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Am J Physiol Gastrointest Liver Physiol 287: G822-G829, 2004. First published June 24, 2004; doi:10.1152/ajpgi.00234.2004
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MUCOSAL BIOLOGY

Characterization of the 5'-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

Svetlana M. Nabokina and Hamid M. Said

Veterans Affairs Medical Center, Long Beach, 90822; and University of California College of Medicine, Irvine, California 92697

Submitted 26 May 2004 ; accepted in final form 16 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transcriptional regulation of expression of the human thiamin transporter-2 (the product of the SLC19A3 gene) is unknown. In this study, we cloned the 5'-regulatory region of the human SLC19A3 gene (2,016 bp), identified the minimal promoter region required for basal activity, demonstrated a critical role for specific cis-regulatory elements in determining the promoter activity, and confirmed activity and physiological relevance of the cloned SLC19A3 promoter in vivo. With the use of transiently transfected human intestinal epithelial Caco-2 cells and 5'-deletion analysis, the minimal promoter region required for basal activity of the SLC19A3 promoter was found to be encoded in a sequence between –77 and +59 by using the start of transcription initiation as position 1. This minimal region was found to contain a number of putative cis-regulatory elements, with a critical role for a stimulating protein-1 (SP1)/GC-box binding site (at position –48/–45 bp) established by means of mutational analysis. With the use of EMSA and supershift assays, the binding of SP1 and SP3 to the minimal promoter region was also demonstrated. In transiently transfected Drosophila SL2 cells, both SP1 and SP3 transactivated the SLC19A3 minimal promoter in a dose-dependent manner and in combination demonstrated an additive stimulatory effect. Functionality of the full-length SLC19A3 promoter was confirmed in vivo in transgenic mice expressing the promoter-luciferase reporter gene. These studies report the first characterization of the SLC19A3 promoter in vitro and in vivo and demonstrate the importance of an SP1 cis-regulatory element in regulating promoter activity of this important human gene.

SLC19A3; thiamin transporter; transcriptional regulation

THIAMIN, A WATER-SOLUBLE vitamin, plays an essential role in normal cellular functions via its involvement in key metabolic reactions (2). Thiamin deficiency leads to a variety of clinical abnormalities including neurological and cardiovascular disorders (2, 33, 34, 36), whereas optimization of its level appears to have the potential for preventing diabetic retinopathy and blocking tissue damage caused by hyperglycemia of diabetes (13). Thiamin deficiency and suboptimal levels represent significant nutritional problems (18) and occur in a large percentage of alcoholic (17, 36, 37) and diabetic patients (32) and in patients with celiac and renal diseases (20, 23, 35). Thus studies that lead to an improvement in our understanding of the mechanisms involved in the maintenance of normal thiamin body homeostasis are of significance.

Because it cannot be synthesized in the body, thiamin must be obtained from exogenous sources by absorption in the intestine, and thus the gut plays a critical role in maintaining normal thiamin body homeostasis. Previous studies (6, 7, 16, 28, 30, 31), including those from our laboratory, have shown that thiamin uptake in the human intestine occurs via a specialized carrier-mediated mechanism. The molecular identity of the systems involved has been recently identified, and both of the human thiamin transporters (hTHTRs) hTHTR-1 (the product of the SLC19A2 gene) and hTHTR-2 (the product of the SLC19A3 gene) were found to be expressed in the human intestine (5, 8–10, 15, 22). Expression of hTHTR-1 was found to be at both the apical and basolateral membranes of the polarized enterocytes, whereas that of hTHTR-2 was found to be restricted only to the apical membrane (29). Studies from our laboratory (29) have also shown that both the hTHTR-1 and the hTHTR-2 are involved in carrier-mediated thiamin uptake in the human intestine and that together they account for the total carrier-mediated thiamin uptake. More recent investigations in our laboratory (24, 25) have shown that the process of thiamin uptake in the intestine is regulated during ontogeny and in thiamin deficiency and that this regulation involves transcriptional regulatory mechanisms. With this knowledge in mind and because expression of these two hTHTRs is tissue/cell specific (9, 26, 27), understanding the basal and regulated transcriptional activity of the involved genes is obviously important. To achieve this aim, we have recently cloned the 5'-regulatory region of the human SLC19A2 gene and confirmed its promoter activity first in vitro and then in vivo using transgenic mice (26, 27). In addition, we have identified the minimal region required for basal activity of the SLC19A2 promoter and identified a role of cis-regulatory elements for gut-enriched Krupple-like factor, nuclear factor-1 (NF1), and stimulating protein-1 (SP1) in the regulation of the promoter activity and confirmed the promoter activity in vivo using transgenic mice. Nothing, however, is currently known about the 5'-regulatory region of the human SLC19A3 gene in vitro and in vivo. The aim of this study was, therefore, to address these issues. Our results report the characterization of the SLC19A3 promoter in vitro with confirmation of its activity in vivo using transgenic mice. The results also assign a critical role for an SP1 cis-regulatory element in regulating the activity of the SLC19A3 promoter.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and reagents. [{alpha}-32P]CTP (3,000 Ci/mmol) was purchased from PerkinElmer Life and Analytical Sciences. Synthetic oligonucleotides were purchased from Sigma Genosys (Woodlands, TX). Lipofectamine reagent was purchased from Life Technologies (Rockville, MD). Routine biochemicals and cell culture reagents were all of molecular biology quality and were purchased from Fisher Scientific (Tustin, CA) and Sigma (St. Louis, MO). SP1-containing plasmid was kindly provided by Dr. Robert Tjian (University of California, Berkeley, CA), the pPacSP3 construct was kindly provided by Dr. Guntram Suske (Philipps-Universität, Marburg, Germany), and pPacSP1 and pPac0 were made in our laboratory (27).

