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Originally published In Press as doi:10.1074/jbc.M412662200 on December 3, 2004

J. Biol. Chem., Vol. 280, Issue 7, 5588-5597, February 18, 2005
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Structure and Regulation of the Murine Reduced Folate Carrier Gene

IDENTIFICATION OF FOUR NONCODING EXONS AND PROMOTERS AND REGULATION BY DIETARY FOLATES*

Mingjun Liu{ddagger}, Yubin Ge{ddagger}§, Diane C. Cabelof¶, Amro Aboukameel{ddagger}, Ahmad R. Heydari¶, Ramzi Mohammad{ddagger}, and Larry H. Matherly{ddagger}§||

From the {ddagger}Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, the §Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, and the Department of Nutrition and Food Science, Wayne State University, Detroit, Michigan 48201

Received for publication, November 9, 2004 , and in revised form, November 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The upstream structure and regulation of the mouse reduced folate carrier (mRFC) gene was characterized. By 5'-rapid amplification of cDNA ends assay and DNA sequencing from mouse tissues and 7–15-day stage embryos, mRFC transcripts with four unique 5' noncoding exons, designated mRFC-a,-b,-c, and -d, were identified mapping over 6300 bp. The 5' noncoding exons were characterized by multiple transcription starts and, for form b, two alternate splice forms. mRFC transcript forms were measured by real-time reverse transcription-PCR in mouse tissues and embryos and in L1210 leukemia and BNL CL.2 liver cell lines. The highest mRFC levels were detected in kidney and brain. mRFC-b and -c were the major transcript forms, with low levels of mRFC-a and -d. The 5'-flanking regions for exons a–d each exhibited promoter activity in reporter gene assays. mRFC transcripts and individual noncoding exons were measured in small intestine and kidney from mice fed folate-deficient or -replete diets. Mice fed the folate-deficient diet exhibited a significant (13.8-fold) increase in total mRFC transcripts and protein in the small intestine, reflecting increases in each of the mRFC-b, -c, and -d forms. Only minor changes in mRFC transcript levels or distributions were detected for kidney. Levels of folate-binding protein {alpha} were also increased in both small intestine and kidney in folate-deficient mice (91- and 2-fold, respectively). Multidrug resistance-associated proteins 1 and 3 were, likewise, elevated in intestine from folate-deficient mice (53- and 168-fold, respectively); however, there were no significant changes in kidney. Our results document the existence of four unique noncoding exons and promoters for mRFC and demonstrate a facile induction of mRNAs for mRFC and multidrug resistance-associated proteins 1 and 3 in intestine in response to changes in dietary folate intake.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tetrahydrofolate cofactors are essential to cell survival, since they participate in one-carbon transfer reactions in biosynthetic pathways leading to the purine nucleotides, thymidylate, serine, and methionine (1). Since mammalian cells cannot synthesize folates de novo, they must obtain these derivatives from the outside environment. A number of transport systems for folates have been described, including: the family of membrane-localized folate-binding proteins (FBPs)1 that mediate folate uptake by an endocytotic process (2); the reduced folate carrier (RFC) (3, 4) and a low pH folate transporter (5), both facilitative uptake carriers; and assorted export proteins typified by the family of multidrug resistance proteins (MRPs), MRP1, -3, and -4 (6–10).

The best characterized route for the membrane transport of tetrahydrofolate cofactors into mammalian cells and tissues is the ubiquitously expressed RFC (3, 4). Reflecting its profound physiologic and pharmacologic importance, there has been intense interest in the molecular biology and regulation of RFC. The mouse RFC (mRFC) cDNA was the first RFC cDNA to be cloned in 1994 (11), and this was soon followed by homologous human RFC (hRFC) cDNAs (12–15). These developments paved the way for the structural characterization of the mRFC gene in 1997 (16, 17) and the hRFC gene in 1998 (18, 19). Both mRFC and hRFC transcripts are characterized by heterogeneous 5'-ends (12, 16, 17, 20), yet, for both species, there is only a single RFC locus, on chromosome 10 for the mouse (21) and chromosome 21 for the human gene (13). The structures of the mRFC and hRFC genes were initially considered to be similar, in terms of their sizes (~23 kb versus 27 kb, respectively), and the numbers (i.e. five) and intron junctions for the coding exons (16–19). Moreover, for both mRFC and hRFC, two alternative upstream noncoding exons and promoters were originally identified (designated 1 and 1a for the mRFC gene and A and B for the hRFC gene). However, other than their high GC contents, there appeared to be no significant sequence similarities in the upstream noncoding exons or regulatory regions between the species.

More recent studies have shown that the hRFC gene locus is significantly larger than originally believed, reflecting the presence of as many as six noncoding regions (designated hRFC-A1/A2, -A, -B, -C, -D, and -E) and multiple promoters, spanning more than 35 kb upstream of the AUG translational start in coding exon 1 (22, 23). Based on these findings, we now reconsider the upstream structure and regulation of the mRFC gene as a pretext to better understanding its in vivo regulation in relation to embryonic development (24, 25) and disease in response to nutritional folate status (26) and to antifolate chemotherapy of malignant and autoimmune diseases (3). Our results document the existence of four unique 5'-untranslated regions (5'-UTRs) and promoters for mRFC. Moreover, we demonstrate a facile in vivo induction/repression of gene expression for both luminal (RFC) and basolateral (MRP1 and MRP3) intestinal folate transporters in response to levels of dietary folate intake.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents—Primers were purchased either from Invitrogen or Sigma. LipofectamineTM 2000 was purchased from Invitrogen. Restriction and modifying enzymes, reporter gene vectors (pGL3-Basic and pRLSV40), and other molecular biologicals were obtained from Promega (Madison, WI). Tissue culture reagents and supplies were purchased from assorted vendors with the exception of iron-supplemented calf serum (Hyclone Laboratories, Inc., Logan, UT). All other chemicals were obtained from commercial sources in the highest available purity. The affinity-purified AE390 antibody to the mouse RFC (27) was a gift of Dr. I. David Goldman (Albert Einstein College of Medicine, Bronx, NY).

