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Humanizing mouse folate metabolism: conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure Chen Yang,*,†,1 Lin-Ying Xie,†,1 Jolene J. Windle,†,‡ Shirley M. Taylor,†,§ and Richard G. Moran*,†,2 *Department of Pharmacology and Toxicology, †Massey Cancer Center, ‡Department of Human and Molecular Genetics, and §Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia, USA The mouse is extensively used to model human folate metabolism and therapeutic outcomes with antifolates. However, the folylpoly-␥-glutamate synthetase (fpgs) gene, whose product determines folate/antifolate intracellular retention and antifolate antitumor activity, displays a pronounced species difference. The human gene uses only a single promoter, whereas the mouse uses two: P2, akin to the human promoter, at low levels in most tissues; and P1, an upstream promoter used extensively in liver and kidney. We deleted the mouse P1 promoter through homologous recombination to study the dual-promoter mouse system and to create a mouse with a humanized fpgs gene structure. Despite the loss of the predominant fpgs mRNA species in liver and kidney (representing 95 and 75% of fpgs transcripts in these tissues, respectively), P1-knockout mice developed and reproduced normally. The survival of these mice was explained by increased P2 transcription due to relief of transcriptional interference, by a 3-fold more efficient translation of P2-derived than P1-derived transcripts, and by 2-fold higher stability of P2-derived FPGS. In combination, all 3 effects reinstated FPGS function, even in liver. By eliminating mouse P1, we created a mouse model that mimicked the human housekeeping pattern of fpgs gene expression.—Yang, C., Xie, L.-Y., Windle, J. J., Taylor, S. M., Moran, R. G. Humanizing mouse folate metabolism: conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure. FASEB J. 28, 1998 –2008 (2014). www.fasebj.org

ABSTRACT

Key Words: knockout mice 䡠 transcription 䡠 translation 䡠 protein translocation and degradation 䡠 transcriptional interference

Abbreviations: CHX, cycloheximide; ES, embryonic stem; FPGS, folylpoly-␥-glutamate synthetase; HA, hemagglutinin; hGH, human ␥-glutamate hydrolase; KO, knockout; nt, nucleotide; MCS, multiple cloning site; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; RACE, rapid amplification of cDNA ends; TSS, transcription start site; UTR, untranslated region; WCL, whole-cell lysate; WT, wild type 1998

The folate cofactors mediate 1-carbon transfer reactions that are essential for all living organisms (1). In mammals, as circulating folates penetrate the plasma membrane, they are promptly converted into polyglutamate metabolites by folylpoly-␥-glutamate synthetase (FPGS), a metabolic conversion that promotes intracellular retention of these essential cofactors and their affinity for some folate cofactor-dependent enzymes (2). Likewise, polyglutamation enhances the affinity of most antifolates for their target enzymes and their intracellular retention to the extent that the parent compounds are considered to be prodrugs (3, 4). The intracellular trapping induced by polyglutamation dramatically affects the pharmacodynamics of antifolates, with bolus administration having a prolonged intracellular effect. Thus, expression of FPGS is a central determinant of tumor sensitivity to antifolates, and decreased FPGS activity is a major cause of antifolate resistance (5–7). The preclinical development of antifolates has relied on the mouse as the major model organism to predict the utility of these compounds in human cancer therapy. However, severe, unexplained toxicities have occurred in early clinic trials of antifolates without warning from prior mouse studies. For instance, the toxic dose of lometrexol varied by ⬃100fold between man and mouse, an effect that could be mimicked by inducing a mildly lower folate economy in mice by brief feeding of a folate-deficient diet (8, 9). Such drastic differences in responses to antifolates between mouse and human may reflect the differences in the tissue-specific expression patterns of the fpgs gene between the species (refs. 10 –13 and Fig. 1). 1

These authors contributed equally to this work. Correspondence: Massey Cancer Center, Virginia Commonwealth University, 401 College St., Richmond, VA 232980035, USA. E-mail: [email protected] doi: 10.1096/fj.13-243261 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2

0892-6638/14/0028-1998 © FASEB

MATERIALS AND METHODS Construction of the P1-A1aA1b targeting vector

Figure 1. Distinct fpgs transcription patterns in mouse and human tissues. These estimates of promoter usage were derived from previously published RNA protection assays (10, 11) and from RT-qPCR (this report).

The human fpgs gene encompasses 15 exons spanning 12 kb, and its transcription is controlled by a single promoter immediately upstream of the first exon (14 –17). In mice, 2 additional exons (A1a and A1b), located 10 kb upstream of the 15 exons homologous to those of the human gene, are used in place of exon 1 when an associated promoter (P1) is activated (11, 13, 18). The mouse fpgs gene transcribes strongly and predominantly from the P1 promoter in liver and kidney, the 2 major folate storage organs, but its transcription initiates exclusively from the proximal promoter (P2) in all other murine tissues, tumors, and cell lines (11), indicating liver- and kidney-specific transcription factors for P1 promoter usage. In mouse P2, the behavior and distribution of transcription factor binding sites are very similar to those of the human promoter (12, 19). The intricate tissuespecific expression patterns of the mouse fpgs gene are coordinated by epigenetic events and also appear to involve transcriptional interference over P2 in tissues expressing transcript from P1 (20). Intriguingly, although a P1 promoter and exon A1a and A1b sequences are conserved in the human genome, human fpgs transcription is exclusively controlled by the P2 promoter, and human P1 transcripts are negligible even in liver (10). Hence, the mouse fpgs depends on a dual-promoter control system to make 2 proteins that differ at their N-terminal sequences and in sensitivity to feedback by folylpolyglutamates (21), whereas the human fpgs uses only a single promoter equivalent to mouse P2 (Fig. 1). In an effort to understand the role of the P1 promoter in mouse development and to create a mouse model that more faithfully reflects human folate metabolism, we deleted the mouse P1 promoter and its associated exons by homologous recombination. The mouse homozygous for the P1 deletion allele [P1knockout (KO)] developed and reproduced normally. There were only minor changes in FPGS activity and folate metabolism in P1-KO mice because of several interacting factors, each of which made the FPGS expressed in mouse a closer mimic of the human enzyme. Thus, the dual-promoter system of mouse fpgs is a retained evolutionary remnant, and the P1-KO mouse is a more representative model for human folate metabolism and, presumably, the responsiveness to antifolates. MOUSE WITH HUMANIZED FPGS PROMOTER USAGE

