Nuclear localization of FRα

To test the hypothesis that FRα translocates to the nucleus, a time course (0 min, 15 min and 30 min) for FRα nuclear localization was performed in DAOY cells treated with FA. The results of FRα immunoblots using mouse monoclonal antibody on nuclear extracts (Fig. 1a, b) showed that FRα translocates to the nucleus within 15 min of FA incubation. It is to be noted that a very faint band of immunoreactivity for FRα (38 kd band) was present in the nucleus even in the absence of FA.

Figure 1 Nuclear localization of FRα. (a) Nuclear extracts from DAOY cells treated with FA (200 μg/ml) for zero, 15 and 30 min at 37°C were subjected to immunoblotting using monoclonal anti-FRα (recognizing a 38 kd band) and polyclonal anti-pRB (recognizing 110 kd band) (see Supplementary Fig S1 ). (b) Ratio of FRα/pRB average band intensity (densitometry data is an average + SEM of three experiments). (c) Subcellular factions from DAOY cells not-treated or treated with FA (200 μg/ml) for 30 min were immunoblotted with NCAM, N-cadherin, ICAM-1 vimentin, hsp90, pRB, H3 and FRα (rabbit polyclonal) antibodies. The FRα polyclonal antibody is made against an epitope corresponding to amino acids 1-257 representing full length FR α of human origin. This antibody is reported to recognize multiple types of FR, α, β and perhaps γ. 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 +/− standard error mean. The ratio of average band intensity of FRα (42 kd and 38 kd) to subcellular fraction markers: FRα/ICAM-1; FRα/hsp90; FRα/vimentin; FRα/pRB; FRα/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 μg/ml) for 30 min at 37°C and immunostained using FRα 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 FRα (red) and pRB (green) in the nucleus (also stained blue with DAPI). The data is a representative of five separate experiments. Full size image

To study FRα distribution in the absence and presence of FA (30 min), we isolated different subcellular fractions of DAOY cells- membrane, cytosol, cytoskeletal, nuclear and chromatin enriched fractions and performed western immunoblots (Fig. 1c) using FRα 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 FRα (42 kd and 38 kd doublet) with the marker of individual subcellular fraction (FRα/ICAM-1, FRα/hsp90, FRα/vimentin, FRα/pRB and FRα/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, FRα was predominantly present in the cytosolic fraction whereas in the presence of FA, the FRα (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 FRα (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 + cytosol + insoluble cytoskeletal pellet) and nuclear fraction (nuclear + chromatin bound) we observe that even in the absence of FA, FRα is present in the nucleus and in the presence of FA, there is a significant increase in the translocation of FRα to the nuclear fraction.

Presence of FRα 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 FRα and pRB was observed in the presence of FA. These results suggest the following: (i) In the absence of FA, there is a more FRα in the cytosolic fraction; (ii) Upon FA treatment, FRα is distributed significantly to the non-nuclear membrane fraction as well to the nuclear enriched and chromatin bound fractions; (iii) Of the two immunostained FRα- 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 FRα appear to translocate to the nucleus, only the 38 kd band translocates significantly to the chromatin bound fraction in the FA treated cells.

FRα binds to cis-regulatory elements of gene promoters

The above studies suggested that FRα translocates to the nucleus and in the presence of FA, it is enriched in the chromatin bound fraction. To determine whether FRα activates FGFR4, FGFR4 promoter-luciferase constructs P-535/+99 from human FGFR4 promoter17 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). FRα 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 FRα activates FGFR4 promoter by binding to cis-regulatory elements.

Figure 2 Activation of Pax3 downstream target genes by FRα. FRα-pcDNA3 or control pcDNA3 (0.2 ng/well) constructs were co-transfected with Hes1 promoter-luciferase construct (a), human FGFR4 promoter-luciferase construct P-535/+9919 (b) or a control PGL3 (promoter-less luciferase gene) into DAOY cells.FA (200 μg/ml) was added 24 h post transfection. Luciferase activity was assayed 48 h post-transfection. pRLnull (5 ng/well) was used as a transfection control in all wells. For both Hes1 promoter luciferase and FGFR4 promoter-luciferase construct P-535/+99 FRα significantly increased promoter activity, with the highest increases observed in the presence of FA. Experiments were performed in quadruplicate with each data point in duplicate. * p<0.05; ** p<0.001; *** p<0.0001 (Student T-test). Full size image