Cell Culture—L1210 murine leukemia cells were provided by Dr. I. David Goldman (Albert Einstein College of Medicine) and were cultured in RPMI 1640 medium (Sigma) containing 10% (v/v) heat-inactivated iron-supplemented calf serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 20 µM 2-mercaptoethanol in a 37 °C humidified atmosphere in the presence of 5% CO2, 95% air. BNL CL.2 mouse liver cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in Dulbecco's modified Eagle's medium with 10% calf serum and antibiotics.

Characterization of mRFC Transcript 5'-UTRs by 5'-Rapid Amplification of cDNA Ends (5'-RACE) assay and RT-PCR—Eleven commercial mouse embryo and tissue total RNAs (7-day embryo, 11-day embryo, 15-day embryo, 17-day embryo, liver, lung, smooth muscle, testis, kidney, brain, and uterus) were purchased from Clontech (Mountain View, CA). mRFC transcripts were analyzed by 5'-RACE with a SMART RACE kit (Clontech) as in our earlier study (23) using gene-specific antisense primers (5'-GATGAAGCTTTCCCCAGGACGCAAC-3' for primary PCR and 5'-GTGGTCCTGCCTGGGCTCTTCGTATG-3' for nested secondary PCR, respectively). Secondary PCR products were ligated into pGEM T-Easy vector (Promega), and, following transformation into competent JM109 cells, plasmid DNAs were prepared from 16–27 colonies, and 5'-RACE inserts were sequenced with the M13 forward primer. Sequence data were aligned to mouse RFC genomic DNA sequence (GenBankTM accession number GI 28497897) by BLAST 2.0.

For rare 5'-RACE products (i.e. exons a and d), additional confirmation of the transcription start sites was performed, using sense 5'-RACE nested universal primer A (Clontech) and an antisense noncoding exon-specific primer (5'-CACCGGGTGAGGAGTCACGTC-3' for exon a and 5'-CACCCCAGCAGGGATGTTCAG-3' for exon d), with first round 5'-RACE products from kidney, brain, and uterus as templates. PCR conditions were 94 °C for 4 min (1 cycle), 94 °C for 30 s, and 62 °C for 45 s and 72 °C for 45 s (35 cycles), followed by 72 °C for 7 min (1 cycle). The amplicons were cloned into pGEM-T Easy vector, and 10 clones were sequenced with M13 forward and reverse primers.

To confirm mRFC noncoding exon sequences identified in murine tissues and cell lines, total RNAs were reverse transcribed from 1 µgof total RNA using random hexamer primers and avian myeloblastosis virus reverse transcriptase. cDNAs were amplified with 5'-UTR-specific sense primers for exons a (5'-CATATTGACGTGACTCCTCAC-3'), b (5'-GAGCTAGGAATCCATTCTATGC-3'), c (5'-GAGCTGAGACCCGGTCAAG-3'), and d (5'-CTGAGCATCAGCTGAACATC-3') and a common antisense primer (5'-GATGAAGCTTTCCCCAGGACGCAAC-3') within coding exon 1. For the rare 5'-UTRs (a and d) that could not be detected by primary PCR, secondary amplifications were performed with the noncoding exon-specific primers and a nested antisense primer within coding exon 1 (5'-GTGGTCCTGCCTGGGCTCTTC-3'). PCR conditions for all amplifications were 94 °C for 4 min (1 cycle), 94 °C for 30 s, 62 °C for 45 s, and 72 °C for 45 s (35 cycles) and 72 °C for 7 min (1 cycle). The amplicons were cloned into pGEM-T Easy vector for transformation and sequencing.