Two contiguous HindIII genomic DNA fragments containing the mouse fpgs promoters were cloned previously from a mouse 129/sv bacterial artificial chromosome (BAC) library (11). One of these, a 8.5-kb HindIII fragment, includes the upstream P1 promoter and exons A1a and A1b (in pGEM1zP1-A1aA1b); the 7.1-kb HindIII genomic fragment includes the downstream P2 promoter and exon 1 (in pGEM1z-P2Ex1; see Fig. 2A). The targeting vector, pKO2lx, was based on pKO Scrambler NTKV-1901 (Agilent Technologies, Santa Clara, CA, USA) in which the neo selection casette was flanked by 2 loxP sites (loxP2 and loxP3). The 5= homologous recombination region, a 3.2-kb HindIII-XhoI fragment upstream of the P1 promoter, was cloned into pBluescript-SK (Agilent Technologies) and modified by insertion of a 48nucleotide (nt) SalI-loxP-XhoI fragment (loxP1) into the XhoI site (oligonucleotide sequences are listed in Supplemental Table S1). The floxed 5= homology region HindIII-XhoI fragment and a 2.6-kb XhoI-XhoI target fragment containing P1-A1aA1b were ligated simultaneously into the pKO2lx vector digested with HindIII and XhoI. The 3= homology region was created by sequentially ligating a 2.7-kb XhoIHindIII fragment from pGEM1z-P1-A1aA1b and a 2.2-kb HindIII-SpeI fragment from pGEM1z-P2-Ex1 into pBluescriptSK. The resulting 4.9-kb fragment was released by Xhol and NotI digestion and inserted into the SalI- and NotI-digested pKO2lx vector containing the 5= homology region and P1A1aA1b fragment. The structure of the final targeting vector was confirmed by sequencing. Generation of embryonic stem (ES) cells with P1 conditional- or complete-KO fpgs allele The linearized targeting vector was electroporated into 129/Sv ES cells, and G418- and ganciclovir-resistant clones harboring the desired homologous recombination event were identified by PCR analysis and DNA sequencing to confirm the retention of all 3 loxP sites. The PCR primers were P1-F (5=-TGAGTCAGTAGGCTCAGTGTGAGA-3=) and a pKO2lx multiple cloning site (MCS) sequence primer MCS-R (5=GCGGTCTAGGAATTCTCTAGGATCG-3=). Clones that had recombined within the A1a/A1b region were discarded. Homologous recombination was confirmed by Southern blot analysis on SacII-digested genomic DNA. The blot was hybridized with a [␣-32P]dCTP (PerkinElmer, Waltham, MA, USA)labeled PCR fragment encompassing exon A1a through A1b generated using primers 9047-F (5=-TGCGAACCTCTCAGGTAAACACTC-3=) and 9322-R (5=-CCTGAAAGGAGGCAGTCTTAGCTT-3=). The wild-type (WT) allele is represented by an 18-kb SacII fragment, whereas the recombined allele is shortened to 11 kb, because of the SacII site introduced with the pKO2lx MCS. Cre recombinase treatment of ES cells was used to eliminate the PGK-Neo cassette and/or the P1-A1aA1b sequence. Correctly targeted ES clones were transiently transfected with the pCMV-Cre plasmid, which also contains a neomycin resistance cassette. G418-sensitive clones were analyzed by PCR using primers P1-F and MCS-R. Cre-mediated loxP1/ loxP3 recombination generated complete knockout clones and resulted in a 175-bp PCR product, whereas the Cre-mediated loxP2/loxP3 recombination resulted in conditional-KO clones, detected by a 2.8-kb PCR band. 1999

