The FU gene and its possible protein isoforms
© Østerlund et al; licensee BioMed Central Ltd. 2004
Received: 22 March 2004
Accepted: 22 July 2004
Published: 22 July 2004
FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci). Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling.
The FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1) maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI.
The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing. Thus, mRNAs may contain different 5'UTRs and encode different protein isoforms. Furthermore, FU is able to enhance the activity of GLI2 but not of GLI1, implicating FU in some aspects of Hedgehog signaling.
The signaling molecule Hedgehog (Hh) and components of its intracellular signaling pathway have been the subject of intensive research in several species from fruit fly to man during recent years. Numerous developmental and morphogenic processes are controlled by the Hedgehog family of proteins. Much effort has been directed at identifying components of the signaling pathway and their respective roles and interactions [for an extensive review see ]. In Drosophila, Hh signaling to the transcription factor Cubitus interruptus (Ci) is mediated by a protein complex consisting of Ci and three other cytosolic proteins. These are the costal 2 (cos2), suppressor of fused (su(fu)) and fused (fu), where fu is a kinase domain containing protein with positive regulatory activities in Hh induction of Ci mediated transcriptional activation. Hh binds to its receptor patched (ptc), a 12 membrane spanning protein, leading to the activation of another membrane protein smoothened (smo) [2, 3]. Smo is a 7 transmembrane protein that, by an unknown mechanism, signals to the Ci containing protein complex leading to activation of Ci. Vertebrate homologues of these Drosophila genes and proteins have been identified during the last decade. To a large extent the signaling pathway has been conserved in vertebrates. However, the picture is more complicated since some of the Drosophila genes have two or more vertebrate homologues. There are three Ci homologues in vertebrates, GLI1, GLI2 and GLI3. GLI1 has activation properties whereas GLI2 and GLI3 have both activation and repression activities [reviewed in ]. It is expected that the human homologue of fu (FU) is a positive regulator of GLI proteins, whereas the su(fu) homologue SUFU is a negative regulator. It has been shown by several groups that SUFU inhibits both GLI1 and GLI2 transcriptional activity and has major effects on the shuttling between cytosol and nucleus [5–7]. In a similar way it was shown in C3H/10T½ cells that FU is a positive regulator of GLI2 but with little effect on GLI1 . FU is a 1315 residue protein with high similarity to fu in the N-terminal kinase domain.
Interestingly, it was discovered that mutations in PTCH1, the human counterpart of ptc, underlie the Nevoid Basal Cell Carcinoma Syndrome (NBCCS) [9, 10]. Patients with NBCCS (also known as Gorlin syndrome) have developmental abnormalities and eventually develop basal cell carcinoma (BCC) and other tumors like medulloblastoma and rhabdomyosarcoma [11, 12]. Also SMO and SUFU mutations as well as overexpression of GLI1 or GLI2 can lead to BCC or medulloblastoma [13–16]. Thus, investigations of this signaling pathway, its genes and protein components, is not only important for understanding development and morphogenesis, but also for cancer biology.
Here three FU cDNA clones have been identified and used for sequence analysis, identification and structural description of the FU gene, as well as for construction and subcloning of FU expression vectors. Using the available public databases the FU gene was found to be present in a sequenced BAC clone from chromosome 2. FU is located in the same region of chromosome 2q34-q36 to which the human limb malformation disorder Syndactyly type 1 (SD1) has recently been mapped in a large German pedigree  and confirmed in an Iranian family . Its possible association with this condition was investigated by sequencing the coding exons of the FU gene in an affected member from the German family . The tissue expression pattern of FU has been determined using an RNA array and Northern blots. FU is expressed in all 72 tested tissues. It is clear that not only a single transcript is expressed. Instead transcripts of different sizes are seen and some tissues apparently express more than one major transcript. From the genomic structure and the cDNA clones it was possible to predict several alternative splicing events and consequently the likely expression of different protein isoforms. Two of the isoforms were expressed in HEK293 cells and tested for their ability to regulate the activity of GLI1 and GLI2, showing positive effects on GLI2 but not on GLI1.