Real Time RT-PCR Quantitation of mRFC Transcripts—Total RNAs were isolated from BNL CL.2 and L1210 cells with Trizol reagent (Invitrogen). Total RNA from mouse small intestine was purchased from BioChain Institute, Inc. (Hayward, CA). RNAs from snap-frozen small intestine and kidney samples from the in vivo folate feeding experiment (see below) were prepared with a RNeasy RNA kit (Qiagen, Valencia, CA), following the protocol provided by the manufacturer. cDNAs were synthesized from 1 µg of RNAs prepared from cell lines or tissues, using random hexamer primers and an RT-PCR kit (PerkinElmer) and purified with the QIAquick PCR Purification Kit (Qiagen). Total mRFC transcripts, mRFC-a,-b,-c, and -d 5'-UTRs, and 18 S RNAs were quantitated with a LightCycler real time PCR machine (Roche Applied Science). PCRs contained 2 µl of purified cDNA or standard plasmid, 4 mM MgCl2, 0.5 µM each sense and antisense primers, and 2 µl of FastStart DNA Master SYBR Green I enzyme-SYBR reaction mix (Roche Applied Science). PCR amplifications for the individual 5'-UTRs were performed with sense primers to 5'-UTR sequences (exon a, 5'-CATATTGACGTGACTCCTCAC-3'; exon b, 5'-GAGCTAGGAATCCATTCTATGC-3'; exon c, 5'-GAGCTGAGACCCGGTCAAG-3'; and exon d, 5'-CTGAGCATCAGCTGAACATC-3') and a common antisense primer located in coding exon 1 (5'-GTGGTCCTGCCTGGGCTCTTCGTATG-3'). Total mRFC transcripts were amplified by sense (5'-GTGGAGTGTCATCTTGGCCCG-3') and antisense (5'-CGTTCCAGGAGGAAGGGTGTG-3') primers within coding exon 1. 18 S RNA was measured with sense (5'-GTAACCCGTTGAACCCCATT-3') and antisense (5'-CCATCCAATCGGTAGTAGCG-3') primers. For all amplifications, PCR conditions consisted of an initial denaturing step of 99 °C for 10 min, followed by 35–55 cycles of 95 °C, 62 °C for 10s, and 72 °C for 5s, with melting curve analysis from 40 to 99 °C to ensure specificity. External standards were prepared by amplification of cDNAs for 18 S RNA, total mRFC, and the individual 5'-UTRs, using the above primers. The amplicons were cloned into pGEM-T Easy vector, and the vectors were linearized with ApaI and used to prepare external standard curves. Total mRFC and transcript levels for each 5'-UTR were normalized to 18 S RNA. All real time PCR results were expressed as mean values from 2–3 separate experiments using the same cDNA preparation.

Transcript levels for MRP1, MRP3, MRP4, and FBP-{alpha} were measured by real time PCR on cDNAs prepared as described above, using the following primers: MRP1, 5'-AGAACACGGTCCTCACATGGGT-3' (sense) and 5'-GCCCAGCAGATGATCCACAGAA-3' (antisense); MRP3, 5'-GCTGTGGAGAGAGTCAAGGAGT-3' (sense) and 5'-CTGCACATGAACAGTCACGTTC-3' (antisense); MRP4, 5'-ACTGGTCATAAGCGGAGACTGG-3' (sense) and 5'-GCCTTTGTTAAGGAGGGCTTCC-3' (antisense); and FBP-{alpha}, 5'-GCTCACCTGATGACTGTGCAGT-3' (sense) and 5'-GGAACAGCAGGAATTCGTCTTC-3' (antisense). PCR conditions were identical to those for mRFC, above. Standards were prepared by PCR with the above primers, cloning the amplicons into pGEM-T-Easy vector and linearizing the plasmids with ApaI, as described above.

Preparation of mRFC-luciferase Reporter Constructs—Genomic sequences (positions –4315 to –7000, relative to the ATG translational start in exon 1), including putative mRFC promoters flanking noncoding exons mRFC-a (positions –5049 to –4315), mRFC-b (positions –5615 to –4945), mRFC-c (positions –6101 to –5616), and mRFC-d (positions –7002 to –6104), were amplified from mouse genomic DNA isolated from whole blood. An XhoI restriction site was added to the sense primer, and a HindIII restriction site was incorporated into the antisense primer. The primers included promoter a sense (5'-GACTACTCGAGTACGGTTCTGTTCCTTCCGGTG-3') and antisense (5'-GACATAAGCTTCACCGGGTGAGGAGTCACGTC-3'), promoter b sense (5'-GAATACTCGAGTAGGGGGCTCGTAAAGGGGCTCAG-3') and antisense (5'-GAATCAAGCTTACCCTGCCTGGAGCTTTGTAG-3'), promoter c sense (5'-GACAGCTCGAGAGTTTGAAAGATTGGGGGA-3') and antisense (5'-GAATCAAGCTTACCCGTCCAGCGCTTGACCGG-3'), and promoter d sense (5'-GAAGTCTCGAGGCCATGCAGACTGGATACCACT-3') and antisense (5'-GCTGTAAGCTTCACCCCAGCAGGGATGTTCAG-3') primers (restriction sites are underlined). PCR was performed with Easy-A high fidelity PCR cloning enzyme (Stratagene) using the following conditions: 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 55 s; and one cycle of 72 °C for 7 min. The amplicons were digested with XhoI and HindIII and subcloned into the multiple cloning site of XhoI/HindIII-digested pGL3-Basic vector.

Transient Transfections and Reporter Gene Assays—BNL CL.2 cells were seeded at 2.5 x 105 cells/well in 6-well poly-L-lysine-coated dishes 24 h before transfection. When cells were 70% confluent, 2 µg of the mRFC promoter constructs or empty pGL3-Basic (negative control) were co-transfected with 10 ng of pRLSV40 plasmid using LipofectamineTM 2000 according to the manufacturer's protocol. Cells were incubated for 6 h and then washed with phosphate-buffered saline and supplemented with complete Dulbecco's modified Eagle's medium for 48 h. Cell lysates were assayed for luciferase activity by the Dual-luciferase Reporter Assay System (Promega) with a Turner BioSystems Luminometer model TD-20/20. Firefly luciferase activities were normalized with Renilla luciferase activities. All transfections were performed in triplicate.