Generation and characterization of homozygous P1-KO mice All experimental procedures were conducted under protocols approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee. Individual ES clones with floxed or deleted P1-A1aA1b alleles were injected into C57BL/6 blastocysts to create chimeric mice; breeding of these chimeras to C57BL/6 mice resulted in germline transmission. Mice heterozygous for the P1 deletion allele were backcrossed to C57BL/6 for 8 generations. Genotypic analysis by tail DNA PCR produced a product of 275 bp from the WT allele with primers 9047-F and 9322-R, and a band of 175 bp from the P1-KO allele with primers P1-F and MCS-R. The fertility and the progeny sex ratio were compared between littermate WT and homozygous P1-KO mice. The growth of WT and P1-KO mice was monitored 2⫻/wk in the first 10 wk while mice were fed a normal-folate diet (Test Diet 5S4Q, with 1300 ␮g of folate/kg of diet; Test Diet, St. Louis, MO, USA) or a low-folate diet (Test Diet 5T2L, with 63 ␮g of folate/kg of diet) in the presence of 1% succinylsulfathiazole, with ⱖ6 mice in each group. The morphology and weights of liver, kidney, spleen, heart, and brain were recorded at 10 wk and compared between WT and P1-KO mice. Analysis of folate metabolism FPGS enzyme assays were performed on extracts of mouse liver, kidney, heart, and brain as described previously (22). A total of 30 ␮g of protein from tissue extracts without ammonium sulfate precipitation was used in these assays, and the 10-␮l reactions were incubated at 37°C for 30 min. The activity of FPGS in tissue extracts was calculated as picomoles per hour per milligram of protein as described previously (23). The folate content and polyglutamation state in liver extracts were measured using a Lactobacillus casei microbiological assay (24) with modifications. Recombinant human ␥-glutamate hydrolase (hGH) was purified from extracts of the BL21(DE3)pLysS strain of Escherichia coli (EMD Millipore, Billerica, MA, USA) transformed with pET24a-hGH (a gift from Dr. T. J. Ryan, Wadsworth Center, Albany, NY, USA; ref. 25). The activity of the purified hGH was determined to be 8.1 IU/ml by the conversion of 5,10-dideazatetrahydropteroylpentaglutamate to 5,10-dideazatetrahydrofolate measured by HPLC (26). Mouse liver extracts (10 mg of protein in a 100-␮l volume) were treated with increasing amounts of hGH, and 30 ␮g of the treated extracts were assayed with L. casei (ATCC 7469; American Type Culture Collection, Manassas, VA, USA) using folinic acid as a standard (24). Mouse serum homocysteine levels in the normal state or after 18 h of food withdrawal were measured using thiol-specific HPLC (27). Northern blot analysis Total RNA from tissues of 10-wk-old mice was extracted with TRIzol (Life Technologies, Carlsbad, CA, USA), and 10-␮g aliquots were used for Northern blot analysis (12). The antisense RNA probe was generated by T3 RNA polymerase (Thermo Scientific, Waltham, MA, USA) on linearized pCR4TOPO-Mmfpgs containing mouse fpgs exons E2⫺E15 in the presence of [␣-32P]CTP (PerkinElmer), and the sense RNA probe was produced by T7 RNA polymerase (Thermo Scientific) on the same plasmid linearized with SpeI. The RNA probes were acid-phenol extracted and ethanol precipitated and used at a concentration of 2 ⫻ 106 cpm/ml of hybridiza2000

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tion solution (ULTRAhyb; Life Technologies). Ethidium bromide staining of ribosomal 18S RNA was used as a loading control. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) RT-qPCR was performed on total RNA extracted from murine, human, (FirstChoice Human Survey Panel; Life Technologies), and rat tissues (BioChain, Newark, CA, USA). Total RNA (5 ␮g) was treated with 1 U of DNase I (Thermo Scientific) in a 10-␮l reaction for 20 min at 37°C, and then 0.5 ␮l of 50 mM EDTA was added. DNase I was inactivated by a 10-min incubation at 65°C. Total RNA was reverse transcribed using the Maxima First Strand cDNA Synthesis Kit (Thermo Scientific) in a 20-␮l reaction according to the manufacturer’s protocol. The reaction was diluted to 100 ␮l, and 1 ␮l of cDNA was used in a 25-␮l qPCR using Maxima SYBR Green qPCR Master Mix (Thermo Scientific). The real-time PCR data were analyzed by Opticon Monitor 3 software (Bio-Rad Laboratories, Hercules, CA, USA). Mouse and rat promoter-specific transcripts were quantified using first exon-specific primer pairs (Supplemental Table S1). Total fpgs transcript levels were determined using a common primer pair covering exons E2⫺E5 or E14⫺E15. pcDNA3.1(⫺) plasmids harboring fpgs exons A1a/A1bE2⫺E15 or exons E1⫺E15 were used as absolute standards. The relative DNA amounts between the 2 standard plasmids were adjusted by using qPCR with the common primer pairs E2⫺E5. 18S rRNA was used as a normalization control. Rapid amplification of cDNA ends (RACE) The 5= end and 3= end of mouse and rat fpgs transcripts were determined using a FirstChoice RNA Ligase Mediated RACE Kit (Life Technologies) on total RNA samples. The reverse transcription reaction products were used for PCR amplification using PfuUltra II DNA Polymerase Master Mix (Agilent Technologies). The blunt-ended PCR fragments were cloned into pJET1.2 (Thermo Scientific) for sequencing. The genespecific primers for RACE PCR are listed in Supplemental Table S1. Construction of pcDNA3.1(ⴚ) fpgs expression vectors To delete the extra 5=-untranslated region (UTR) from the pCMV promoter, NdeI/NheI double-digested pcDNA3.1(⫺) (Life Technologies) was ligated with a PCR fragment amplified with primers pcDNA-Nde-F (5=-GTATCATATGCCAAGTACGCC-3=) and pcDNA-Nhe-pCMV-R (5=-GCTAGCAGCCAGAGAGCTCTGCTTATATA-3=) using pcDNA3.1(⫺) as a template. Coding sequences for mouse and rat fpgs were amplified from 5= RACE cDNA with promoter-specific forward primers and a reverse primer containing the C-terminal hemagglutinin (HA) tag coding sequence (Supplemental Table S1). After NheI/XhoI digestion, PCR products containing C-terminal HAtagged fpgs coding sequences were ligated into a 5=-UTR-deleted pcDNA3.1(⫺). An expression vector (pcDNA3) containing human FPGS without an HA tag (clone M7.2) was constructed previously (16). QuikChange Site-Directed Mutagenesis (Agilent Technologies) was used to introduce mutations with the primer pairs listed in Supplemental Table S1. Transient transfection of AuxB1 cells The Chinese hamster ovary cell line AuxB1 was maintained in minimal essential medium-␣ (Life Technologies) plus 10%

