To directly examine the role of these putative regulatory sites, we mutated the GKLF/SP-1 (the sites have some shared core elements, see Fig. 3A), NF-1, or SP-1 regulatory sites and then examined the effect of these mutations on the minimal promoter activity in Caco-2 cells (see EXPERIMENTAL PROCEDURES). Our results showed that individual mutation of the GKLF/SP-1, NF-1, or SP-1 site decreased the promoter activity by 70% compared with activity of the control (unmutated) minimal SLC19A2 promoter. For comparison, mutating the AP-1 site or introducing a mutation at a random site did not change the promoter activity (Fig. 2).
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Identification of a DNA/protein profile for specific deletions of the SLC19A2 promoter using EMSA. To further confirm the importance of the cis elements described above (shown in Fig. 3A), we performed EMSA with specific labeled DNA fragments of the minimal SLC19A2 promoter using nuclear extract isolated from Caco-2 cells. The typical mobility shift pattern that we observed for the minimal promoter region included four major DNA/protein complexes (Fig. 3B, lanes 1-4, bands indicated by numbers and arrows). We found that the deletion of the NF-1 and AP-1 sites in the 5' region of the minimal promoter (Fig. 3B, lane 4, fragment 305 to 121) changed the mobility shift pattern of bands 3 and 4, bringing them closer together. Deletion of the SP-1 site in the 3' region of the minimal promoter (Fig. 3B, lane 5, fragment 397 to 251) eliminated either band 3 or 4. Further 5' deletions of the 397 to 251 fragment did not alter the mobility shift pattern (Fig. 3B, lanes 6 and 7).
Competition analysis of the observed SLC19A2 DNA/protein interactions using EMSA. The specificity of the DNA/protein interactions was established using EMSA and competition analysis with unlabeled SLC19A2 minimal promoter DNA or consensus oligonucleotides to specific cis-regulatory elements. All four of the DNA/protein complexes disappear when a 100-fold molar excess of unlabeled fragment of the minimal promoter DNA (397 to 121) is added to the binding mixture (Fig. 4A). When we added a 50-fold molar excess of unlabeled oligonucleotides specific for consensus NF-1 or SP-1 sites to the binding mixture, we observed a decrease in intensity of DNA/protein complexes 3 and 4 (Fig. 4B, lanes 3 and 4). However, oligonucleotides corresponding to consensus binding sites for AP-1, Myc-Max, or a mutated SP-1 did not change the mobility shift pattern (Fig. 4B, lanes 2, 5, and 6). The results were identical; however, the intensity of bands 3 and 4 decreased more dramatically when the amount of competing oligonucleotides was increased 10 times to a 500-fold molar excess (Fig. 4B, lanes 8-11).
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Confirmation of regulatory factor binding to cis elements using EMSA and supershift analysis. To assess the identity of the proteins involved in the EMSA pattern, we used supershift analysis with specific antibodies to GKLF, NF-1, and SP-1. The addition of each antibody to separate reaction mixtures caused the appearance of new supershifted DNA/protein complexes (Fig. 5). The GKLF and NF-1 antibodies caused the appearance of a single supershifted band (Fig. 5A, lanes 3 and 4, bottom arrow on right), whereas the SP-1 antibody caused the appearance of an additional supershifted band (Fig. 5A, lane 5, indicated by both arrows on right). The addition of an equal amount of nonspecific IgG did not alter the binding pattern (Fig. 5A, lane 2). When a labeled SLC19A2 DNA fragment containing only the NF-1 site was used in the binding mixture, a single shifted band was observed (Fig. 5B, lane 1). The band was supershifted when a NF-1-specific antibody was added to the mixture (Fig. 5B, lane 3, indicated at arrow) but not by nonspecific IgG (Fig. 5B, lane 2).
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Activation of the SLC19A2 promoter in Drosophila SL2 cells by the human SP-1 transcription factor. Because SP-1 is expressed in virtually all mammalian cells, we utilized Drosophila SL2 cells, an established in vitro model lacking endogenous SP-1 activity (3), to clearly determine whether the transcription factor specifically activates the SLC19A2 promoter. We cotransfected either the minimal (356 to 36) or the full-length (2,250 to 36) promoter-Luciferase construct along with the Drosophila expression vector pPac-SP1 into Drosophila SL2 cells. Neither the minimal nor full-length promoter alone had strong activity in SL2 cells (Fig. 6). However, addition of the SP-1-containing expression vector led to a significant increase in promoter activity in a dose-dependant manner, resulting in an approximately fourfold activation at 1 µg, approximately fivefold activation at 2 µg, and approximately sixfold activation at 4 µg of pPac-SP1 (Fig. 6).
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Confirmation of promoter activity in vivo using transgenic mice. To further our characterization of the human SLC19A2 promoter, we examined the activities of the full-length and minimal promoter-Luciferase constructs in vivo utilizing transgenic mice. The expertise of the UCI-TGF was used to provide us with transgenic mice. Using PCR on genomic DNA, three mice were determined to be carrying the full-length promoter-Luciferase transgene and seven to be carrying the minimal promoter-Luciferase transgene (Fig. 7). We examined Luciferase activity and RNA levels in specific tissues of transgenic mice carrying the full-length or minimal promoter-Luciferase constructs (Fig. 8). We found that the highest level of Luciferase activity and RNA was in the skeletal muscle, followed (in descending order) by the, heart, brain, liver, kidney, stomach, lung, jejunum, ileum, colon, and duodenum. The results were similar for both the full-length promoter and minimal promoter constructs; however, the Luciferase activity was found to be higher for the minimal compared with full-length promoter. Southern analysis revealed that the higher activity was due to the fact that the minimal construct had multiple insertions into the mouse genome, whereas the full-length construct had a single insertion (data not shown). We performed Luciferase activity and RNA analysis on three founders for each of the transgenic lines and obtained identical results. The data described are representative of the results from one founder for each line.