Mouse Feeding Experiments—Experiments were performed with male C57BL/6-specific pathogen-free mice (3–4 months old) maintained on a 12-h light/dark cycle and fed standard mouse chow and water ad libitum. Mice were assigned to two dietary groups and were fed AIN93G-purified isoenergetic diets (Dyets, Inc., Lehigh Valley, PA). The control group received a folate-replete diet containing 2 mg/kg folic acid. The experimental group received a folate-deficient diet containing 0 mg/kg folic acid. All diets were supplemented with 1% succinyl sulfathiazole. The experimental diets were continued for 8 weeks, at which time animals were anesthetized under CO2 and sacrificed by cervical dislocation, and tissues were collected (see below). Serum folates were measured with the SimulTRAC-SNB radioassay kit for vitamin B12 (57Co) and folate (125I) (ICN Diagnostics, Orangeburg, NY), as described previously (28).

Eighteen kidney and small intestine samples (nine from mice fed folate-deficient diets, nine from mice fed normal folate-replete diets) were collected and snap-frozen in liquid nitrogen. For the small intestine, a 4-cm section was collected and rinsed with ice-cold phosphate-buffered saline prior to freezing; a 1-cm segment ~1 cm proximal to the stomach was used for preparing RNA, as described above. RNAs were used to prepare cDNAs for real time RT-PCR analysis of total mRFC transcripts and mRFC 5'-UTRs, along with transcript levels for MRP1, -3, and -4 and FBP-{alpha} (see above).

Immunohistochemistry—Cryosections were prepared from small intestines, fixed, and treated with affinity-purified primary mouse RFC antibody (AE390) (27). Slides were developed with a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) with 3,3'-diaminobenzidine tetrahydrochloride. Slides were counterstained with Mayer's hematoxylin.

Comparative Promoter Analysis of Human and Mouse RFC—Homology analysis of the mouse and human RFC upstream regulatory regions was performed with the mVISTA program (available on the World Wide Web at www-gsd.lbl.gov/vista/index.shtml). Putative or confirmed regulatory sequences for mouse (a, b, c, and d) and human (A1/A2, A, B, C, D) RFC promoters were analyzed for transfactor binding sites with the GenomatiX MatInspector program (available on the World Wide Web at www.genomatix.de/cgi-bin/./eldorado/main.pl).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Heterogeneous 5'-UTRs Transcribed from Four Distinct Noncoding Exons in the mRFC Gene—5'-RACE was used to characterize the extent of 5'-UTR heterogeneity for mRFC transcripts from seven mouse tissues (liver, lung, muscle, testis, brain, kidney, and uterus) and four mouse stage embryos (7, 11, 15, and 17-day). 16–27 5'-RACE clones for each tissue/embryo were subjected to DNA sequencing for a total of 234 5'-RACE clones. DNA sequences were aligned with a mouse chromosome 10 genomic contig (positions 2860047–2904354; accession number GI28497897). 5'-RACE clones clustered over four regions (designated a–d), spanning 2055 bp and up to 6270 bp upstream of the translation start in coding exon 1 (see schematic in Fig. 1A). The composite sequences for the a, b, c, and d groups of 5'-RACE clones are shown in Fig. 1B and are characterized by multiple and reproducible 5' termini in separate tissues, consistent with the presence of multiple transcriptional start sites.



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  		FIG. 1.
Schematic of mRFC gene upstream region and DNA sequences for the mRFC 5'-UTRs. A, the upstream region of the mRFC gene is shown, including four noncoding exons (designated a, b, c, and d) and the first coding exon (exon 1) including the ATG translational start site. The approximate distances between each of the noncoding exons were determined from those in mouse chromosome 10 genomic sequence (accession number GI 284978). B, 5'-RACE was performed as described under "Materials and Methods" with RNAs prepared from mouse tissues. Sequence data are shown for the four unique 5'-UTRs (mRFC-a, -b, -c, and -d), including probable transcription start sites (closed circles) and, for mRFC-b, alternate overlapping splice forms. The latter is composed of two apparent splice forms, including a major form designated bi (underlined) and a less frequent form, designated bii, including an additional 105 bp of 3' sequence in addition to that in bi.

 
The distribution of 5'-RACE clones showed tissue/embryo specificity (not shown). Over 95% of the clones fell into groups b and c, corresponding to the mRFC noncoding exons 1 and 1a previously reported by Tolner et al. (16). Group b is composed of two apparent splice forms including a major form (designated bi and underlined in Fig. 1B) that has a 3'-end identical to that for the major mRFC noncoding exon 1 previously reported (16), along with 51 additional 5' nucleotides. A less frequent form, designated bii, included an additional 105 bp of 3' sequence (Fig. 1B). The majority of the 5'-RACE clones in group c had 5'-ends within 20 bp of that originally reported for exon 1a by Sirotnak and co-workers (16), although another form included 70 bp of additional upstream sequence (Fig. 1B). Finally, two novel groups of 5'-RACE clones were detected in assorted tissues, including group a in brain and testis (147 and 132 bp, respectively), mapping between group b and the mRFC coding exon 1, and group d, identified in nine clones from assorted tissues and embryos and mapping upstream of group c (Fig. 1A).

