Human Reduced Folate Carrier Gene Research Articles

Knockout of the folate transporter folt-1 causes germline and somatic defects in C. elegans.

BackgroundThe C. elegans gene folt-1 is an ortholog of the human reduced folate carrier gene. The FOLT-1 protein has been shown to transport folate and to be involved in uptake of exogenous folate by worms. A knockout mutation of the gene, folt-1(ok1460), was shown to cause sterility, and here we investigate the source of the sterility and the effect of the folt-1 knockout on somatic function.ResultsOur results show that folt-1(ok1460) knockout hermaphrodites have a substantially reduced germline, generate a small number of functional sperm, and only rarely produce a functional oocyte. We found no evidence of increased apoptosis in the germline of folt-1 knockout mutants, suggesting that germline proliferation is defective. While folt-1 knockout males are fertile, their rate of spermatogenesis was severely diminished, and the males were very poor maters. The mating defect is likely due to compromised metabolism and/or other somatic functions, as folt-1 knockout hermaphrodites displayed a shortened lifespan and elongated defecation intervals.ConclusionsThe FOLT-1 protein function affects both the soma and the germline. folt-1(ok1460) hermaphrodites suffer severely diminished lifespan and germline defects that result in sterility. Germline defects associated with folate deficiency appear widespread in animals, being found in humans, mice, fruit flies, and here, nematodes.

Human Reduced Folate Carrier Gene and Transcript Variants: Functional, Physiologic, and Pharmacologic Consequences

Human Reduced Folate Carrier Gene and Transcript Variants: Functional, Physiologic, and Pharmacologic Consequences

The Human Reduced Folate Carrier Gene Is Regulated by the AP2 and Sp1 Transcription Factor Families and a Functional 61-Base Pair Polymorphism

Recently, our laboratory reported an intricate regulation of the human reduced folate carrier (hRFC) gene, involving multiple promoters and noncoding exons. We localized promoter activity to a 452-bp GC-rich region upstream of noncoding exon A, including a 47-bp basal promoter with a CRE/AP-1-like consensus element that bound the bZip family of DNA-binding proteins (e.g. CREB-1 and c-Jun). We now report that three nearly identical tandem repeats (49-61 bp) in the hRFC-A upstream region are involved in regulating promoter activity. By in vitro binding assays, multiple transcription factors (e.g. AP2 and Sp1/Sp3) bound this region. When AP2 was cotransfected with the hRFC-A reporter construct into HT1080 cells, promoter activity increased 3-fold. In Drosophila SL2 cells, Sp1 transactivated promoter A and showed synergism with CREB-1. However, c-Jun was antagonistic to the effects of Sp1. A sequence variant in the hRFC-A repeated region was identified, involving an exact duplication of a 61-bp sequence. This variant had an allelic frequency of 78% in 72 genomic DNAs and resulted in a 63% increase in promoter activity. These results identify important regions in the hRFC-A promoter and critical roles for AP2 and Sp1, in combination with the bZip transcription factors. Moreover, they document a functionally novel polymorphism that increases promoter activity and may contribute to interpatient variations in hRFC expression and effects on tissue folate homeostasis and antitumor response to antifolates.

The human reduced folate carrier gene is ubiquitously and differentially expressed in normal human tissues: identification of seven non-coding exons and characterization of a novel promoter.

Our previous study identified two alternate non-coding upstream exons (A and B) in the human reduced folate carrier (hRFC) gene, each controlled by a separate promoter. Each minimal promoter was regulated by unique cis -elements and transcription factors, including stimulating protein (Sp) 1 and Sp3 and the basic leucine zipper family of proteins, suggesting opportunities for cell- and tissue-specific regulation. Studies were performed to explore the expression patterns of hRFC in human tissues and cell lines. Levels of hRFC transcripts were measured on a multi-tissue mRNA array from 76 human tissues and tumour cell lines and on a multi-tissue Northern blot of representative tissues, each probed with full-length hRFC cDNA. hRFC transcripts were ubiquitously expressed, with the highest level in placenta and the lowest level in skeletal muscle. By rapid amplification of cDNA 5'-ends assay from nine tissues and two cell lines, hRFC transcripts containing both A and B 5'-untranslated regions (UTRs) were identified. However, five additional 5'-UTRs (designated A1, A2, C, D and E) were detected, mapping over 35 kb upstream from the hRFC translation start site. The 5'-UTRs were characterized by multiple transcription start sites and/or alternative splice forms. At least 18 unique hRFC transcripts were detected. A novel promoter was localized to a 453 bp fragment, including 442 upstream of exon C and 11 bp of exon C. A 346 bp repressor flanked the 3'-end of this promoter. Our results suggest an intricate regulation of hRFC gene expression involving multiple promoters and non-coding exons. Moreover, they provide a transcriptional framework for understanding the role of hRFC in the pathophysiology of folate deficiency and antifolate drug selectivity.