Cell culture. The Caco-2 (human colonic adenocarsinoma) and Scheider’s Drosophila SL2 cell lines were purchased from the American Type Culture Collection (Manassas, VA). Caco-2 cells were grown in DMEM supplemented with 10% fetal bovine serum, glutamine (0.29 g/l), sodium bicarbonate (2.2 g/l), penicillin (100,000 U/l), and streptomycin (10 mg/l) in an atmosphere of 5% CO2-95% air at 37°C. SL2 cells were grown at room temperature in Schneider’s insect medium (Sigma) containing 10% fetal bovine serum and antibiotics.

5'-Rapid amplification of the cDNA ends. Transcription initiation site(s) for SLC19A3 in Caco-2 cells was identified with the rapid amplification of the cDNA ends (RACE) technique using 5'-RACE kit version 2.0 (Life Technologies). The sequence information for the human SLC19A3 cDNA deposited in GenBank (accession no. AF283317) was used as a guide for primer design. Five micrograms of total RNA isolated from Caco-2 cells was used with gene-specific reverse primer 5'-GCACCGACCCTGCTGTGTA-3' in the initial RT-PCR. The first-strand cDNAs were isolated and tailed. The PCR of tailed cDNAs was then performed by using the gene-specific reverse primer 5'-CAACAGCAGCAGCCAGGTAATGATGAAACT-3' and the manufacturer’s Abridged Anchor primer. A subsequent nested amplification was performed by using the manufacturer’s Abridged Universal Amplification primer and the gene-specific primer 5'-CCAGGAACTGCTTAGTGAAGTTCTGTAA-3'. PCR products were analyzed on a 2% agarose gel and subcloned into the pGEM-T Easy vector (Promega, Madison, WI). The DNA sequence was verified by the Laragen Sequencing Facility (Los Angeles, CA).

Cloning of the 5'-regulatory region for the SLC19A3 gene. The sequence information for the SLC19A3 gene and flanking sequence deposited in GenBank (accession no. AC064853; definition-Homo sapiens bacterial artificial chromosome clone RP11–90L9 from 2 complete sequences) was used to design PCR cloning primers. A PCR was performed by using two gene-specific primers (forward: 5'-CGGGGTACCAGACTGTGGCTATGAGATATTAGA-3' and reverse: 5'-CTAGCTAGCATCGCTCACTTGCCGCACGA-3') and 100 ng of human genomic DNA (Clontech). PCR conditions were denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 4 min, and then a final extension at 72°C for 10 min. The 2,016-bp product was isolated on a 0.7% agarose gel, cloned into the pGEM-T Easy vector, digested with KpnI and NheI (these sites were designed into the primers), and subcloned into the KpnI/NheI cut pGL3-Basic luciferase reporter vector (Promega) upstream of the luciferase gene. The DNA sequence was verified by the Laragen Sequencing Facility.

SLC19A3 promoter-luciferase reporter constructs. 5'-Deleted constructs were made by PCR using gene-specific primers (Table 1) followed by subcloning of the PCR products into the KpnI/NheI cut pGL3-Basic vector. 3'-Deletion (construct –1957/–287) was introduced by restriction endonuclease digestion with HindIII and NheI followed by Klenow fill-in and self-ligation. Mutations of consensus sites for transcriptional factors were introduced into the minimal promoter region with QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the primers containing mutated nucleotides (Table 2). Sequence of all constructs was verified by sequencing (Laragen Sequencing Facility) before use.