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dialyzed fetal bovine serum (Gemini, Irvine, CA, USA). In vitro transfection was performed with linear polyethylenimine (molecular mass 25 kDa, 23966-2; Polysciences, Warrington, PA, USA) as described previously (28), with modifications. In brief, AuxB1 cells (1.2⫻106/60-mm dish) were seeded 24 h before transfection. The medium was changed 30 min before transfection. Expression plasmids (5 ␮g) were mixed with 250 ␮l of Opti-MEM (Life Technologies), and 15 ␮l of 1 ␮g/␮l polyethylenimine was added. After 15⫺20 min of incubation at room temperature, the mixture was added dropwise into the culture. Cells were harvested by scraping on ice 20 h later. Immunoblotting Transfected AuxB1 cells were pelleted and then were resuspended in 2% SDS buffer and sonicated using a bath sonicator (Diagenode, Denville, NJ, USA). An equivalent sample was resuspended in buffer containing 50 mmol/L Tris (pH 7.5), 50 mmol/L NaCl, 1% Nonidet P-40, 0.5 mM EDTA, and protease inhibitor complete mixture (Roche Applied Science, Indianapolis, IN, USA) and incubated for 30 min on ice. After a 10-min centrifugation, the supernatant containing cytosolic and mitochondria fractions (postnuclear fraction) was saved, and the pellet was treated with 2% SDS buffer and sonication to release nuclear protein. Protein lysates were resolved on 7.5% SDS-polyacrylamide gels and were transferred to a nitrocellulose membrane. Membranes were probed with antibodies against human FPGS C-terminal peptides (clone 4-18, 1:400; Eli Lilly & Co., Indianapolis, IN, USA; ref. 29), HA (clone HA-7, 1:5000, Sigma-Aldrich, St. Louis, MO, USA), ␤-actin (1:5000; Abcam, Cambridge, MA, USA), or ␤-tubulin (1:1000, Cell Signaling Technology, Danvers, MA, USA).

RESULTS Construction of homologous recombination vectors for conditional and complete deletion of the fpgs P1 promoter and the associated exons Because the P1 promoter was used so predominantly in liver and kidney, there was a considerable risk that deletion of P1 would cause embryonic lethality; hence, we initially pursued a conditional gene-KO strategy (Fig. 2A). The targeting vector was based on vector pKO2lx with a neomycin selection cassette flanked by 2 loxP sites (loxP2 and loxP3), into which was inserted 5= and 3= homologous recombination sequences and a floxed P1-A1aA1b fragment, as described in the Materials and Methods. The targeting construct was electroporated into ES cells, and G418- and ganciclovir-resistant clones harboring the desired homologous recombination event and retaining all 3 loxP sites (Fig. 2A) were identified by PCR and by DNA sequencing and confirmed by Southern blot analysis (Fig. 2C). To produce a homologous recombination allele in which P1 and exons A1a and A1b were universally deleted (termed P1-KO hereafter), a correctly targeted ES clone was transiently transfected with the pCMV-Cre plasmid to induce loxP recombination by Cre recombinase. Whereas recombination between loxP2 and loxP3 generated a neomycin-sensitive conditional KO clone (Fig. 2B, right), recombination between loxP1 and loxP3 resulted in the P1-KO allele (Fig. 2B, left). Clones sensitive to G418 were further analyzed by PCR to determine the site of recombination driven by Cre recombinase (Fig. 2D).

Protein half-life assay Pulse-chase labeling of protein was performed on transfected AuxB1 cells (30). In brief, 14 h after transfection, AuxB1 cells (5⫻106 in 60-mm dishes) were labeled with 120 ␮Ci of 35S protein labeling mix (PerkinElmer) for 40 min (240 ␮Ci for a 10-min short labeling). Cells were washed and chased with unlabeled 4 mM methionine and 1 mM cysteine. Cells were harvested, solubilized in lysis buffer containing 50 mM Tris (pH 7.5), 0.5 mM EDTA, 1% SDS, and 1 mM dithiothreitol, and sonicated for 10 min. The lysates were diluted with an equal volume of 1% Nonidet P-40 buffer, and immunoprecipitated with anti-HA monoclonal antibody. The precipitates were resuspended and an equal volume was applied to SDS-PAGE gels. The gel was fixed and dried, and the signal intensities were acquired by phosphoimager analysis using ImageQuant software (GE Healthcare, Piscataway, NJ, USA). This experiment was performed 3 times on different days. Protein turnover inhibition assay To block protein synthesis, 50 ␮g/ml cycloheximide (CHX) was added to AuxB1 cells 16 h after transfection. Inhibitors of lysozyme (50 mM ammonia chloride or 100 ␮M chloroquine) or of the proteasome (50 ␮M bortezomib or 50 ␮M MG-132) were added at the same time. After 6 h of treatment, cells were harvested, and protein was extracted with 1% SDS buffer and resolved for immunoblotting analysis. MOUSE WITH HUMANIZED FPGS PROMOTER USAGE

P1-KO mice had a normal phenotype and only minor changes in folate metabolism Individual ES cell clones with correctly recombined conditional KO or P1-KO alleles (Fig. 2B) were injected into C57BL/6 blastocysts to create chimeric mice, and further breeding of these chimeras to C57BL/6 mice resulted in germline transmission. When heterozygous mice were mated, homozygous mice were born for both conditional-KO and P1-KO alleles. Genotypic analysis (Fig. 2E) indicated that homozygous P1-KO mice, heterozygous animals, and WT pups, were obtained in a 1:2:1 mendelian frequency from heterozygous matings. The homozygous P1-KO mice exhibited a normal phenotype. They showed no gross morphological defects, and their growth in the first 10 wk was not distinguishable from that of WT littermate mice with feeding of either standard mouse chow or a low-folate diet in both sexes (data not shown). Both male and female homozygous P1-KO mice were fertile, and their reproduction capacities were the same as those of WT mice. The absence of phenotypic changes in homozygous P1-KO mice was surprising, given the lack of the P1-derived mRNA in these animals, which is the dominant fpgs transcript in WT liver and kidney (see below). 2001