To further confirm the presence of the four unique 5'-UTRs identified by 5'-RACE in mRFC transcripts, RNAs from testis and a 15-day embryo that expressed particular sequences by 5'-RACE (forms a, b, and c and forms b, c, and d, respectively) were reverse transcribed and PCR-amplified with upstream primers to each of the mRFC noncoding exons and downstream primers to the mRFC coding sequence in exon 1. For the a and d 5'-UTRs, a secondary amplification step was also performed. For all four 5'-UTRs, specific products of the expected sizes were amplified (mRFC-a, 135 bp; mRFC-b, 245 and 348 bp; mRFC-c, 223 bp; and mRFC-d, 140 bp; results not shown). These were confirmed by DNA sequence analysis.

Tissue-specific Expression of Total mRFC Transcripts and mRFC 5'-UTRs by Real Time RT-PCR—5'-RACE has an inherent statistical bias, since 5'-RACE results can be significantly impacted by template secondary structures (29). Thus, to better quantitate mRFC transcript levels and the individual 5'-UTRs identified by 5'-RACE, we used real time RT-PCR. Individual 5'-UTRs were screened with upstream primers to the noncoding sequences and a common downstream coding sequence primer, whereas total mRFC transcripts were assayed with sense and antisense coding sequence primers. Transcripts were normalized to levels of 18 S RNA. Commercial RNAs were analyzed for the assorted mouse tissues used for 5'-RACE, along with RNAs from mouse small intestine and L1210 murine leukemia and BNL CL.2 murine liver cells in culture. mRFC transcripts were detected in all RNA samples yet spanned a 36-fold range (Fig. 2). The highest mRFC levels were detected in kidney and brain, and low but still detectable levels were detected in lung, small intestine, and uterus. These results generally parallel those for mRFC transcripts on Northern blots of total RNAs from select normal mouse tissues (30). For the results shown in Fig. 2, appreciable levels of mRFC transcripts were present in the four-stage (7-, 11-, 15-, and 17-day) embryos; however, changes in mRFC levels were insignificant as a function of embryo stage.



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  		FIG. 2.
Real time RT-PCR analysis of mRFC total transcripts. Data are shown for mRFC transcript levels in tissues, stage embryos, and cell lines based on real time PCR analysis. mRFC transcript levels were normalized to 18 S RNA and are presented as mean values ± S.E. from three separate analyses.

 
Among the mRFC-a, -b, -c, and -d 5'-UTRs, exon b was the predominant form in 7 of 10 tissues and cell lines examined (Fig. 3). Exon b also predominated in mRFC transcripts from the early stage embryos. However, there was no change in 5'-UTR usage with embryo stage. For the lung and kidney RNAs, 5'-UTR sequence derived from exon c clearly predominated, although these tissues expressed the lowest and highest mRFC transcripts, respectively, among our tissue cohort. An appreciable amount of the mRFC-c 5'-UTR (>50% of exon b) was also detected in other tissues. Low levels of exons a and d were detected in assorted tissues by real time PCR (≤15% of total mRFC transcripts), with the highest level of mRFC-a transcripts in testis, and in L1210 and BNL CL.2 cells. The highest levels of mRFC-d transcripts were detected in kidney and small intestine.



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  		FIG. 3.
Real time RT-PCR analysis of mRFC 5'-UTRs. Data are shown for mRFC 5'-UTR levels in mouse tissues, stage embryos, and cell lines based on real time PCR analysis. mRFC 5'-UTR levels are normalized to 18 S RNA and are presented as mean values ± S.E. from three separate analyses.

 
Identification of Four mRFC Promoters—By analogy with hRFC and other multipromoter genes, the unique noncoding exons for mRFC are likely to be transcribed from unique promoters. In previous studies, promoter activity was localized to the 5'-flanking regions immediately upstream of the mRFC-b and -c noncoding exons (31).

We similarly demonstrated high level promoter activity (60- and 10-fold, respectively, over pGL3-Basic) in transient transfections of BNL CL.2 cells with mRFC-b and -c promoter constructs, including 671 bp (positions –5615 to –4945) and 487 bp (positions –6101 to –5616), respectively, of 5'-flanking and noncoding exon sequence (Fig. 4A). For the mRFC-a and –d 5'-flanking regions (positions –5049 to –4314 and –7002 to –6104, respectively), promoter activities were, likewise, detected, albeit at much lower levels (4- and 3-fold, respectively, over pGL3-Basic). The nucleotide sequences for the active mRFC-a and mRFC-d promoters are shown in Fig. 4B. The major cis elements are summarized in Table I.



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  		FIG. 4.
Promoter activities for the mRFC-a,-b,-c, and –d promoters: Nucleotide sequences for the mRFC-a and –d promoters. A, 5'-flanking regions proximal to exons a–d, as described under "Results," in pGL3-Basic reporter plasmid were transiently co-transfected into BNL CL.2 mouse liver cells with pRLSV40 plasmid. After 48 h, cell lysates were prepared and assayed for luciferase activity. Firefly luciferase activities were normalized with Renilla luciferase activities. All transfections were performed in triplicate. Results with the promoter constructs are compared with that for empty pGL3-Basic. B, the nucleotide sequences for the mRFC promoters a and d are shown. The 5'-ends of the mRFC-a and -d noncoding exons are indicated by italic type.