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  		Table 1. Sequence of primers used in PCR reactions for preparation of 5' deleted mutants of SLC19A3 promoter

 

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  		Table 2. Sequence of primers used in PCR for developing mutant SLC19A3 minimal promoter constructs

 
Transfection and reporter gene assay. Caco-2 cells were cotransfected in 12-well plates at 70–80% confluency with 2 µg of each test construct and 100 ng of the Renilla transfection control plasmid Renilla luciferase-thymidine kinase (pRL-TK) (Promega). Transfection was performed with Lipofectamine reagent (Life Technologies) according to the manufacturer’s instructions. Cells were harvested at 2–3 days after transfection and Renilla-normalized firefly luciferase activity was measured by using the Dual Luciferase Assay system (Promega). Data are presented as means ± SE of at least three independent experiments and given as fold over pGL3-Basic expression set arbitrarily at one. Drosophila SL2 cells were seeded at ~5 x 105 cells per well in 12-well plates 24 h before transfection with Lipofectamine reagent. Two micrograms of pGL3-Basic or SLC19A3 promoter construct were used along with varying amounts of the control plasmid pPac0 and 0, 0.1, 1, or 2 µg of the Drosophila SP expression plasmid (pPacSP1 or pPacSP3), keeping the total amount of DNA constant at 4 µg (4). At 48-h posttransfection, cells were harvested and firefly luciferase activity was assayed with the Luciferase Assay system (Promega). Luciferase activity was normalized to protein concentrations of all the cell lysates. Data presented are means ± SE of at least three independent determinations and given as fold over the level of luciferase expression for each construct in the absence of coexpression of exogenous SP protein that was set arbitrarily at one.

EMSA. Nuclear extract from Caco-2 cells were prepared by standard methods (1). Gel shift assays were performed with a commercially optimized kit (SP1 Gelshift kit; Active Motif, Carlsbad, CA) designed for characterization of SP1-like promoter elements. Binding reactions were carried out with 5–8 µg of nuclear extract and [{alpha}-32P]CTP end-labeled DNA fragment at 4°C. Oligonucleotide competition analysis was conducted with a 100- to 200-fold molar excess of commercial competitor oligonucleotides (Santa Cruz Biotechnologies, Santa Cruz, CA) or a 50-fold molar excess of unlabeled SLC19A3 DNA fragment. For the supershift assays, the nuclear extract was pretreated with nonspecific IgG, anti-SP1 (Active Motif), or anti-SP3 (Santa Cruz Biotechnologies) antibodies. DNA-protein complexes were separated on 3.2% nondenaturing polyacrylamide gel in 0.25x Tris borate-EDTA (pH 8.4) at 4°C and at 200 V.

Generation of transgenic mice and luciferase analysis. The 2,016-bp cloned 5'-regulatory region of the SLC19A3 gene fused to the firefly luciferase reporter gene was used to generate transgenic founders utilizing the expertise of the transgenic mouse facility of our university as described by us recently (27). Pups were genotyped by performing a PCR with specific primers for the human SLC19A3 promoter (forward 5'-CGGGGTACCGTGCAGAGTGATTATAAC-3') and luciferase gene (reverse 5'-ATGCGAGAATCTCACGCAG-3') that would yield a 592-bp product. The transgenic mice were paired with wild-type CB6F2 littermates to ensure that the transgene was passed to the F1 progeny and to establish transgenic human SLC19A3 promoter-luciferase mouse colonies.

For transgene assays, specific mouse tissues were removed and homogenized in ice-cold passive lysis buffer (Promega) using a PowerGen125 (Fisher, Pittsburgh, PA) hand blender, and clarified (25,000 g; 10 min) homogenates were taken for measurement of firefly luciferase activity using a Luciferase Assay system. Luciferase activity was normalized to total protein concentration for each sample.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Determination of the transcriptional start site of SLC19A3 in Caco-2 cells. In this study, we used the rapid amplification of the cDNA ends (RACE) technique to identify the transcription start site(s) of SLC19A3 in Caco-2 cells to help locate the putative promoter for further investigation. Several clones for the single prominent PCR product of ~170 bp were obtained from 5'-RACE and were sequenced. The results showed the potential existence of only one transcription initiation site for SLC19A3 in Caco-2 cells, which begins at nt-88 using the A in the initiator ATG sequence as position 1.

Identification of the SLC19A3 promoter. In these studies, we sought to identify the 5'-regulatory region of the SLC19A3 gene and focused the promoter search in the area upstream of the transcription initiation site. With the use of the originally reported sequence information for the SLC19A3 gene (9) and flanking sequence as a guide for primer design, a 2,016-bp genomic fragment was cloned from human genomic DNA. The fragment begins inside exon I of the SLC19A3 gene at the position +59 bp (using transcription initiation site as +1) and extends to –1,957 bp upstream of the transcription initiation site. Because the translation initiator ATG sequence is located inside exon II of the SLC19A3 gene, which is separated from exon I by an intron of ~15 kb, the isolated 5'-flanking region of SLC19A3 gene did not contain the translation initiation site. The identity of the genomic DNA fragment was established by sequencing. Activity of the cloned putative human SLC19A3 promoter was examined in a transient transfection experiment, using the firefly luciferase reporter construct and the human intestinal epithelial Caco-2 cells. As shown in Fig. 1, the cloned 5'-flanking region of the SLC19A3 gene was found to have significant promoter activity in transiently transfected Caco-2 cells.



