Nuclear localization of folate receptor alpha: a new role as a transcription factor

Folic acid (FA) has traditionally been associated with prevention of neural tube defects; more recent work suggests that it may also be involved in in the prevention of adult onset diseases. As the role of FA in human health and disease expands, it also becomes more critical to understand the mechanisms behind FA action. In this work we examined the hypothesis that folate receptor alpha (FRα) acts as a transcription factor. FRα is a GPI-anchored protein and a component of the caveolae fraction. The work described here shows that FRα translocates to the nucleus, where it binds to cis-regulatory elements at promoter regions of Fgfr4 and Hes1, and regulates their expression. The FRα recognition domain mapped to AT rich regions on the promoters. Until this time FRα has only been considered as a folate transporter, these studies describe a novel role for FRα as a transcription factor.

translocates to the nucleus within 15 min of FA incubation. It is to be noted that a very faint band of immunoreactivity for FRa (38 kd band) was present in the nucleus even in the absence of FA.
To study FRa distribution in the absence and presence of FA (30 min), we isolated different subcellular fractions of DAOY cellsmembrane, cytosol, cytoskeletal, nuclear and chromatin enriched fractions and performed western immunoblots (Fig. 1c) using FRa antibody along with antibodies against subcellular markers NCAM, N-cadherin and I-CAM1 (for membrane enriched fraction), hsp90 (for cytosolic enriched fraction), vimentin (for cytoskeletal enriched fraction), pRB (for nuclear enriched fraction) and histone H3 (for chromatin bound fraction). The ratio of the average band intensities of the two immunoreactive bands of FRa (42 kd and 38 kd doublet) with the marker of individual subcellular fraction (FRa/ICAM-1, FRa/hsp90, FRa/vimentin, FRa/pRB, and FRa/H3 bands) were determined using densitometry (Fig. 1d). It is to be noted that all the membrane markers used here also showed strong immunoreactivity in the nuclear enriched fraction. The hsp90 immunoreactivity was highest in the cytosolic enriched fractions (C) and the vimentin antibody cross-reacted with the insoluble cytoskeletal pellet (P). The pRB immunoreactivity was highest in the nuclear enriched fractions (N) and histone H3 antibody immunoreacted with the chromatin bound fraction (CB). In the absence of FA, FRa was predominantly present in the cytosolic fraction whereas in the presence of FA, the FRa (42 kd band) appeared to translocate to the non-nuclear fraction (membrane, and cytoskeletal pellet fraction) and the 38 kd band to the nucleus. In the nucleus the FRa (38 kd band) appeared to be present in the chromatin bound fraction in the presence of FA. When the data in Fig. 1e is presented as total non-nuclear fraction (membrane 1 cytosol 1 insoluble cytoskeletal pellet) and nuclear fraction (nuclear 1 chromatin bound) we observe that even in the absence of FA, FRa is present in the nucleus and in the presence of FA, there is a significant increase in the translocation of FRa to the nuclear fraction.
Presence of FRa in the nucleus was further confirmed by confocal microscopy in DAOY cells (Fig. 1f). pRB was chosen as a nuclear marker. Increased co-localization of FRa and pRB was observed in the presence of FA. These results suggest the following: (i) In the absence of FA, there is a more FRa in the cytosolic fraction; (ii) Upon FA treatment, FRa is distributed significantly to the non-nuclear membrane fraction as well to the nuclear enriched and chromatin bound fractions; (iii) Of the two immunostained FRa-42 kd and 38 kd bands, the 42 kd band seems to translocate to the membrane enriched fraction in the presence of FA. Although both 42 kd and 38 kd bands of FRa appear to translocate to the nucleus, only the 38 kd band translocates to immunoblotting using monoclonal anti-FRa (recognizing a 38 kd band) and polyclonal anti-pRB (recognizing 110 kd band) (see Supplementary Fig  S1). (b) Ratio of FRa/pRB average band intensity (densitometry data is an average 1 SEM of three experiments). (c) Subcellular factions from DAOY cells not-treated or treated with FA (200 mg/ml) for 30 min were immunoblotted with NCAM, N-cadherin, ICAM-1 vimentin, hsp90, pRB, H3 and FRa (rabbit polyclonal) antibodies. The