 


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  		TABLE I
Major cis elements in mRFC and hRFC promoters

Shown is a summary of the major confirmed and putative cis elements in the mRFC and hRFC promoters, based on data base analysis. For both mRFC and hRFC, the numbering is relative to the ATG translational start in the first coding exon 1 and is based on genomic sequence data in the data base (GenBankTM accession numbers GI 28497897 and AL163302 [GenBank] ). Experimental characterization of the hRFC promoters are described in the following references: hRFC-A1/A2 (23, 24), hRFC-A (32), hRFC-B (33), and hRFC-C (35).

 
Effects of Dietary Folates on Tissue Levels of mRFC, MRP1, -3, and -4, and FBP-{alpha} Transcripts—As an extension of these experiments, we characterized the effects of folate deficiency on mRFC transcripts and 5'-UTRs in the proximal end of the small intestine and in kidney from two groups of mice fed folate-replete (2 mg/kg folic acid) and -deficient (0 mg/kg folic acid) diets over 8 weeks. The folate-deficient diet in these mice was previously reported to result in a substantial (>95%) decrease in serum folates (10.29 nmol/liter versus 139.13 nmol/liter for the folate-replete diet) (28). In nine mice from each group, decreased serum folate was accompanied by a highly significant increase (13.8-fold; p = 0.0056 by Mann-Whitney test) in median total mRFC transcripts in the intestine (Fig. 5A). These changes were accompanied by increased levels of mRFC protein by immunohistochemistry of frozen sections of small intestine from folate-deficient mice (Fig. 6). Interestingly, these changes in intestinal mRFC levels reflected increases in each of the mRFC-b, -c, and -d 5'-UTRs (7.9–19.6-fold; p ≤ 0.03) (Fig. 5A). However, levels of mRFC transcripts with exon a sequence were largely unchanged between folate-depleted and -replete groups.



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  		FIG. 5.
Modulation of mRFC levels by dietary folates. The design of the feeding experiments is described under "Materials and Methods." The results for small intestine RNAs (A) and kidney RNAs (B), normalized to 18 S RNAs, are presented as -fold change values relative to individual median values for total mRFC and the mRFC-a, -b, -c, and –d noncoding exons for the animals fed the folate replete diet (assigned a valued of 1). Relative median values are noted by the horizontal bars. Absolute levels for total mRFC and 5'-UTRs for mice fed a standard folate replete diet are presented in Figs. 3 and 4. FR, folate replete; FD, folate-deficient.

 



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  		FIG. 6.
Immunohistochemical staining of small intestine. Representative cryosections from mice fed folate-replete and -deficient diets were prepared and stained for RFC using affinity-purified AE390 mRFC-specific antibody, as described under "Materials and Methods." The sections shown were viewed with a x400 objective.

 
Since mRFC has been localized to the basolateral membranes of cortical and medullary renal tubular epithelial cells (27) and may participate in net folate reabsorption in the kidney, kidneys were also harvested from folate-replete and -deficient animals and assayed for mRFC transcripts. Median total mRFC transcripts and individual mRFC 5'-UTRs in kidney were slightly increased in mice fed the folate-deficient diet (~2-fold) compared with folate-replete mice (Fig. 5B); however, these differences were not statistically significant.

We assessed the levels of other systems documented to transport folates, including the FBP-{alpha} (Fig. 7) and MRP1, -3, and -4 in kidney and intestinal RNAs (Fig. 8). In intestine, FBP-{alpha} levels were exceedingly low and near the limit of detection. Conversely, in kidney, FBP-{alpha} expression was appreciable. For both intestine and kidney, FBP-{alpha} transcripts were significantly increased (91- and 2-fold, respectively; p = 0.0002 and p = 0.04, respectively) for mice on the folate-deficient diet (Fig. 7). Intestinal levels of MRP1 and MRP3 were also dramatically increased in mice fed low folate diets (median increases of 53- and 168-fold, respectively), although there was no statistically significant difference between folate-replete and -depleted diets in the levels of MRP4 (Fig. 8A). For kidney, moreover, there were no statistically significant changes in the levels of MRP1, -3, or -4 between the folate-replete and -deficient groups (Fig. 8B).



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  		FIG. 7.
Modulation of folate-binding protein {alpha} levels by dietary folates. RNAs identical to those used in Fig. 5 were assayed for levels of FBP-{alpha} by real time RT-PCR. Data are shown for FBP-{alpha} transcript levels in small intestine RNAs (A) and kidney RNAs (B), normalized to 18 S RNA, and are presented as median values ± S.E. from three separate analyses. Median values are noted by the horizontal bars. FR, folate-replete; FD, folate-deficient.

 



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  		FIG. 8.
Modulation of MRP levels by dietary folates. Identical RNAs to those used in Fig. 5 were assayed for MRP1, -3, and -4 by real time RT-PCR. Results for small intestine (A) and kidney (B) MRP transcripts are presented as -fold change values relative to individual median values for MRP1, -3, and -4 for the animals fed the folate-replete diet (assigned a value of 1). Relative median values are noted by the horizontal bars. FR, folate-replete; FD, folate-deficient.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our earlier studies suggested a complex regulation of hRFC gene expression (22, 23, 32–34). We previously reported that hRFC is ubiquitously but differentially expressed in human tissues and cell lines, and we identified up to six alternate noncoding exons (designated hRFC-A1/A2, -A, -B, -C, -D, and -E) and multiple promoters for the hRFC gene in assorted normal tissues spanning ~35 kb upstream of the major translational start site (22). Promoter usage was tissue-specific in assorted tissues and tumors (22, 23) and probably reflects multiple transcription factors and/or epigenetic events that respond to particular stimuli to ensure sufficient hRFC and folate uptake for cell proliferation and tissue regeneration. By the use of alternate noncoding exons, posttranscriptional controls are manifest through effects on transcript stabilities and translational efficiencies and/or through the synthesis of modified hRFCs translated from upstream AUGs in the 5'-UTRs (23).