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  		Fig. 1. SLC19A3 promoter activity in Caco-2 cells and deletion analysis of the SLC19A3 promoter. 5' or 3' deletions were introduced into the 2,016-bp full-length SLC19A3 promoter as described in MATERIALS AND METHODS. Promoter constructs in pGL3-Basic were transiently expressed in Caco-2 cells for luciferase assays. Data are reported as relative firefly luciferase activity normalized to Renilla luciferase activity and represent means ± SE of at least 3 independent experiments.

 
Determination of the minimal SLC19A3 promoter and identification of putative cis-regulatory elements. To determine the minimal region required for basal activity of the SLC19A3 promoter, a series of reporter plasmids containing specific lengths of the SLC19A3 5'-flanking region (ranging from nt –1957 to nt –13) upstream of the firefly luciferase gene were transiently transfected into Caco-2 cells. Analysis of luciferase activity (Fig. 1) showed that a short promoter fragment spanning nt –77 to +59 bp conferred ~70% of the activity of the cloned 2,016-bp promoter fragment. A further deletion of 48 bp resulted in a drastic reduction in promoter activity, whereas deletion of an additional 16 bp virtually abolished the activity. We concluded that the minimal 5'-flanking region of the SLC19A3 gene required for basal promoter activity is encoded in a sequence between –77 and +59.

To investigate the possible existence of putative cis-regulatory elements in the minimal SLC19A3 promoter, we subjected the DNA genomic fragment to computer analysis using MatInspector (21). Results of the search showed the existence of a number of putative cis-regulatory elements for several transcription factors that include octamer factor 1 (OCT1), NF1, early growth response gene (EGR)2/EGR3, and SP1 guanosine cytidine (GC) box (Fig. 2). To investigate the possible role of these cis-elements in governing the basal activity of the SLC19A3 promoter, we examined the functional consequences of introducing a specific mutation at these distinct sites (by means of site-directed mutagenesis) on promoter activity after transfection into Caco-2 cells. The results showed that mutating of the OCT1, EGR2/EGR3, NF1, and the proximal (–15/–12 bp) SP1/GC box sites had no negative effect on activity of the SLC19A3 minimal promoter. In contrast, mutating the distal (–48/–45 bp) SP1/GC box binding site led to a significant (P < 0.01) reduction in promoter activity to <45% that of the control (i.e., unmutated minimal promoter). These findings suggest a role for the distal SP1/GC box binding site in regulating the activity of the minimal SLC19A3 promoter. It is of interest to note here that mutating of the OCT1 site led to some increase in SLC19A3 promoter activity suggesting possible suppressive role of this site in the regulation of SLC19A3 promoter.



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  		Fig. 2. Schematic of the SLC19A3 minimal promoter region and mutational analysis of cis-regulatory elements. A: schematic is shown depicting the minimal promoter sequence (–77 to +59) with annotated putative cis-elements (bold letters). Cis-elements on the complementary strand are shown in italicized letters. The nucleotides that were altered for mutational analysis are underlined. The transcription initiation site is underlined and numbered as +1. B: two conserved nucleotides in the core consensus binding site for transcriptional factors were changed as described in MATERIALS AND METHODS. The mutated minimal promoter constructs in pGL3-Basic were transiently expressed into Caco-2 cells for luciferase assays. Data are reported as relative firefly luciferase activity normalized to Renilla luciferase activity and represent means ± SE of at least 3 independent experiments. NF1, nuclear factor 1; SP1, stimulating protein-1; OCT1, octamer factor 1; EGR, early growth response gene.

 
SP1/SP3 binding in the minimal SLC19A3 promoter. To demonstrate whether or not SP1 binds in the SLC19A3 core promoter, the promoter region was tested by using EMSA with nuclear extract from Caco-2 cells. The minimal promoter formed several complexes resulting in two major bands, bands 1 and 2, with decreased gel mobility (Fig. 3). Interestingly, the similar mobility shift pattern was obtained for a shorter promoter fragment –52/+59 bp, lacking potential sites for OCT1 and EGR2/EGR3, but still containing SP1/GC box sites. These two major DNA/protein complexes are specific, because both of them were competed away with unlabeled probe (SLC19A3 minimal promoter) and unlabeled consensus SP1 oligonucleotides (Fig. 4). However, these DNA/protein complexes were not competed away with the oligonucleotides corresponding to consensus binding sites for other transcription factors (AP1, AP2, NF-{kappa}B, Myc-Max). Furthermore, a mutated SP1 oligonucleotide was found to be ineffective in competing with the minimal promoter for formation of complexes 1 and 2.



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  		Fig. 3. DNA/protein profile from EMSA using the SLC19A3 minimal promoter region. Gel shift assays were conducted with Caco-2 nuclear extracts as described in MATERIALS AND METHODS. The labeled fragment from the SLC19A3 promoter region used in binding reaction is shown at top with the numbered position indicative of the distance from the transcription initiation site represented as position +1. Major DNA/protein complexes are numbered and indicated with arrows.