Repression of Human Reduced Folate Carrier Gene Expression by Wild Type p53

The relationship between loss of functional p53 and human reduced folate carrier (hRFC) levels and function was examined in REH lymphoblastic leukemia cells, which express wild type p53, and in p53-null K562 cells (K562(pTet-on/p53)) engineered to express wild type p53 under control of a tetracycline-inducible promoter. Activation of p53 in REH cells by treatment with daunorubicin was accompanied by decreased ( approximately 5-fold) levels of hRFC transcripts and methotrexate transport. Treatment of K562(pTet-on/p53) cells with doxycycline resulted in a dose-dependent expression of p53 protein and transcripts, increased p21 protein, decreased dihydrofolate reductase, and G(1) arrest with decreased numbers of cells in S-phase. p53 induction was accompanied by up to 3-fold decreases in hRFC transcripts transcribed from the upstream hRFC-B promoter and similar losses of hRFC protein and methotrexate uptake capacity. Expression of p15 in an analogous inducible system in K562 cells resulted in a nearly identical decrease of S-phase cells and dihydrofolate reductase without effects on hRFC levels or activity. When the hRFC-B promoter was expressed as full-length and basal promoter-luciferase reporter constructs in K562(pTet-on/p53) cells, induction of p53 with doxycycline resulted in a 3-fold loss of promoter activity, which was reversed by cotransfection with a trans-dominant-negative p53. These studies show that wild type p53 acts as a repressor of hRFC gene expression, via a mechanism that is independent of its effects on cell cycle progression.

The Basal Promoters for the Human Reduced Folate Carrier Gene Are Regulated by a GC-box and a cAMP-response Element/AP-1-like Element: BASIS FOR TISSUE-SPECIFIC GENE EXPRESSION

Our laboratory previously identified two functional promoters (designated A and B) for the human reduced folate carrier (hRFC) gene that result in hRFC transcripts with differing 5'-untranslated regions. By transiently transfecting HT1080 and HepG2 cells with a series of 5' and 3' deletions in the hRFC-B and -A promoters, the minimal promoters were localized within 46 and 47 base pairs, respectively. Gel mobility shift assays with the hRFC-B basal promoter region revealed specific DNA-protein complexes involving a highly conserved GC-box and Sp1 or Sp3. In Drosophila SL2 cells, both Sp1 and the long Sp3 isoform potently transactivated the hRFC-B basal promoter; however, the short Sp3 isoforms were transcriptionally inert and resulted in a potent inhibition of Sp1 transactivation. For the hRFC-A basal promoter, a CRE/AP-1-like element was bound by the bZip superfamily of DNA-binding proteins. Cell-specific DNA-protein complexes were identified for hRFC-A (CREB-1 and c-Jun in HT1080 cells; CREB-1 and ATF-1 in HepG2 cells). When the GC-box and CRE/AP-1-like elements were mutated, a 60--90% decrease in promoter activity was observed in both cell lines. These results identify the critical regulatory regions for the hRFC basal promoters and stress the functional importance of the Sp and bZip families of transcription factors in regulating hRFC expression.

Isolation and characterization of genomic sequences involved in the regulation of the human reduced folate carrier gene (RFC1).

Decreased reduced folate carrier (RFC) activity has been associated with MTX resistance in experimental models of transport-mediated MTX resistance, and has been attributed to changes in the expression of RFC1, the gene that encodes a protein with this activity. RNA transcripts of RFC1 may contain any one of four distinct 5' untranslated regions (UTRs). We cloned a human genomic DNA fragment upstream from the RFC1 translation start site and determined the nucleotide sequence of a 4.8kb region that contained the exons corresponding to each of the reported UTRs. To identify regulatory elements that may be involved in RFC1 transcription, we first developed a semi-quantitative RT-PCR assay using primers specific for each of the 5' UTRs to amplify RNA transcripts containing each of the RFC1 5' exons, and found evidence for differential transcription of RFC1 non-coding exons in tissues, during development, and in MTX-resistant, transport-deficient cells. We also found that RFC1 RNA levels are cell cycle regulated and peak at the G1 to S transition. Then, using a series of RFC1 promoter-reporter fusion constructs, we identified genomic sequences that may be involved in the regulation of expression of exons 1b and 1c of the RFC1 gene. These studies therefore identify regulatory regions of RFC1 promoters and potential models of regulation in which these regions may exert control.

Structure and organization of the human reduced folate carrier gene

The human reduced folate carrier gene was found to contain 7 exons, including two alternative non-coding exons (exons 1 and 2), spanning ∼29 kb. Two transcript variants involving exon 7 were detected in K562 cells by RT-PCR, distinguishable from the wild-type transcript by deletions of 625 bp (KS32) and 988 bp (KS1). The presence of consensus splice donor and acceptor elements in the deleted KS1 isoform suggested that this form was likely a splice variant; however, KS32 likely arose during reverse transcription rather than by alternative splicing.

Structural organization of the human reduced folate carrier gene: evidence for 5' heterogeneity in lymphoblast mRNA.

The reduced folate carrier (rfc1) gene encodes a protein that is involved in the intracellular accumulation of folates. Point mutations in this gene and alterations resulting in the down regulation of its message are major factors involved in the resistance to antifolate chemotherapeutic compounds. As a framework for understanding the significance of such changes in relation to gene expression and function, in this report we describe the organization of the rfc gene from human lymphoblasts. The gene contains 5 exons (2 to 6) coding for protein. At least four 5' exons, used in a mutually exclusive manner in the production of the rfc message from lymphoblast cells, are spliced to exon 2, which contains the translational start site. "Semi-quantitative" PCR indicates that exon 1 is preferentially used. The major transcriptional start site has been mapped by RACE and RNase protection to a region 109 to 135 base pairs 5' to the start of exon 1. The 5' region of the gene has no TATA box-like sequence but contains several consensus binding sites for transcriptional factors such as SP-1, MZF1, CREB, AP-1, ETS, GATA-1 and GATA-2. The overall organization of the human gene is similar to that of the hamster and mouse genes.