Abstract
Folates are essential vitamins that play a key role as one-carbon donors in a spectrum of biosynthetic pathways including RNA and DNA synthesis. The proton-coupled folate transporter (PCFT/SLC46A1) mediates obligatory intestinal folate absorption. Loss-of-function mutations in PCFT result in hereditary folate malabsorption, an autosomal recessive disorder characterized by very low folate levels in the blood and cerebrospinal fluid. Hereditary folate malabsorption manifests within the first months after birth with anemia, immune deficiency, and neurological deficits. Here we studied the role of inducible trans-activators of PCFT gene expression. Bioinformatics identified three putative nuclear respiratory factor 1 (NRF-1) binding sites in the minimal promoter. The following evidence establish that PCFT is an NRF-1-responsive gene; electrophoretic mobility shift assay showed NRF-1 binding to native but not mutant NRF-1 sites, whereas antibody-mediated supershift analysis and chromatin immunoprecipitation revealed NRF-1 binding to its consensus sites within the PCFT promoter. Moreover, mutational inactivation of individual or all NRF-1 binding sites resulted in 40-60% decrease in luciferase reporter activity. Consistently, overexpression of NRF-1 or a constitutively active NRF-1 VP-16 construct resulted in increased reporter activity and PCFT mRNA levels. Conversely, introduction of a dominant-negative NRF-1 construct markedly repressed reporter activity and PCFT mRNA levels; likewise, introduction of NRF-1 siRNA duplexes to cells resulted in decreased PCFT transcript levels. Moreover, NRF-1 silencing down-regulated genes encoding for key folate transporters and enzymes in folate metabolism. These novel findings identify NRF-1 as a major inducible transcriptional regulator of PCFT gene expression. The implications of this linkage between folate transport and metabolism with mitochondria biogenesis and respiration are discussed.
Highlights
Ological pH and cannot traverse the plasma membrane by passive diffusion
We examined the impact of Nuclear respiratory factor-1 (NRF-1) knockdown on the expression status of genes encoding for key folate-dependent enzymes that are central to, and crucial for the folate metabolism including: dihydrofolate reductase (DHFR), thymidylate synthase (TS), glycinamide ribonucleotide transformylase (GARTF), 5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICARTF), 5,10-methylenetetrahydrofolate reductase (MTHFR), folylpoly-␥-glutamate synthetase (FPGS), and ␥-glutmayl hydrolase (GGH)
This presumption is further substantiated by the fact that the promoters of some of these genes encoding for folate transporters and folate-dependent enzymes lacked consensus NRF-1 binding sites (Fig. 5); upon bioinformatic examination of the presence of putative NRF-1 binding sites in the promoters of all studied genes encoding for folate transporters and folatedependent enzymes that were down-regulated by NRF-1 knockdown, only reduced folate carrier (RFC), dihydrofolate reductase, ␥-glutmayl hydrolase, cytosolic SHMT (cSHMT), and mitochondrial enzyme serine transhydroxymethylase (mSHMT) had one or more consensus NRF-1 binding site(s), whereas Folate receptors (FRs)␣ and 5,10-methylenetetrahydrofolate reductase were devoid of NRF-1 binding sites in their 2-kb promoter region (Fig. 5)
Summary
Cell Culture—Human cervical cancer HeLa cells were maintained in RPMI 1640 medium (Invitrogen) containing 10% fetal calf serum, 2 mM glutamine, 100 g/ml of penicillin, and 100 g/ml of streptomycin (Biological Industries, Israel) in a humidified atmosphere of 5% CO2. An aliquot of nuclear proteins (6 g) was incubated for 20 min on ice either with the unpurified polyclonal anti-NRF-1 antibody Complexes consisting of DNA, nuclear protein(s), and a specific antibody were resolved by electrophoresis on 6% nondenaturing polyacrylamide gels in Tris borate-EDTA, pH 8.4, at 4 °C. Nuclear proteins (Յ30 g) were resolved by electrophoresis on 10% polyacrylamide gels containing SDS, electroblotted onto Protran BA83 cellulose nitrate membranes (Schleicher & Schuell), and reacted with rabbit anti-NRF-1 (1:2,500) Following three 10-min washes in Tris-buffered saline supplemented with 0.5% Tween 20 (TBST) at room temperature, blots were reacted with a goat anti-rabbit secondary antibody (Jackson ImmunoResearch), rewashed, and enhanced chemiluminescence (ECL) detection was performed according to the manufacturer’s instructions (Biological Industries). Two-tailed p values Յ0.05 were considered to be statistically significant
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