 


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  		Fig. 4. EMSA with oligonucleotide competition. Gel shift assays were performed with Caco-2 nuclear extracts as described in MATERIALS AND METHODS. A: gel mobility shift assays were conducted with a labeled (–77 to +59) SLC19A3 minimal promoter (no oligo) or 50-fold molar excess of an identical unlabeled minimal promoter (50X self). B: gel mobility shift assays were conducted with a labeled (–77 to +59) SLC19A3 minimal promoter and specific oligonucleotides to the consensus sequences for specific cis-regulatory elements shown at top.

 
Previous studies (11, 19) have shown that the SP1 consensus sequence binds not only SP1 but also SP3 with similar affinity. To assess the identity of the proteins involved in our EMSA pattern, we used supershift analysis with the specific antibodies to SP1 and SP3. Antibodies were added to Caco-2 nuclear extract before the binding reactions. As shown in Fig. 5, both of the SP1 and SP3 antibodies cause supershift in bands 1 and 2. In contrast, a nonspecific IgG antibody had no effect on DNA/protein complex mobility. These results suggest that both of the NFs SP1 and SP3 are able to bind to the 136-bp SLC19A3 minimal promoter region.



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  		Fig. 5. Binding of SP1 and SP3 transcription factors to the SLC19A3 minimal promoter. Gel shift assays were conducted with Caco-2 nuclear extracts as described in MATERIALS AND METHODS. Labeled (–77 to +59) minimal promoter was used as a probe in the absence (no antibody) or presence of anti-SP1 (SP1), anti-SP3 (SP3) polyclonal antibodies, or nonspecific IgG (IgG). Arrows and numbers indicate the two major DNA/protein complexes with arrows at top indicating supershifted bands.

 
Transactivation of the SLC19A3 minimal promoter by SP1 and SP3. SP1 has been shown to be primarily a positive transactivating factor (19), whereas SP3 acts as a transcription activator or a transcription repressor depending on cell/promoter type (11, 12, 14, 19). To compare the effects of SP1 and SP3 on activity of the SLC19A3 minimal promoter, we performed cotransfection assays in Drosophila SL2 cells. These cells represent an established in vitro model system that lacks endogenous SP activity, thus allowing testing of exogenous SP expression on activity of SP-dependent promoters (4). A promoter-luciferase construct containing the minimal SLC19A3 promoter region was transfected into SL2 cells with varying amounts of Drosophila expression vectors pPacSP1 and pPacSP3, containing human SP1 or SP3, respectively. The level of reporter gene expression detected for the construct in the absence of coexpression of exogenous SP protein was assigned the arbitrary value of 1. Addition of the individual SP-containing expression vector, i.e., pPacSP1 and pPacSP3, led to a significant increase in SLC19A3 promoter activity in a dose-dependent manner for both SP1 (P < 0.01, 3- to 22-fold)- and SP3 (P < 0.01, 3- to 20-fold)-transfected cDNAs (Fig. 6). We also examined the effect of cotransfecting of the SL2 cells with a combination of pPacSP1 and pPacSP3 plasmids on activity of the minimal SLC19A3 promoter. The results showed an additive stimulatory effect of both transcription factors on promoter activity.



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  		Fig. 6. cotransfection assays using either SP1- or SP3-containing vectors and SLC19A3 minimal promoter-luciferase construct in Drosophila SL2 cells. SL2 cells were cotransfected with 2 µg of the minimal promoter construct in pGL3-Basic and 0.1 µg, 1 µg, or 2 µg of either control plasmid pPac0 or Drosophila SP expression plasmids. At 48 h posttransfection, cells were harvested and firefly luciferase activity was assayed as described in MATERIALS AND METHODS. A: dose-dependent transactivation of the minimal promoter with pPacSP1. B: dose-dependent transactivation of the minimal promoter with pPacSP3. C: combined transactivation of the minimal promoter with pPacSP1 and pPacSP3. Data are reported as relative firefly luciferase activity normalized to protein concentration and represent means ± SE of at least 3 independent determinations.

 
Confirmation of SLC19A3 promoter activity in vivo. In these studies, our aim was to confirm the activity of the cloned SLC19A3 promoter in vivo and to establish its physiological relevance. For this aim, we generated transgenic mice that carry the full-length SLC19A3 promoter fused to the firefly luciferase reporter gene. Three founders were obtained and used to generate the transgenic mouse colony. Luciferase activity was examined in specific tissues of these mice (identical tissues from nontransgenic mice served as controls). The results showed the cloned SLC19A3 promoter to be indeed functional in vivo in specific mouse tissues. The level of expression of the reporter gene, however, was found to vary from one tissue to another with the level being highest in the brain but less (in descending order) in the heart, skeletal muscles, colon, ileum, jejunum, lungs, kidney, and liver (Fig. 7).