FRa polyclonal antibody is made against an epitope corresponding to amino acids 1-257 representing full length FR a of human origin. This antibody is reported to recognize multiple types of FR, a, b and perhaps c. Rabbit IgG was used as a negative control. M, membrane enriched; C, cytosolic enriched; P, insoluble cytoskeletal enriched; N, nuclear enriched; CB, chromatin bound fraction. The data above is a representative example of 5 different western blots. (d) The data is the average of 5 different western blot experiments 1/2 standard error mean. The ratio of average band intensity of FRa (42 kd and 38 kd) to subcellular fraction markers: FRa/ICAM-1; FRa/hsp90; FRa/vimentin; FRa/pRB; FRa/H3 was determined using densitometry. Statistical significance was calculated using Student's t test. (e) The data in ''d'' is presented as total non-nuclear fraction comprising of membrane, cytosol and insoluble cytoskeletal pellet, and total nuclear fraction comprised of nuclear and chromatin bound fractions. Statistical significance was calculated using Student's t test. (f) DAOY cells were grown in 8 well chamber slides in DMEM with 10% FBS for 24 h, then switched to serum free media in the absence or presence of FA (200 mg/ml) for 30 min at 37uC and immunostained using FRa monoclonal antibody and polyclonal pRB antibody and subjected to confocal microscopy. Secondary antibodies were donkey anti-rabbit Cy3 (red) and donkey anti-mouse Alexa488 (green). Yellow signals indicate co-localization of FRa (red) and pRB (green) in the nucleus (also stained blue with DAPI). The data is a representative of five separate experiments. significantly to the chromatin bound fraction in the FA treated cells.
FRa binds to cis-regulatory elements of gene promoters. The above studies suggested that FRa translocates to the nucleus and in the presence of FA, it is enriched in the chromatin bound fraction. To determine whether FRa activates FGFR4, FGFR4 promoter-luciferase constructs P-535/199 from human FGFR4 promoter 17 were transiently transfected into DAOY cells, treated or not treated with FA. FGFR4 promoter-luciferase reporter activity increased (p,0.05) in the presence of FA (Fig. 2a). FRa significantly increased FGFR4 promoter-luciferase in the absence of FA (p,0.001), with a further increase in the presence of FA (p,0.0001). This demonstrates that FRa activates FGFR4 promoter by binding to cis-regulatory elements.
To establish if FRa activates other FA modulated genes by binding to cis-regulatory regions, mouse Hes1 promoter-luciferase construct 18 was co-transfected with FRa expression vector into DAOY cells treated or not treated with FA. Hes1 is a Pax3 downstream target gene 18 , FA increases Hes1 mRNA and protein levels 14 . Hes1-promoter-luciferase reporter activity increased (p,0.05) in response to FA. FRa significantly increased (p,0.001) Hes1-promoter-luciferase without FA (Fig. 2b), with a further increase with FA treatment. These data indicate that FRa transcriptional activation is not limited to FGFR4.
To confirm FRa binding to cis-regulatory elements of Hes1 and Fgfr4 promoters in intact embryos, chromatin immunoprecipitation (ChIP) experiments were performed using the lower lumbar region of the neural tube from wild-type (WT) mouse embryos (E10.0, 30 somite stage), an area where both of these genes are expressed. FRa bound to cis-regulatory regions of Hes1 and Fgfr4 promoters in vivo (Fig. 3a).
FRa binds to AANTT consensus sequence on Hes1 or FGFR4 promoter. To identify putative FRa binding sequences in Hes1 and Fgfr4 promoters, 32 P-labeled oligonucleotides were made from appropriate promoter regions and EMSA was performed using affinity-purified GST-FRa fusion protein (Fig. 3b, c). GST-FRa fusion protein bound the Hes1 oligonucleotide 59-AAAATTAT-TTTTTTTTTGCGTGAAG-39 which had AANTT or NAAAAN and/or NTTTTN sequences. When this sequence was mutated as in 59-AACCCTTATTCCCCTTTGCGTGAAG-39 there was no shift. Similarly, on the Fgfr4 promoter the GST-FRa binding site mapped to AANTT or NAAAAN and/or NTTTTN in the oligonucleotide 59-CAAACAAACAAAAAGAAACAACAAAAA-AACTTTTTA-39 and NTTTTTN in the oligonucleotide 59-ATA-AAAGCACAACTTTTTACAAAGTTTAAAGTTTTTT-39. When the oligonucleotide sequence did not have the AANTT or TTNAA and NAAAAN consensus GST-FRa did not show a shift as in the case of 59-CGTTCGCGTGCAGTCCGAGATAT-39.