Figure 2. Deletion of the fpgs upstream P1 promoter and exons A1a and A1b in mice through homologous recombination. A) Structural landmarks of the mouse genomic locus surrounding the fpgs P1 promoter after the desired homologous recombination event with retention of all 3 loxP sites (arrowheads). B) Diagram for targeted mouse genomic DNA after Cre-mediated recombination in ES cells. Structure of the fpgs locus in cells selected as full (left) and conditional (right) P1 KO is shown, along with the positions of screening PCR primers and a vector-introduced SacII site. C) Southern blot analysis of ES cell genomic DNA digested with SacII. Blot was hybridized with the indicated labeled PCR probe. WT allele retained an 18-kb fragment, whereas the recombinant allele showed hybridization to an 11-kb fragment due to the introduced SacII. D) PCR genotyping of ES cell clones after loxP recombination mediated by transient Cre expression. Complete KO generated a 175-bp PCR product, whereas a conditional-KO clone resulted in a 2.8-kb PCR fragment. E) Representative PCR genotyping of mouse tail DNA. WT allele produced a PCR band of 275 bp (lane 2), whereas the complete-KO allele generated a PCR band of 175 bp (lane 3). Heterozygous mice had both bands (lane 1). M, markers.

We examined the levels of FPGS in liver and kidney in these P1-KO animals. In our hands, multiple antibodies raised against human FPGS failed to cross-react with mouse FPGS. One well-established previously published (28) monoclonal antibody (clone 4-18) detected a clean band of the expected size from liver and kidney lysates of mice (and rats), but this turned out to be a cross-reacting protein (Supplemental Fig. S1). Therefore, FPGS enzyme assays were performed as an estimate of protein levels; these assays detected ⬃70% FPGS activity in liver and ⬃80% in kidney of P1-KO mice compared with that in WT littermates (Fig. 3A). The classic assay for tissue folates involves a microbiological assay using L. casei. This bacterium requires exogenous folates for growth but responds only to

folates with a single glutamate side chain; folates with longer side chains can only be used after conversion to monoglutamates by folylpoly-␥-glutamate hydrolase. After treatment of extracts with recombinant hGH before assay, liver extracts from WT and P1-KO mice contained 8⫺10 and 6⫺7 times more usable folates than untreated extracts, respectively (Fig. 3B). Therefore, liver folates were predominantly in polyglutamate forms and were at similar levels in WT and P1-KO mice. Serum homocysteine levels are a sensitive indicator of whole-body folate economy; there were similar levels of serum homocysteine in WT and P1-KO mice of both sexes, and serum homocysteine decreased similarly by ⬃50% after 18 h of food withdrawal in P1-KO and matched WT mice (Fig. 3C).

Figure 3. Functional assays illustrate minor decreases in folate metabolism in P1-KO mice compared with those in WT mice. A) FPGS enzyme activity in lysates of mouse liver and kidney. B) Folate levels and polyglutamation state in lysates of mouse liver. Liver extracts were incubated with increasing amounts of hGH, and then aliquots were assayed for the ability to support the growth of folate-deficient L. casei. C) Homocysteine levels in sera from sibling untreated mice or animals denied access to food for 18 h. 2002

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Therefore, the level of FPGS enzyme activity in liver and kidney was only minimally disturbed by the deletion of the fpgs P1 promoter, and there was a correspondingly normal pattern of tissue folate polyglutamates in P1-KO mice, explaining the normal development and growth of these animals. P1-KO mice assumed a humanized fpgs expression pattern The levels of fpgs transcripts were measured in mouse tissues by Northern blots using strand-specific probes covering exons E2⫺E15. The sense RNA probe did not detect signal in any tissue tested, suggesting no cryptic antisense transcription (data not shown). With the antisense probe, liver and kidney of WT mice showed far greater fpgs signals than the heart and brain (Fig. 4A). In contrast, the levels of total fpgs transcripts in liver and kidney were much lower in P1-KO mice than in WT mice, and the levels of fpgs transcripts in P1-KO mice

were more uniform across tissues than those in WT mice (Fig. 4A). This humanized fpgs expression pattern in tissues of P1-KO mice was verified by RT-qPCR. Comparing the total fpgs transcript levels in liver, kidney, and heart with those in brain illustrated the shift from a tissue-specific pattern in WT mice to a more homogenous lower level expression in P1-KO mice, mimicking the human fpgs expression pattern across tissues (Fig. 4B). We confirmed that the P1 promoter had been inactivated in P1-KO mice. Using exon-specific primers, we quantitated the contributions of promoter-specific transcripts in various tissues. The fpgs transcripts in liver of WT mice originate predominantly from P1 (90⫺95%), with only 4⫺5% coming from P2 (Fig. 4C). With the elimination of upstream P1 transcription in P1-KO mice, transcription from P2 in liver increased by a factor of 1.5⫺2.6 fold (Fig. 4C), reflecting the relief of transcription interference of P2 mediated by active

Figure 4. P1-KO mice assumed a human-like fpgs expression pattern. A) Northern blot of total RNA from tissues of WT and P1-KO mice. Formamide-treated total RNA was loaded onto a formaldehyde denaturing gel. In vitro transcribed fpgs antisense RNA covering exons 2⫺15 was used as a probe. Expected size for fpgs mRNA is 2400 nt, and ethidium bromide staining of 18S rRNA was used as a loading control. B) Total fpgs transcripts from WT and P1-KO mouse and human tissues quantitated with RTqPCR. PCR primer pairs for both mouse and human tissues covered exons E2⫺E4. Total fpgs transcript levels in different tissues were normalized to 18S rRNA content and were expressed relative to the levels in brain. C) Relative contributions of P1 and P2 transcripts in liver and kidney were compared between WT and P1-KO mice by RT-qPCR. P1 and P2 promoter-specific PCR primer pairs cover exons A1b-E3 and E1⫺E3, respectively. Total fpgs transcript level in WT tissues (determined by qPCR with a primer pair covering exons E2⫺E4) was set at 100%. Plasmid pcDNA3.1(⫺) containing cDNA from P1 or P2 transcripts was used as an absolute standard. D) Relative contribution of P1 and P2 transcripts in heart and brain was compared between WT and P1-KO mice with RT-qPCR.