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  		Fig. 7. Determination of SLC19A3 promoter-luciferase activity in transgenic mice. Full-length (–1957 to +59) promoter-luciferase activity in intestine (A) or other specific tissues (B) of transgenic mice was determined as described in MATERIALS AND METHODS. Data are presented in relative light units over nontransgenic control tissues, represent 6 mice from 1 founder line, and are shown as means ± SE.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The aim of this study was to identify the 5'-regulatory region of the SLC19A3 gene and to characterize its activity both in vitro and in vivo. To locate the putative promoter region, we first determined the transcription initiation site(s) of SLC19A3 in Caco-2 cells and found one that begins at position –88 (using the A in the initiator ATG sequence as position 1). On the basis of this information, we cloned a 2,016-bp upstream DNA genomic fragment and showed that this region has significant promoter activity in transiently transfected Caco-2 cells. We then characterized the cloned promoter and determined the minimal region required for its basal activity, examined the role of putative cis-regulatory elements encoded in this minimal region, and determined the NFs that interact with them. We also confirmed functionality and physiological relevance of the cloned promoter in vivo in transgenic mice.

The cloned SLC19A3 promoter region was found to have characteristics typical of a housekeeping gene, such as high GC content, lack of obvious TATA or CCAAT regulatory sequences, and the presence of two SP1/GC box cis-elements (3). With the use of 5'-deletion analysis, the minimal SLC19A3 promoter was localized to position –77 to +59, using the transcription start site as position 1. This region was found to have a number of putative cis-regulatory elements. To examine the role of these cis-elements in regulating the activity of the SLC19A3 promoter, we mutated these sites individually and tested the effect of such mutations on the activity of the minimal promoter. Our results showed that mutating of the OCT1, EGR2/EGR3, NF1, and the proximal (–15/–12 bp) SP1/GC box sites had no negative effect on promoter activity. On the other hand, mutating of the distal (–48/–45 bp) SP1/GC box site led to a significant reduction in promoter activity. On the basis of these observations, we concluded that the distal SP1/GC box binding site of the minimal SLC19A3 promoter region is important for governing basal activity. The inability to completely abolish promoter activity in mutational experiments may reflect the partial binding of the transcription factor to the mutated promoter region and/or the possible contribution of accessory proteins that coordinate the binding of transcription factor to promoter.

We continued our analysis of the minimal SLC19A3 promoter region by performing EMSA and oligonucleotide competition analysis using nuclear extracts from Caco-2 cells. We identified two major specific DNA/protein complexes. Both of these complexes were competed away with unlabeled consensus SP1 oligonucleotides, but the oligonucleotides corresponding to consensus binding sites for other transcription factors as well as a mutated SP1 oligonucleotide were found to be ineffective in competing with the minimal promoter for formation of the DNA/protein complexes. From these results we concluded that SP1-related proteins from Caco-2 cells appear to be involved in binding with the minimal SLC19A3 promoter region.

To date, two SP1-related proteins (SP3 and SP4) containing highly conserved zinc finger DNA binding domains have been found to recognize the consensus GC box with specificity and affinity similar to that of SP1 (11, 19). The other member of the zinc finger SP1 multigene family, SP2, binds to the GC box with a much lower affinity. Moreover, SP1 and SP3 are ubiquitously expressed in many cell types, whereas SP4 expression is restricted to certain cell types of the brain (11). To determine whether SP1 and/or SP3 bind to the GC box of the SLC19A3 minimal promoter, we performed supershift analysis using the specific anti-SP1 and anti-SP3 antibodies. The results showed a clear supershift of the identified DNA/protein complexes using either antibody suggesting the specific binding of SP1 as well as SP3 to the minimal promoter region using a Caco-2 cell nuclear extract.

Whereas SP1 has only been implicated as an activator of transcription, SP3 is a bifunctional transcriptional regulator and can act as activator or repressor of transcription depending on the cell/promoter type (11, 12, 14, 19). We found that in SP-deficient Drosophila SL2 cells, SP1 and SP3 were both able to stimulate transcriptional activity of the SLC19A3 minimal promoter in a dose-dependent manner. When added together in combination transfection experiments, the stimulatory effects of SP1 and SP3 transcription factors was found to be additive. Because, as mentioned above, SP3 recognizes the SP1/GC box with specificity and affinity similar to that of SP1 (11, 19), our latter finding from combination transfection experiment may suggest that SP1 and SP3 bind to the same SP1/GC box in basal SLC19A3 promoter. Taken together, our results on characterization of SLC19A3 minimal promoter region show that SP1/GC box is the major site of regulation and this regulation may involve SP1 and/or SP3. These findings, however, do not eliminate the possibility that other cis- and trans-acting elements may also contribute to minimal promoter activity. Previously, we (27) have shown that the minimal promoter region of the other thiamin transporter SLC19A2 contains several cis-regulatory elements important for promoter activity, including SP1. In this regard, it is interesting to note that SP1 is involved in the regulation of basal promoter activity of both thiamin transporters, and thus this NF appears be important for regulation of both thiamin transporter genes.