To further confirm the identity of FRa binding sites on Hes1 and FGFR4 promoters, AANTT sites were mutated on Hes1 and FGFR4 promoter-luciferase reporter constructs P-535/199. Mutated constructs were transfected into DAOY cells as above. Luciferase activity did not increase with these constructs for either Hes1 or FGFR4 promoters (Fig. 4a). The results confirm that FRa binds Hes1 and FGFR4 promoters at AANTT or TTNAA and NTTTTN or NAAAAN sites.

Discussion
Previous work from our lab demonstrated that in the absence of functional Pax3, FA increased KDM6B, through up-regulation of KDM6B targeting micro-RNAs. This in turn altered H3K27 methylation marks on the promoters of Pax3 downstream targets, Hes1 and Neurog2, and affected gene transcription 14 . Thus one mechanism of FA action is through remodeling of chromatin structures. In this study we have elucidated a second mechanism for FA action, through activation of FRa and its' subsequent action as a transcription factor. A hypothetical model showing FRa internalization is presented in Fig. 4b. FRa is internalized in a caveolar structure as early endosome 19 . The endosome becomes increasingly acidic 20 and fuses with a lysosome 21 . In the lysosome FA is released 22 and lysosomal GPIspecific phospholipase D 23 cleaves off the GPI anchor on FRa, which is then set free. Free FRa translocates into the nucleus where it binds to cis-regulatory elements of target genes and directly activates transcription. This model does not take into account FRa recycling and it is still unclear exactly how FRa translocates into the nucleus.
Binding to Hes1 and Fgfr4 promoters suggests FRa involvement in stem cell maintenance and skeletal muscle differentiation, respectively. The list of putative FRa targets shown in Table 1, suggests that FRa may be involved in regulating a plethora of developmental genes involved in myogenesis, skeletonogenesis, cell mobility, neural crest cell migration, cranial and cardiac neural crest formation, hair morphogenesis, oligodendrogenesis, spermatogenesis 24 , melanogenesis 25 , and epithelial to mesenchymal transformation 26      or NTTTTN and/or NAAAAN map close to Pax3, a transcription factor and multifunctional regulatory protein, expressed early in embryogenesis, binding sites, which map to ATTA, GTTCC, TAAT, CCTTG, CAAGG, GTTAT, TATTG, GTGTGA, and CAGTGT 27 . These observations suggest that FRa and Pax3 might appear as a complex regulating target genes synergistically. However, up-regulation of Hes1 or Neurog2 in FA-rescued Sp 2/2 embryos 14 suggests that FRa may also have a role independent of Pax3. Future work shall test this hypothesis.
The observation that FRa acts like a transcription factor is relevant to our understanding of the mechanisms of FA action during development and has significant implications for disorders associated with FA deficiency and FRa misregulation and for management of human cancers which express FRa as a tumor antigen. FA has been associated with the prevention of adult onset diseases (reviewed in ref. 2). In some of these cases FA deficiency may not be the problem. The issue may be an inability to respond to FA due to misregulation of FRa. Cazzaniga and colleagues 28 compared levels of serum folate and assessed differences in folate binding ability with primary fibroblast cultures, from Alzheimer's disease (AD) patients and agematched healthy subject. Circulating folate was significantly lower in AD patients, whereas folate binding to fibroblasts was significantly higher, possibly due to enhanced expression of FRa in AD fibroblasts. Cerebral folate transport deficiency is an inherited brain-specific folate transport defect caused by mutations in the folate receptor 1 (FOLR1) gene which codes for FRa 29 . This disorder generally has a late infantile onset and symptoms include progressive movement disturbance, psychomotor decline, epilepsy and disturbed brain myelination, as well as a depletion of white matter choline and inositol 30 . Grapp and colleagues 29 reported that whereas WT FRa was localized in the plasma membrane, in cerebral folate deficiency FRa mutants were mistargeted to intracellular compartments. The data presented in this paper provides relevant insight to these clinical situations. If FA interaction with FRa is misregulated, key transcriptional events may be affected. This in turn can lead to a series of developmental consequences or to adult onset disease associated with FA levels. Further work needs to be done to examine direct transcriptional activation of FRa responsive genes by FRa and its' role in these multifactorial diseases.