The present results demonstrate that the upstream gene structure for mRFC, like its human counterpart, is complex and involves multiple alternate noncoding exons and promoters. Thus, by 5'-RACE analysis of RNAs prepared from mouse tissues, cell lines, and stage embryos, we identified mRFC transcripts with four distinct 5'-UTRs designated mRFC-a, -b, -c, and -d that, when aligned to chromosome 10 genomic sequence, mapped within ~6300 bp upstream of the major translational start site in exon 1 of the mRFC gene. All four 5'-UTRs were characterized by multiple transcriptional start sites, and, for the mRFC-b transcript form, 2 alternative splice forms were identified. The mRFC-b and -c 5'-UTRs corresponded to those previously reported (16, 17); however, we found slight differences in their 5' and 3' boundaries. The novel a and d 5'-UTRs reported herein have not been described previously. The existence of four separate 5'-UTRs was confirmed by standard RT-PCR from RNAs prepared from testis and the 15-day stage embryo, and relative levels of these alternate transcript forms were accurately measured by real time RT-PCR with specific primers to the individual 5'-UTRs. Although mRFC-b and -c were the predominant 5'-UTR forms in all tissues, relative levels were variable, since mRFC-b was the major transcript form in liver, muscle, testis, brain, and uterus, whereas exon c was the major 5'-UTR identified in lung, small intestine, and kidney. mRFC-d was detected in multiple tissues, and its range spanned more than 800-fold, with the highest level in kidney and lowest level detected in liver. For mRFC-a transcripts, there was a 96-fold difference between the samples with the highest (BNL CL.2 cells) and with the lowest (uterus) transcript levels. Another potential level of tissue specificity could involve the distributions of the alternately spliced mRFC-bi and –bii transcript forms. For instance, in our 5'-RACE analysis, bi was the major form identified in testis and bii predominated in lung (results not shown). Promoter activities were demonstrated for the 5'-flanking regions proximal to mRFC-a, -b,-c, and -d exons by transient transfections of BNL CL.2 cells.

What is particularly striking, given the close homologies between the coding regions for hRFC and mRFC, is the nearly complete absence of obvious sequence homologies in the upstream noncoding exons or regulatory regions for the human and rodent genes. However, upon closer examination of the distributions of putative cis elements in the mRFC promoters in comparison with results of laboratory studies of hRFC-A1/A2, -B, and -C minimal promoters (23, 33–35), similarities are clearly discernible between the mouse and human genes (Fig. 9 and Table I). For instance, the pattern of functional E-box, GATA1, and Ikaros binding sites within a 244-bp span of the hRFC-A1/A2 minimal promoter flanking the transcriptional start sites (23, 34) is distinctly similar to 250 bp of flanking sequence for mRFC-a. For mRFC-b, the series of putative GC-box, E-box, and Ikaros elements in a 309-bp stretch flanking the transcriptional start sites parallels these functional elements documented in the 145-bp segment of the hRFC-B core promoter (33). Finally, for hRFC-C, functional CEBP/{beta} and Sp1 consensus binding elements are localized to within a 110-bp region (35); likewise, a CCAAT-box and GC-box can be identified within 68 bp flanking the transcriptional start sites for mRFC-c. Although the functional features of the hRFC-D promoter have not been published,2 by data base analysis, both the hRFC-D and mRFC-d flanking regions include putative binding elements for Sp1, Ikaros, USF1, and GATA1 (also depicted in Fig. 9 and summarized in Table I). Interestingly, no functional homologs of the hRFC-A promoter and 5'-UTR were identified for mRFC.



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  		FIG. 9.
Comparison of cis elements in hRFC and mRFC promoters. A schematic summarizes the similarities in the confirmed and putative cis elements in the hRFC and mRFC promoters, based on direct experiment and data base analysis, as described under "Materials and Methods." The major elements depicted in this figure are summarized in Table I. For both the mRFC and hRFC genes, the numbering is relative to the ATGs in coding exon 1 and is based on genomic sequence data in the data base (GenBankTM accession numbers GI 284978 and AL163302 [GenBank] , respectively).

 

Dietary folates are transported across the enterocyte brush border membrane in the proximal part of the small intestine by a saturable process, at least in part, identical to RFC. Thus, it was of particular interest to examine the effects of dietary folate intake on the expression of mRFC and 5'-UTR/promoter usage in small intestine from mice fed folate-deficient and -replete diets. Under these conditions, serum folates are depleted more than 95% (28). In our study, substantially increased total mRFC transcripts were detected in the small intestine by real time PCR analysis in folate-deficient compared with folate-replete mice. This was attributed to increased usage of three of the four major mRFC 5'-UTRs (and promoters), including forms b, c, and d. However, the low levels of mRFC-a transcripts were unaffected by the level of dietary folate.