To extend our characterization of the SLC19A3 promoter, we studied the activity of a 2,016-bp full-length SLC19A3 promoter region fused to the firefly luciferase reporter gene in vivo using transgenic mice. Our results confirmed the functionality of the full-length SLC19A3 promoter in vivo. The pattern of expression of the SLC19A3 promoter-luciferase transgene in mice was found to be similar to that of the reported SLC19A3 RNA expression in specific native human tissues (9) with a considerable level of expression in heart and skeletal muscles and low levels in lung, colon, and small intestine. However, in contrast to the human SLC19A3 expression pattern, the level of transgene expression in mice was very low in kidney and liver. Interestingly, the highest level of the SLC19A3 promoter-luciferase transgene expression was found in mouse brain. This observation is in agreement with the previously reported high expression of mouse Slc19a3 in the brain (9). Expression of the human SLC19A3 promoter-luciferase transgene in vivo was also found to be higher in mouse ileum compared with jejunum. This finding corresponds with the higher maximum velocity value observed for thiamin uptake in human ileal brush-border membrane vesicles (BBMV) compared with maximum velocity of thiamin uptake in human jejunal BBMV (7).

In conclusion, this study represents the first characterization of the SLC19A3 promoter in vitro and in vivo and reports the identification of transcription factors critical for maintaining basal activity of the promoter. These findings should serve as a base for future studies in the physiology of thiamin homeostasis and the pathophysiology of thiamin-related disorders.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Department of Veterans Affairs Grants and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56061 and DK-58057.