To establish if FRα activates other FA modulated genes by binding to cis-regulatory regions, mouse Hes1 promoter-luciferase construct18 was co-transfected with FRα expression vector into DAOY cells treated or not treated with FA. Hes1 is a Pax3 downstream target gene18, FA increases Hes1 mRNA and protein levels14. Hes1-promoter-luciferase reporter activity increased (p<0.05) in response to FA. FRα significantly increased (p<0.001) Hes1-promoter-luciferase without FA (Fig. 2b), with a further increase with FA treatment. These data indicate that FRα transcriptional activation is not limited to FGFR4.

To confirm FRα 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. FRα bound to cis-regulatory regions of Hes1 and Fgfr4 promoters in vivo (Fig. 3a).

Figure 3 FRα binds to murine Hes1 and Fgfr4 promoter cis-regulatory elements. (a) ChIP assays was performed using E10.0 (30 somite) lumbar neural tube. Anti-FRα polyclonal antibody was used to immunoprecipitate (IP) the protein–DNA complex. This antibody is made against epitope corresponding to amino acids 1-257 representing full length FR α of human origin. This antibody is reported to recognize multiple types of FR, α, β and perhaps γ. Primers used to amplify cis-regulatory elements in Hes1 and Fgfr4 promoters are shown in Supplementary Information Table 1. Rabbit IgG was used as an IP negative control. ChIP experiments were performed in triplicate using one lumbar neural tube region per assay with a total of n = 4. (b) EMSA of binding reactions performed using GST-FRα fusion protein and 32P-labeled double-stranded oligonucleotides. Mouse Hes1 oligo #1 (with AANTT): 5′-AAAAAATTATTTTTTTTTTGCGTGAAG-3′; Mouse Hes1 oligo #2 (mutant AAA>CCC): 5′-AAACCCTATTTCCCCTTTGCGTGAAG-3′: (c)Mouse Fgfr4 oligo #3 (with AANTT): 5′-CAAACAAACAAAAAGAAACAACAAAAAAACTTTTTA-3′; Mouse Fgfr4 oligo #4 (with AANTT): 5′-ATAAAAGCACAACTTTTTACAAAGTTTAAAGTTTTTT-3; Mouse Fgfr4 oligo #5 (deletion mutant without AANTT) 5′-CGTTCGCGTGCAGTCCGAGATAT-3′. The arrow shows GST-FRα binding to oligonucleotides which have the AANTT sequence. Full size image

FRα binds to AANTT consensus sequence on Hes1 or FGFR4 promoter

To identify putative FRα binding sequences in Hes1 and Fgfr4 promoters, 32P-labeled oligonucleotides were made from appropriate promoter regions and EMSA was performed using affinity-purified GST-FRα fusion protein (Fig. 3b, c). GST-FRα fusion protein bound the Hes1 oligonucleotide 5′-AA AAT TATT TTTT TTTTGCGTGAAG-3′ which had AANTT or NAAAAN and/or NTTTTN sequences. When this sequence was mutated as in 5′-AA CCC TTATT CCCC TTTGCGTGAAG-3′ there was no shift. Similarly, on the Fgfr4 promoter the GST-FRα binding site mapped to AANTT or NAAAAN and/or NTTTTN in the oligonucleotide 5′-CAAACAAA CAAAAAG AAACAA CAAAAAAACTTTTTA -3′ and NTTTTTN in the oligonucleotide 5′-ATAAAAGCACAA CTTTTTA CAAAGTTTA AAGTTTTTT -3′. When the oligonucleotide sequence did not have the AANTT or TTNAA and NAAAAN consensus GST-FRα did not show a shift as in the case of 5′-CGTTCGCGTGCAGTCCGAGATAT-3′.

To further confirm the identity of FRα binding sites on Hes1 and FGFR4 promoters, AANTT sites were mutated on Hes1 and FGFR4 promoter-luciferase reporter constructs P-535/+99. 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 FRα binds Hes1 and FGFR4 promoters at AANTT or TTNAA and NTTTTN or NAAAAN sites.