A similar finding of increased intestinal RFC in rats fed folate-deficient diets was reported by Said et al. (26). In this report, it was shown that levels of transepithelial transport of folate across the brush border membrane of intestinal absorptive cells were also increased in rats fed folate-deficient diets and were accompanied by an increased steady state RFC mRNA and protein (26). However, it was not previously possible to associate these changes with levels of individual 5'-UTRs and RFC promoters.

In our study, we extended our study in folate-deficient and -replete animals to include kidney RNAs, based on the localization of mRFC to the basolateral membranes of cortical and medullary renal tubular epithelial cells (27), which suggests its role in folate reabsorption from kidney. However, unlike intestine, the changes in kidney mRFC were modest and statistically insignificant. Conversely, levels of FBP-{alpha}, localized to brush border membranes, were significantly increased in kidneys from mice fed the low folate diet. Whereas FBP-{alpha} transcripts were detected at extremely low levels in intestines from mice fed the normal diet, suggesting a minor contribution of FBP-{alpha} to transepithelial folate transport, with folate deficiency FBP-{alpha} was, nonetheless, increased more than 90-fold.

Since in vivo homeostasis of folates depends on both efflux and influx processes, we also measured intestinal levels of transcripts for MRP1, -3, and -4, reported to transport folate and antifolate substrates in cultured cells (6–10). Interestingly, levels of both MRP1 and MRP3 in intestine were dramatically increased in folate-deficient mice, although the level of MRP4 was largely unchanged. There were no significant changes in the levels of MRP transcripts in kidney with dietary folate status.

Thus, our results suggest the existence of a facile homeo-static response of intestinal folate transport processes in response to levels of dietary folate intake involving both mRFC, localized to the brush border compartment of enterocytes (27), and MRP1 and -3, localized to enterocyte basolateral membranes (36, 37). When faced with folate deficiency, the combined increases in mRFC and MRP1 and -3 should serve to maximize net uptake of dietary folates from the intestinal lumen across both the brush border and basolateral membranes and into the bloodstream. Likewise, levels of dietary folates could profoundly influence oral bioavailability of antifolate drugs in this manner. Clearly, a better understanding of the regulation of folate cofactor transport by RFC and MRPs should foster new insights into the effects of folate deficiency in relation to the pathophysiology of a number of health-related problems, ranging from fetal abnormalities to cardiovascular disease and cancer (38). This may also lead to new approaches for administering antifolate chemotherapy for cancer (39) and autoimmune diseases (40).

Several mechanisms can be envisaged to explain the substantial changes in tissue mRFC, MRP, and FBP-{alpha} transcripts in response to folate deprivation/repletion. Possibilities include transcriptional activation or repression via effects on binding of individual transcription factors at cis elements in the mRFC, MRP, and/or FBP-{alpha} promoters. Alternatively, expression could be regulated via epigenetic alterations or posttranscriptionally. Folates are directly involved in the methylation of homocysteine to form methionine, a precursor of S-adenosylmethionine required for DNA methyltransferase. Moreover, it has been shown that extracellular concentrations of folates influence intracellular S-adenosylmethionine (41, 42), and depletion of S-adenosylmethionine by methyl deficiency reduces both global and gene-specific methylation patterns (43). Indeed, based on previous studies in KB human nasopharyngeal epidermoid cells treated with low levels of extracellular folates, increased FBP-{alpha} mRNA and transcription (44) may be due to altered patterns of methylation (45). However, increased FBP-{alpha} mRNA has also been associated with increased transcript stability in folate-deficient cells (44, 46).

DNA methylation appears to regulate levels of both hRFC and MRP1 and -3 in MDA-MB-231 human breast cancer cells, since transcripts are increased by treatment with 5-aza-2'-deoxycytidine (47). Further, methylation of the hRFC-B promoter in primary central nervous system lymphomas was reported to be associated with a lower complete remission to methotrexate-based chemotherapy (48). Preliminary bisulfite sequencing studies of genomic DNAs from small intestines obtained from mice fed folate-deficient and -replete diets strongly imply a causal role for changes in DNA methylation of CpG islands in increased activity of at least one mRFC promoter (i.e. promoter c).3 However, in other cases, these effects may not necessarily occur in cis via direct effects on promoter methylation and may involve modulation of one or more transactivating factors (47, 49). Future studies will focus on the mechanistic bases for this adaptive in vivo regulation of intestinal folate transport involving both apical and basolateral localized folate transporters.


   FOOTNOTES
 
* This study was supported by NCI, National Institutes of Health, Grant CA53535. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY73445, AY734486 [GenBank] , and AY734487 [GenBank] , and U57780 [GenBank] . Back

|| To whom correspondence should be addressed: Experimental and Clinical Therapeutics Program, Karmanos Cancer Institute, 110 E. Warren Ave., Detroit, MI 48201. Tel.: 313-833-0715 (ext. 2407); Fax: 313-832-7294; E-mail: matherly{at}karmanos.org.

1 The abbreviations used are: FBP, folate-binding protein; RFC, reduced folate carrier; hRFC, human RFC; mRFC, mouse RFC; 5'-UTR, 5'-untranslated region; 5'-RACE, 5'-rapid amplification of cDNA ends; RT, reverse transcription; MRP, multidrug resistance-associated protein; contig, group of overlapping clones. Back

2 M. Liu and L. H. Matherly, manuscript in preparation. Back

3 M. Liu and L. H. Matherly, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. I. David Goldman for the AE390 antibody to the mouse RFC.



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 DISCUSSION
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