Another clinical role for FRa is in cancer, where it is recognized as a tumor antigen/biomarker 31 . Because of this, diagnostic and therapeutic methods which exploit FRa are being developed for cancer treatment, including the use of folate-drug conjugates 31 . The knowledge that FRa acts as a transcription factor can be exploited to target FA-siRNA or FA-drug conjugates to silence downstream targets in appropriate cancers. For instance, two Pax3 downstream targets c-MET and MITF are associated with melanoma 32 . MET promotes the melanoma phenotype by stimulating migration, invasion, resistance to apoptosis, and tumor cell growth. PAX3 mediates MET induction through direct activation of the gene, and indirect regulation through MITF. FA-drug conjugates exploiting the proximity of Pax3 and FRa binding sites could potentially silence c-met and/or MITF expression.
In summary, our study shows that FRa is localized in the nucleus, where it binds to cis-regulatory elements (AANTT or TTNAA and NTTTTN or NAAAAN) on FA modulated genes and activates their transcription. This novel role of FRa as a transcription factor provides insight into developmental mechanisms associated with FA responsiveness. It also provides an exciting new avenue to explore for treatment of diseases associated with FA deficiency, FRa misregulation and cancers which express FRa as a biomarker.
Statistical analysis. Values given are means: SEM. Probabilities (p) were calculated with Student's unpaired t test using GraphPad Prism version 4.0. p values , 0.05 were considered statistically significant. One-way ANOVA with Bonferroni's multiple comparison tests were used for multiple comparisons between data.
Nuclear localization of FRa. DAOY cells were treated with FA (200 mg/ml) for zero, 15 and 30 minutes at 37uC. For the western blots studies, the DAOY cells not-treated or treated with FA for 30 min were used. Subcellular fractions, membrane enriched, cytosolic, insoluble pellet cytoskeletal fraction, nuclear and chromatin bound fractions (30 mg) were immunoblotted with antibodies against FRa (rabbit polyclonal, 15500), NCAM (15500); N-Cadherin (151000); I-CAM-1 (151000); vimentin (1510,000); pRB (15500) and Histone H3 (15500). This rabbit polyclonal anti-FRa antibody is made against epitope corresponding to amino acids 1-257 representing full length FR a of human origin. This antibody is reported to recognize multiple types of FR, a, b and perhaps c. The average band intensity of FRa/ICAM-1; FRa/hsp90; FRa/vimentin; FRa/pRB; FRa/H3 was determined using densitometry. For immunostaining, DAOY cells were plated and grown in DMEM with 10% FBS for 24 hours and then changed to serum-free media. FA (200 mg/ml) was added to appropriate wells. The cells were allowed to grow for an additional 30 min. Cells were immunostained with anti-FRa (mouse monoclonal antibody; 15100) and pRB (rabbit polyclonal; 15100). This mouse monoclonal FRa antibody recognizes only the 38 kd band of FRa. Rabbit IgG was used as a negative control. Secondary antibodies were donkey anti-rabbit Cy3 (red) (15200) and donkey anti-mouse Alexa488 (green) (15200). Confocal microscopy was done with a Zeiss 510 META Confocal Laser Scanning Microscope.
Real time quantitative RT-PCR. Real time quantitative RT-PCR was done as described earlier 27 . Primers and probes used in this study were designed using Primer Express software (PerkinElmer Life Sciences). Primers were synthesized by Operon Inc. probes were synthesized by MegaBases Inc (Refer Supplementary Information- Table 1 for primers).
Chromatin immunoprecipitation (ChIP) assays. ChIP assays using lumbar neural tube from WT embryos (E10.0) were performed as described earlier 34 . PCR was performed with primers for murine Hes1 and Fgfr4 promoter regions (Supplementary Information- Table S1 for primers). All ChIP samples were tested for false-positive PCR amplification by sequencing the 200-bp amplified product to ascertain the specificity of FRa binding to cis-regulatory elements.