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DISCUSSION |
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Our in vitro results confirmed that the activity of the SLC19A2 promoter was significantly higher in liver HepG2 cells compared with intestinal epithelial Caco-2 cells. In addition, and in agreement with the transfected promoter activity findings, the level of the endogenous SLC19A2 promoter was found to be more active in HepG2 cells compared with Caco-2 cells when the SLC19A2 RNA level was used as a gauge to measure promoter activity. The results tend to validate the use of our cell culture system for studying effects on SLC19A2 promoter activity in vitro. Furthermore, we utilized Caco-2 cells and promoter mutational analysis to confirm the importance of specific cis-regulatory elements and found that GKLF/SP-1, NF-1, and SP-1 sites are involved in regulating SLC19A2 minimal promoter activity. When we performed mutational analysis with the identical mutated minimal promoter-Luciferase constructs in HepG2 cells, we observed the same reduction in activity for the GKLF/SP-1 and NF-1 site; however, mutation of the SP-1 site did not affect activity (18). These observations suggest that some similar cis elements are important for the promoter activity observed in Caco-2 and HepG2 cells. The difference in the levels of activity in the two cell types may involve the SP-1 site and/or the levels of transcription factors available to the promoter. We continued our analysis of the SLC19A2 minimal promoter in Caco-2 cells by performing EMSA and oligonucleotide competition analysis. We identified four specific DNA/protein complexes, two of which (3 and 4) decreased in intensity in the presence of NF-1 or SP-1 oligonucleotides, confirming the importance of the NF-1 and SP-1 cis-regulatory sites. In addition, we used supershift analysis to add to the study, showing that the transcription factors GKLF, NF-1, and SP-1 form specific DNA/protein complexes with the human SLC19A2 promoter in vitro. The importance of the role of the SP-1 site in promoter activation was further confirmed by cotransfection studies with Drosophila SL2 cells, an established in vitro model lacking endogenous SP-1 activity (3). Our results showed a dose-dependent activation of both the minimal and full-length SLC19A2 promoter by SP-1. Taken together, our in vitro results suggest the possibility that the transcription factors GKLF, NF-1, and SP-1 activate gene expression in vivo, potentially playing a role in the tissue-specific expression pattern observed for the SLC19A2 message, and may further imply that these factors participate in transcriptional regulatory mechanisms that determine thiamin transporter levels and, ultimately, human thiamin homeostasis.
Although we were able to show GKLF, NF-1, and SP-1 directly interacting with the SLC19A2 minimal promoter region, we must mention that we were unable to identify two of the major DNA/protein complexes formed (DNA/protein complexes 1 and 2) and have no information regarding their contribution to promoter activity. We were also unable to definitively determine which specific factor, GKLF, NF-1, SP-1, or some combination, is specifically responsible for the formation of DNA/protein complexes 3 and 4. Work currently being performed in our laboratory will attempt to address these issues.
Our in vivo results using transgenic mice established the functionality of both the full-length and minimal SLC19A2 promoter and determined that the pattern of expression of the SLC19A2 promoter-Luciferase transgene was similar to the reported SLC19A2 RNA expression pattern in native human tissues. Interestingly, the pattern of promoter-Luciferase activity in transgenic mice resembled both the results reported for SLC19A2 RNA tissue expression in humans and mice (4, 6, 7, 9, 12, 13). One important difference, however, is that the promoter-Luciferase activity in transgenic mice more closely mimics the human SLC19A2 RNA pattern in that the highest level of activity is observed in skeletal muscle in contrast to the mouse Slc19a2 RNA expression pattern, in which levels in skeletal muscle are very low.
Our studies support the idea that understanding transcriptional regulation and tissue-specific expression contributes to our knowledge of the physiology of thiamin homeostasis. An important point supporting this idea is that a defect in the gene for the thiamin transporter, SLC19A2, has been shown to specifically affect certain tissues in TRMA patients, suggesting that specific cells require a higher level of expression of the transporter. Therefore, clearly identifying the expression pattern and, more importantly, the transcriptional mechanisms that regulate that pattern will not only be informative in understanding the development of the component disorders present in the disease but also the physiology of specific cells that are influenced by SLC19A2 expression. Many laboratories, including ours, have performed detailed studies to determine the expression patterns for the human and mouse thiamin transporters, allowing one to speculate on the metabolic requirements for thiamin in specific tissues. Our current study expands these investigations by determining transcriptional elements that control expression and establishing a model system that allows the regulatory element of expression, the promoter, to be carefully analyzed in vivo.
In summary, our data show that the activity of the SLC19A2 minimal promoter varies in Caco-2 and HepG2 cells and that this activity correlates well with the endogenous RNA levels. We also demonstrate that the cis elements for GKLF, NF-1, and SP-1 contribute to promoter activity in intestinal epithelial cells, with SP-1 specifically increasing promoter activity, and that the transcription factors are also involved in forming specific DNA/protein complexes with the SLC19A2 minimal promoter. Both the full-length and minimal promoter regions are active in vivo, and the activity mimics the RNA expression pattern observed in humans, as well as our cell culture data. Our conclusion is that human tissues differentially regulate expression levels of SLC19A2 through the promoter and that the cis-regulatory elements and the factors GKLF, NF-1, and/or SP-1 may play a role in the regulation.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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