   ACKNOWLEDGMENTS
 
We thank Dr. J. C. Reidling for critical discussion and helpful review of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. M. Said, Veterans Affairs Medical Center-151, Long Beach, CA 90822 (E-mail: hmsaid{at}uci.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ausubel FM. Current Protocols in Molecular Biology. New York: Wiley, 1997.
  2. Berdanier CD. Advanced Nutrition Micronutrients. New York: CRC, 1998. p. 80–88,
  3. Bonham K and Fujita DJ. Organization and analysis of the promoter region and 5' non-coding exons of the human c-src proto-oncogene. Oncogene 8: 1973–1981, 1993.[Web of Science][Medline]
  4. Courey AJ and Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell 55: 887–888, 1988.[CrossRef][Web of Science][Medline]
  5. Diaz GA, Banikazemi M, Oishi K, Desnick RJ, and Gelb BD. Mutations in a new gene encoding a thiamine transporter cause thiamine-responsive megaloblastic anemia syndrome. Nat Genet 22: 309–312, 1999.[CrossRef][Web of Science][Medline]
  6. Dudeja PK, Tyagi S, Gill R, and Said HM. Evidence for carrier-mediated mechanism for thiamine transport to human jejunal basolateral membrane vesicles. Dig Dis Sci 48: 109–115, 2003.[CrossRef][Web of Science][Medline]
  7. Dudeja PK, Tyagi S, Kavilaveettil RJ, Gill R, and Said HM. Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles. Am J Physiol Cell Physiol 281: C786–C792, 2001.[Abstract/Free Full Text]
  8. Dutta B, Huang W, Molero M, Kekuda R, Leibach FH, Devoe LD, Ganapathy V, and Prasad PD. Cloning of the human thiamine transporter, a member of the folate transporter family. J Biol Chem 274: 31925–31929, 1999.[Abstract/Free Full Text]
  9. Eudy JD, Spiegelstein O, Barber RC, Wlodarczyk BJ, Talbot J, and Finnell RH. Identification and characterization of human and mouse SLC19A3 gene: a novel member of the reduced folate family of micronutrient transporter genes. Mol Genet Metab 71: 581–590, 2000.[CrossRef][Web of Science][Medline]
  10. Fleming JC, Tartaglini E, Steinkamp M, Schorderit DF, Cohen N, and Neufeld EJ. The gene mutated in thiamine-responsive anemia with diabetes and deafness (TRMA) encodes a functional thiamine transporter. Nat Genet 22: 305–308, 1999.[CrossRef][Web of Science][Medline]
  11. Hagen G, Muller S, Beato M, and Suske G. Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res 20: 5519–5525, 1992.[Abstract/Free Full Text]
  12. Hagen G, Muller S, Beato M, and Suske G. Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J 13: 3843–3851, 1994.[Web of Science][Medline]
  13. Hammes HP, Du X, Edelstein D, Taguchi T, Matsumura T, Ju Q, Lin J, Bierhaus A, Nawroth P, Hannak D, Neumaier M, Bergfeld R, Giardino I, and Brownlee M. Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nat Med 9: 294–299, 2003.[CrossRef][Web of Science][Medline]
  14. Kiela PR, LeSueur J, Collins JF, and Ghishan FK. Transcriptional regulation of the rat NHE3 gene. J Biol Chem 278: 5659–5668, 2003.[Abstract/Free Full Text]
  15. Labay V, Raz T, Baron D, Mandel H, Williams H, Barrett T, Szargel R, McDonald L, Shalata A, Nosaka K, Gregory S, and Cohen N. Mutation in SLC19A2 cause thiamine-responsive megaloblastic anemia associated with diabetes mellitus and deafness. Nat Genet 22: 300–304, 1999.[CrossRef][Web of Science][Medline]
  16. Laforenza U, Patrini C, Alvisi C, Faelli A, Licandro A, and Rindi G. Thiamine uptake in human intestinal biopsy specimens, including observations from a patient with acute thiamin deficiency. Am J Clin Nutr 66: 320–326, 1997.[Abstract/Free Full Text]
  17. Leevy CM and Baker H. Vitamins and alcoholism. Am J Clin Nutr 21: 1325–1328, 1968.[Web of Science][Medline]
  18. Nichols HK and Basu TK. Thiamin status of the elderly: dietary intake and thiamin pyrophosphate response. J Am Coll Nutr 13: 57–61, 1994.[Abstract]
  19. Philipsen S and Suske G. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res 27: 2991–3000, 1999.[Abstract/Free Full Text]
  20. Pietrzak I, Baczyk K, and Mlynarczyk M. Content of thiamin in plasma and erythrocytes in patients with end stage renal disease. Przegl Lek 53: 423–426, 1996.[Medline]
  21. Quandt K, Frech K, Karas H, Wingender E, and Werner T. MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 23: 4878–4884, 1995.[Abstract/Free Full Text]
  22. Rajgopal A, Edmondson A, Goldman D, and Zhao R. SLC19A3 encodes a second thiamine transporter ThTr2. Biochim Biophys Acta 1537: 175–178, 2001.[Medline]
  23. Raskin NH and Fishman RA. Neurological disorders in renal failure. N Engl J Med 294: 204–210, 1976.[Web of Science][Medline]
  24. Reidling JC, Balamurugan K, Nabokina S, and Said HM. Adaptive regulation of intestinal thiamin uptake: molecular mechanisms in wild type and transgenic mice (Abstract). Gastroenterology 126, Suppl 2: A–61, 2004.
  25. Reidling JC, Balamurugan K, Nabokina S, and Said HM. Ontogenic regulation of intestinal thiamin uptake: molecular mechanisms in wild type and transgenic mice (Abstract). Gastroenterology 126, Suppl 2: A–659, 2004.
  26. Reidling JC, Subramanian V, Dudeja P, and Said HM. Expression and promoter analysis of SLC19A2 in the human intestine. Biochim Biophys Acta 1561: 180–187, 2002.[Medline]
  27. Reidling JC and Said HM. In vitro and in vivo characterization of the minimal promoter region of the human thiamin transporter SLC19A2. Am J Physiol Cell Physiol 285: C633–C641, 2003.[Abstract/Free Full Text]
  28. Rindi G and Ferrari G. Thiamine transport by human intestine in vitro. Experientia 33: 211–213, 1977.[CrossRef][Web of Science][Medline]
  29. Said HM, Balamurugan K, Subramanian VS, and Marchant JS. Expression and functional contribution of hTHTR-2 in thiamin absorption in human intestine. Am J Physiol Gastrointest Liver Physiol 286: G491–G498, 2004.[Abstract/Free Full Text]
  30. Said HM, Ortiz A, Kumar C, Chatterjee N, Dudeja P, and Rubin S. Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2. Am J Physiol Cell Physiol 277: C645–C651, 1999.[Abstract/Free Full Text]
  31. Said HM, Ortiz A, Subramanian VS, Neufeld EJ, Moyer MP, and Dudeja PK. Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460. Am J Physiol Gastrointest Liver Physiol 281: G144–G150, 2001.[Abstract/Free Full Text]
  32. Saito N, Kimura M, Kuchiba A, and Itokawa Y. Blood thiamine levels in outpatients with diabetes mellitus. J Nutr Sci Vitaminol (Tokyo) 33: 421–430, 1987.[Medline]
  33. Tallaksen CM, Bell H, and Bohmer T. Thiamine and thiamine phosphate ester deficiency assessed by high performance liquid chromatography in four clinical cases of Wenicke encephalopathy. Alcohol Clin Exp Res 17: 712–716, 1993.[CrossRef][Web of Science][Medline]
  34. Tanphaichirt V. Modern Nutrition in Health and Disease, edited by Shils ME, Olsen JA, and Shike M. New York: Lea and Febiger, 1994, p. 359–375,
  35. Thomson AD. The absorption of sulfur-labeled thiamin hydrochloride in control subjects and in patients with intestinal malabsorption. Clin Sci 31: 167–179, 1966.[Web of Science][Medline]
  36. Tomasulo PA, Kater RMH, and Iber FL. Impairment of thiamine absorption in alcoholism. Am J Clin Nutr 21: 1341–1344, 1968.[Abstract]
  37. Victor M, Adams RD, and Collins GH. The Wernicke-Korsakoff Syndrome and Related Neurological Disorders Due to Alcoholism and MalNutrition. Philadelphia, PA: Davis, 1989.



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