Analysis of Hes1 and Fgfr4 promoter activity. A 2.5 kb Hes1 promoter-luciferase construct was provided by Dr. R. Kageyama, Institute for Virus Research, Kyoto University Kyoto, Japan. Human FGFR4 promoter constructs were provided by Dr Shereen Ezzat, Departments of Medicine, Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada. The FRa expression construct in pcDNA3 was kindly provided by Dr Asok Antony, Indiana University Medical School Indianapolis, IN, USA. Renilla luciferase plasmid, pRL-null (0.5 mg) (dual luciferase system, Promega), was simultaneously transfected as an insertional control for transfection efficiency in all of the studies. DAOY cells were seeded at 5 3 104 cells/ 60 mm diameter dish in DMEM supplemented with 10% fetal calf serum for 24 h prior to transfection. For co-transfections: human FRa cDNA and mouse Hes1-Luc promoter or human FRa cDNA and human FGFR4-Luc promoter were transfected into the cells using MegaTran 1.0 from OriGene; Cat#: TT200003. After 24 h, FA was added to the treatment wells. The cells were washed 3 times with phosphate-buffered saline (PBS) and lysed with Passive Lysis Buffer (PLB) 48 h post transfection. Luciferase activity was measured using a Vector 2 Luminometer (PerkinElmer Life Sciences). The AA.CC substitution mutations of the putative FRa binding sites on murine Hes1 or human FGFR4 promoters were made with the QuickChangeXL sitedirected mutagenesis kit (Stratagene). Hes1 promoter-luciferase containing plasmid or plasmids containing the mutated sequences (20 ng) were transiently cotransfected with FRa-pcDNA3 or pcDNA3 vector control into DAOY cells and luciferase assays were done as described earlier 34 using the Dual Luciferase kit from Promega. Renilla luciferase plasmid, pRL-null (5 ng/well) (Dual Luciferase System Promega), was simultaneously transfected as an insertional control for transfection efficiency.
Purification of GST-FRa fusion protein. GST-FRa fusion plasmid was kindly provided by Dr. Asok Antony. Escherichia coli was transformed with GST-FRa fusion www.nature.com/scientificreports plasmid, and the cells were grown overnight in LB medium. GST-FRa fusion protein production was induced with 0.1 mM isopropyl-D-thiogalactopyranoside (Sigma) for 2 hr. Cells were pelleted and sonicated to release the protein in 50 mM Tris-HCl pH7.8 buffer. Supernatant containing GST-FRa fusion protein among other proteins was loaded onto glutathione-Sepharose 4B column (Amersham Pharmacia Biotech). Unbound proteins were washed with 50 mM Tris-HCl pH7.8 buffer and eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 9.5. Eluted protein was concentrated using Centricon 10 (Amicon) as per the manufacturer's instruction.
Electro-mobility shift assays (EMSA). Probes were prepared for EMSA by annealing complementary oligonucleotides representing selected regions of murine Hes1 and Fgfr4 promoters, followed by 59-end labeling with [c-32 P] ATP by T4 polynucleotide kinase. EMSA was done as described earlier 18 . Double-stranded oligonucleotide probes produced, covered the following regions: WT Hes1 promoter oligo: 59-AAAAAATTATTTTTTTTTTGCGTGAAG-39 and mutant Hes1 promoter oligo: 59-AAACCCTTATTCCCCTTTTGCGTGAAG-39; WT Fgfr4 promoter oligo #1: 59-CAAACAAACAAAAAGAAACAACAAAAAAACTTTTTA-39 WT promoter oligo #2: 59-ATAAAAGCACAACTTTTTACAAAGTTTAAAGTTTTTT-39 and an oligo without AANTT sequence #3: 59-CGTTCGCGTGCAGTCCGAGATAT-39. For shift assays 4 mg GST-FRa fusion protein was pre-incubated at room temperature for 30 min with 4 M urea and EMSA reaction buffer (100 mM Tris-HCl PH7.5, 500 mM KCl, 6.5% glycerol, 50 mM pyrophosphate, 25 mM DTT in 2.5% Tween 20, 1 mg/ml salmon sperm DNA) prior to addition of labeled oligonucleotides. For hot reactions GST-FRa fusion protein and radio-labeled oligonucleotides were added and preincubated for 30 min at room temperature. For cold reactions GST-FRa fusion protein and unlabeled oligonucleotides were added, and labeled oligonucleotides were added after 30 min. After pre-incubation, free DNA and DNA protein complexes were resolved in 6% polyacrylamide gels (these gels were pre-run at 1000 V and 5 mA for 16 h in the continuous cooling system) using 0.253 TBE as the running buffer. Electrophoresis was performed at 1000 V and 25 mA for 2 h in a continuous cooling system. To visualize shifted bands, gels were dried at RT and transferred to Phosphor Imager Screens (Amersham Biosciences). Gels were exposed overnight at 4uC.