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transcription from the upstream P1 promoter (20). A small change in the promoter-specific transcription pattern was also observed in kidney between WT and P1-KO mice (Fig. 4C). Conversely, the low levels of exclusively P2 transcripts in WT heart and brain were not changed by P1 deletion (Fig. 4D). Notably, we also discovered an fpgs dual-promoter system with a similar expression pattern in rat, i.e., a predominant P1 transcript in liver and kidney and exclusive utilization of the P2 promoter in heart and brain (Supplemental Fig. S2A). Therefore, even though the normal phenotype of P1-KO mice implies that the fpgs P1 promoter is not required for embryonic development and adult survival of rodents under laboratory conditions, the conservation of the dual promoter system in rodents suggests that the P1-derived transcripts and/or enzyme have some unique characteristics that provide a survival advantage in the wild. Mouse fpgs transcription start sites (TSSs) were recapitulated by in vitro transfection The fact that the P1-KO mice had normal folate homeostasis and FPGS levels in liver similar to those in WT mice, yet had much lower (10-fold) levels of fpgs transcripts in liver, needed explanation. We studied the post-transcriptional processes for fpgs transcripts made from P1 and P2 after transfection of cDNAs for these transcripts into AUXB1, a hamster cell line with codon usage almost identical to that of mouse cell lines. Initially, we determined the 5=-UTR sequences of mouse P1- and P2-derived transcripts in WT mouse tissues. Cap-dependent RACE illustrated a single TSS 57 nt upstream of the in-frame ATG1 for P1 transcripts from liver and kidney, but multiple TSSs for P2 promoter-controlled transcription in liver, kidney, and brain (Fig. 5A, C), similar to that seen in human tissues (10), with a dominant TSS 9 nt upstream of ATG1 in exon 1. Abnormal splicing events were minimal for both P1 and P2 transcripts. Notably, 5= cap-dependent RACE on RNA from rat tissues also showed multiple TSSs for P2 transcripts (Supplemental Fig. S2B), and the predominant TSS for rat P1 transcript was located in the intron before A1b (where the first in-frame ATG is located), a feature previously found for the vestigial human P1 (10). The 3=-UTR (⬃450 nt) and the poly(A) sites were identical for all fpgs transcripts from mouse tissues. The pcDNA3.1(–) expression vector was modified to delete the intervening T7 promoter sequences, placing the CMV TATA box 30 bp upstream from the NheI site used for insertion of the cDNAs (Fig. 5B). This modification led to initiation of CMV promoter-controlled transcription exactly at the beginning of the cloned cDNAs, a placement found critical for assessing the effect of 5=-UTR sequences on translation. When the constructs containing cDNAs for the P1 transcript (TSS at ⫺57A) and for the major P2 transcript (TSS at ⫺9A) were transfected into AUXB1 cells, 5= cap-dependent RACE analysis indicated TSSs for transcriptional initia2004

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Figure 5. TSSs of fpgs used in mouse tissues was recapitulated in transfected AuxB1 cells. A) Mapping of TSSs from the mouse P1 and P2 promoters to the sequence of the mouse genomic locus. Major TSSs are indicated by arrows. The 3 ATG codons that are potential TSSs are boxed in each promoter, and the first nucleotide in ATG1 was designated as ⫹1. In the 5=-UTR of the P1 transcript, there are 2 upstream out-of-frame ATG codons, which could generate a short 10-aa peptide due to 3 consecutive termination codons overlapping with ATG1. B) Transcription from the CMV promoter in pcDNA3.1(⫺) starts ⬃28⫺33 nt downstream (dashed boxed region) of the TATA box. Deletion of the extra sequence following the CMV promoter allowed transcription to initiate at the beginning of the cloned cDNA after the NheI site. C) Transfection of AuxB1 cells with the P1 and P2 transcripts cloned into this truncated pcDNA3.1(⫺) recapitulated the TSS utilization in vivo. Cap-dependent 5=-RACE was performed on total RNA from liver, kidney, and brain tissues of WT mice and also on those from AuxB1 cells transfected with expression vectors containing cDNA from the major P1 (beginning with ⫺57) and P2 TSS (beginning with ⫺9).

tion in AUXB1 cells precisely the same as those seen with the mouse in vivo transcripts (Fig. 5C). Because the 5= end cap structure facilitates eukaryotic translation (31), we questioned whether the predominant P1 transcript in WT liver has a 5=-m7G cap defect compared with the P2 transcript. Two independent methods were used to isolate 5= capped mRNAs (an immunoprecipitation with monoclonal antibody against m7G-cap and a GST-eIF4E pulldown), and the level of capped fpgs mRNA was estimated by RT-qPCR. A consistent pattern was observed with ⬃10 times

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higher total fpgs transcript in cap-positive mRNA from WT liver than in P1-KO liver and WT brain (data not shown), suggesting no 5= cap defect in P1 transcripts. P2-derived transcripts produce much higher levels of steady-state protein than P1-derived transcripts The levels of mRNAs made in AUXB1 cells transfected with expression vectors containing P2, P1, and a modified P1 cDNA, in which 2 upstream out-of-frame ATG triplets (Fig. 5A) had been mutated, were determined by RTqPCR (Supplemental Fig. S3A). The levels of mRNAs for these 3 fpgs transcripts were identical over an extended time course when equivalent amounts of the different cDNAs were transfected (Supplemental Fig. S3B). Hence, the transcription efficiencies and RNA stabilities for these cDNAs were equivalent in AUXB1 cells. The level of FPGS protein made from these transfected cDNAs was determined. Because none of the available antibodies against human FPGS recognized mouse FPGS (Supplemental Fig. S1), cDNA clones from mouse transcripts were modified to add a C-terminal HA epitope (Supplemental Fig. S1C) to allow this analysis. More than 10 times higher levels of FPGS were detected in whole-cell lysates (WCLs) of AUXB1 cells transfected with mouse P2 cDNA than in cells containing the same levels of P1 transcript (Fig. 6A, B), suggesting a major difference in the efficiency of translation of P1 and P2 transcripts and/or the stability of the proteins made from these transcripts. Two protein bands were observed from P1derived transcripts (Fig. 6A, B); these were identified to be made from ATG1 and ATG3 (Fig. 5A) by mutation of each of these potential start codons (data not shown).

When the 2 upstream out-of-frame ATGs in the P1 transcript (Fig. 5A) were mutated (P1-upATG), translation from ATG1 was significantly enhanced, revealing an interfering effect of the 5=-UTR out-of-frame ATGs on utilization of the downstream in-frame ATG1 (Fig. 6A). This analysis also revealed differences in the subcellular distribution of FPGS made from P1 and P2. Thus, when hypotonic Nonidet P-40-containing buffer was used to separate a nuclear fraction and a postnuclear supernatant, P2-derived FPGS was not found in the nuclear fraction, but a significant amount of P1-derived protein and an even higher fraction of P1-upATG-derived enzyme were nuclear (Fig. 6A, B). Further differential centrifugation analysis of P1-upATG transfectants indicated that the majority of the protein initiated from ATG1 translocated to the nucleus (Fig. 6C), in contrast to the almost exclusive localization of P2-derived FPGS to mitochondria and cytosol (10, 11). This difference in the subcellular compartmentation of P1- and P2-derived FPGS was consistent with the prediction from WoLF PSORT software (National Institute of Advanced Industrial Science and Technology, Tokyo, Japan) analysis of N-terminal amino acid sequences of these proteins. Notably, human FPGS was found to distribute only to the mitochondria and cytosol compartments, directly paralleling mouse P2-derived FPGS (data not shown and ref. 11). Determination of the stability and translation efficiency of the proteins made from P1 and P2 promoters The relative translation efficiencies and stabilities of the protein products from P1 and P2 transcripts were

Figure 6. Levels of P1- and P2-derived FPGS and the subcellular localization of these proteins. AuxB1 cells were transfected with the modified expression constructs containing P2, P1, or P1-upATG cDNA with the endogenous 5=-UTR. P1-upATG had the two 5=-UTR out-of-frame ATGs mutated to ACG and GTG, respectively. Transfected cells were solubilized with SDS buffer to yield a WCL or in Nonidet P-40-containing buffer to produce a postnuclear supernatant (PNS) after centrifugation. Nuclear pellet from Nonidet P-40 treatment was solubilized with SDS buffer to extract a nuclear fraction (N). Western blots were performed on WCL (60 ␮g), postnuclear supernatant (45 ␮g), and nuclear fraction (15 ␮g). A) Levels of FPGS produced by transfection with P2, P1, or P1-upATG constructs. B) Estimation of FPGS levels made from constructs. After transfection with the P2 construct, AuxB1 lysates were diluted 10 times, and loaded in parallel with those from P1 or P1-upATG constructs to allow direct comparison of the relative level of FPGS produced. C) Majority of P1-upATG-derived FPGS translocated to nuclei. Transfected AuxB1 cells were Dounce-homogenized and differential centrifugation was used to separate nuclear (N), cytosolic (C), and mitochondrial (M) fractions. WCL and postnuclear supernatant lysate were prepared with SDS buffer and Nonidet P-40 buffer, respectively. ␤-Tubulin, voltage-dependent anion channel (VDAC), and histone H3 lysine 4 trimethylation (H3K4Me3) were used as markers for cytosolic, mitochondria, and nuclear fractions, respectively. MOUSE WITH HUMANIZED FPGS PROMOTER USAGE

2005

Figure 7. Translational efficiency and stability of P1- and P2-derived proteins. After transfection with cDNAs for P1 or P2 transcripts, AuxB1 cells were labeled with 35S protein labeling mix for either 10 min (pulse) or 40 min (for pulse-chase); in turnover experiments, the label was chased with excess unlabeled methionine/cysteine for the specified times. Cell lysates were immunoprecipitated with anti-HA monoclonal antibody, resolved on SDS-PAGE and FPGS band intensities were acquired from a phosphoimager scan. The experiment was performed 3 times with similar results. A) Estimation of the relative protein synthesis rates from P2 and P1 transcripts. A short pulse period without a chase was used to estimate synthesis rates, shown as the relative band intensities below the gel. B, C) Pulse-chase experiments to determine the stability of FPGS isoforms made from P2 and P1 transcripts. Band intensities were normalized to initial values, and t1/2 values were calculated. D) The ubiquitinproteasome system is largely responsible for FPGS protein degradation. After 16 h of transfection, AuxB1 cells were treated with protein synthesis inhibitor CHX for 6 h. Inhibitors of lysosome (ammonium chloride or chloroquine) and inhibitors of proteasome (bortezomib or MG-132) were applied at the same time with CHX treatment. WCL from AuxB1 cells collected after 16 h of transfection before CHX treatment was used as starting point control (lane 1). ␤-Actin was used as a loading control.

determined by [35S]methionine/cysteine labeling. When short labeling was used, protein turnover did not contribute appreciably to the levels of labeled proteins, allowing an estimate of the relative rate of protein production or translation efficiency at equivalent levels of mRNA. With use of this approach, P2-derived mRNA had a 3-fold higher translation rate in transfected AUXB1 cells than did the P1-derived mRNA (Fig. 7A). A pulse-chase analysis following 35S labeling determined that the FPGS isoform made from the P2 transcript was about twice as stable as that made from the P1 transcript (Fig. 7B, C). Although the ubiquitin-proteasome system is responsible for the degradation of the majority of short-lived proteins, lysosomal degradation can also contribute to protein turnover in a specific manner through chaperone-mediated autophagy (32). Using inhibitors of these 2 degradation pathways, we found that the proteasome was the major pathway for the degradation of both P1and P2-derived FPGS (Fig. 7D). However, P2-derived protein was degraded at a much slower rate than P1 protein, similar to that seen for human FPGS (data not shown). 2006

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DISCUSSION Herein, we report that the rodent-specific dual-promoter control of the fpgs gene was nonessential under laboratory conditions and that mice engineered to have a single P2 promoter adopted an fpgs expression pattern typical of humans. This report and our previous studies illustrate that the tissue-specific expression of the mouse P1 FPGS isoform is under multiple layers of sophisticated regulation, drastically different from the control of expression of the human-like P2 isoform. These results support the value of the P1-KO mouse as a more accurate model system of human folate/antifolate metabolism. Quantitative analysis of transcription from P1 and P2 suggested that mouse fpgs P1 resembles promoters typical for tissue-specific regulation, whereas P2 resembles a housekeeping gene promoter with reasonably constant transcription across tissues, similar to that found for the expression pattern of human FPGS. Like humans, the P1-KO mice express FPGS transcripts at low levels in many tissues. This finding is consistent with our previous epigenetic analysis (20), which demonstrated a DNA methylation state of the sparse CpG

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dinucleotides surrounding mouse P1 promoter only in tissues that suppressed P1 transcription and active chromatin marks around the TSS for P1 only in P1expressing tissues. In contrast, P2 contained a classic CpG island that was not methylated in any tissue and encompassed open chromatin marks in all tissues examined (20). Hence, by deleting P1 in mice, we have removed a rodent-specific multicomponent epigenetic system associated with tissue specificity of FPGS levels. In addition, we found an increase in P2 transcriptional activity in liver of P1-KO mice on removal of P1. This finding is consistent with our previous hypothesis that the mouse fpgs P2 promoter was under transcriptional interference from upstream P1 utilization in liver (20). We know of no other case in which transcriptional interference and relief for such interference by deletion of the interfering promoter has been demonstrated on an endogenous gene. Removal of P1 revealed several differences between the P1 and P2 transcripts and protein products that were previously not recognized. The translation efficiency of the P1 transcript is ⬃3 times lower than that initiated from P2, an effect caused by 2 out-of-frame ATGs in the 5=-UTR made from P1. The P1-derived FPGS is significantly less stable than the P2-derived enzyme, due to a facilitated proteasome-dependent degradation. Finally, the P1-derived enzyme largely translocates to the nucleus, in contrast to the cytosolic and mitochondrial localization of mouse P2-derived FPGS. Therefore, through P1 promoter deletion, we created a mouse model devoid of the complexity of P1-derived FPGS expression control and subcellular localization. The survival and minimally altered folate metabolism of P1-KO mice can be explained as a combined result of the several post-transcriptional factors that resulted in an ⬃10-fold greater steady-state level of FPGS protein made from P2 transcripts, combined with the relief of transcriptional interference. We had previously discovered a difference in the sensitivity of the 2 FPGS isoforms made from P1 and P2 to feedback inhibition by long-chain folylpolyglutamates. The enzyme made from P1 transcripts was insensitive, whereas that made from P2 was sensitive to their enzymatic products (21). As a result, the P2 enzyme would make sufficient folylpolyglutamates and then be shut down by accumulating product, whereas the P1 enzyme made in mouse liver and kidney would allow the continued production of long-chain products despite the accumulation of higher levels of folylpolyglutamates. This would allow the accumulation of folates in liver and kidney under conditions when nutrition was adequate and give a buffer of hepatic/renal folates when the wild mouse was surviving on a less-thanadequate food supply. In the study of antifolate metabolism in mice as a surrogate for humans, the abundant storage and subsequent release of folates from mouse liver and kidney may mask the toxicity of antifolate drugs; this would not happen in the P1-KO mouse or in the single-promoter human system. Mouse and human tumors of all types studied to date uniformly express MOUSE WITH HUMANIZED FPGS PROMOTER USAGE

FPGS protein at high levels from the promoter equivalent to mouse P2. In humans, FPGS made in all normal tissues and tumors is identical in sequence; in the P1 deletion mouse that would also be the case. An important but mechanistically poorly understood observation has been that the antitumor potency of antifolates is substantially affected by the intracellular pool of folylpolyglutamates and that this relationship is vastly different for different antifolate drugs (33). Pemetrexed, which is currently first-line therapy against most non-small-cell lung carcinomas and also is used for mesothelioma (3, 34), is severely affected by folate status, yet current dosage regimens for pemetrexed include a daily dose of folic acid, shown to ameliorate the human toxicity of this drug. These relationships between dietary folate sufficiency and the efficacy of established and newer generation antifolates need to be further investigated. The P1-KO mouse appears to provide an improved model for such preclinical studies. The authors acknowledge the contributions of the Knockout and Transgenic Mouse Core and the Biological Macromolecule Core Laboratories of the Massey Cancer Center that were supported in part by U.S. National Institutes of Health (NIH) Cancer Center Support grant P30 CA16059. The authors also thank Prof. Michael Miles for helpful conversations. This research was supported in part by NIH grant R01-CA39687.

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Received for publication December 17, 2013. Accepted for publication January 23, 2014.

YANG ET AL.