Chromosomal localization of FU
The sequence information derived from the FU cDNA clones 1HFU, 2HFU and Ngo3689 (see Methods) allowed the identification of the FU gene in a 200 kb BAC clone (AC009974) from chromosome 2. The gene is localized to 2q35 at 217.56 Mb using the Ensembl  annotation. The Ensembl gene prediction programs have identified most, but not all (21 of 29 exons; the published FU  predicts 26 exons) of the FU structure and named the gene STK36 (serine/threonine protein kinase 36). Chromosome 2q35 is the locus of several genetically based disorders. Both Syndactyly type 1 (SD1) and Brachydactyly type A1 (BDA1) have been mapped to this region [17, 18, 20]. Recently, the gene responsible for BDA1 has been identified as IHH (Indian Hedgehog) one of the vertebrate Hh homologues . IHH is located in the vicinity of FU on chromosome 2 (217.94 Mb) less than 400 kb away. In order to determine if alterations of FU are responsible for SD1, the FU coding region (exons 3–29) and the flanking intronic regions were sequenced using genomic DNA from an affected member of an SD1 family whose trait maps to the 2q34-q36 region  and an unrelated control individual. No FU mutations were detected in this study, although three single nucleotide polymorphisms were identified. These included a T to C transition in intron 10, 17 bp 5' of exon 11 (IVS10-17T>C), causing gain of a BstNI site, and a G to A transition in exon 16, 17 bp 5' of the end of the exon (1748G>A), causing substitution of glutamine for arginine at amino acid 583 (R583Q) and loss of an AciI site. The altered restriction sites created by these sequence changes were tested in 44 CEPH unrelated individuals. The results showed that both changes are normal sequence variations as previously reported in the NCBI SNP database. The third change was a G to A transition in exon 27 (3008G>A), causing substitution of aspartic acid for glycine at amino acid 1003 (G1003D). By sequencing exon 27 in 8 affected and 6 unaffected members of the SD1 family , the disease variant could be observed in affected and unaffected members of the family, and a homozygous healthy individual was found. This variant has also been reported previously as a single nucleotide polymorphism in the NCBI SNP database.
Multi tissue array and northern blot analyses
Nested PCR analyses
Functional analyses of FU isoforms
The FU gene
In the present paper, the FU gene was identified and its structure determined. FU consists of 29 exons of which exons 1 and 2 encode 5'UTRs, exon 3 contains the initiating ATG codon and exons 13 and 29 contain in frame stop codons (Fig. 1). Exon 1 and 2 may serve as alternative first exons, like the alternative exons 1, 1A and 1B found in the PTCH1 gene . Exons 3 to 9 encode the kinase domain. This segment has strong similarity to Drosophila fu, whereas the remaining C-terminal part has a much weaker similarity . Using the DIALIGN program  it is possible to align fu to L-FU in two regions in the C-terminal part (not shown). These are largely encoded by exons 15–16 and 22–29. This indicates that exons 10–14 and 17–21 may have been recruited to the FU gene during evolution.
Investigations of Syndactyly patient material
We investigated the possibility that FU underlies SD1 based upon the fact that FU lies within the localization interval for SD1 and that it is part of the Sonic Hedgehog signaling pathway, which participates in digital patterning . Although three previously reported single nucleotide polymorphisms were identified, we did not detect any mutation in the FU coding region or flanking intronic regions. While these results do not implicate FU in the causation of SD1, it is possible that this disorder is caused by mutations in the noncoding regions not screened in this study. Alternatively, SD1 could be caused by a genomic rearrangement not identified by sequence analysis, although no altered bands were detected in an affected member of the SD1 family by Southern analysis using a FU cDNA clone as probe (data not shown).
Analyses of FU expression have shown that transcripts are detected in all tissues examined. For the first time evidence is presented showing that more than one transcript can be expressed from this gene. The Northern blots clearly show that FU transcripts of different sizes indeed exist. Here the transcripts are estimated to be slightly bigger than previously reported and in some tissues more than one transcript is evident. It is clear from the available cDNAs and RT/PCR based transcript analyses that alternative splicing occurs. Additionally, it is also clear that different 5'UTRs are present in the transcripts. At least two protein isoforms, besides the previously described L-FU , may be produced. The S-FU isoform is the one that most dramatically differs from L-FU, consisting only of the N-terminal one third of L-FU. S-FU expression results from inclusion of exon 13 in the mature transcript. This alternative splicing event was detected in all tissues examined and at an apparently constant ratio. Also a case of regulated alternative splicing was detected by RT/PCR, but with a much less dramatic impact at the protein level, since it only results in the loss of 21 residues encoded by exon 24. However, the expression reveals a possible tissue specific regulation of this alternative splicing event. This may well reflect that L-FUΔ24 plays a biological role different from L-FU. Since it appears that the mRNA for L-FUΔ24 is not expressed in small intestine and prostate it can be speculated that FU has a different role there, if a leucine zipper is truly lost in L-FUΔ24. It is intriguing that testis appears to express transcripts both with and without the 63 bp segment and is also the tissue with strongest expression. Perhaps the expression of L-FUΔ24 and L-FU together is linked to the function of Desert Hedgehog which has been shown to have a particular role in spermatogenesis . Whether interactions with GLI proteins, SUFU or other components of the signaling pathway are altered, and if this has any impact on GLI or SUFU activities, remains to be investigated. Certainly this adds another variable to the complicated picture of Hedgehog signaling and GLI regulation in vertebrates.
Functional investigations and perspectives
The assessment of functionality revealed that S-FU was not able to regulate GLI1 or GLI2 when expressed in 293 cells. In contrast, both L-FU and a variant lacking a full kinase domain (2HFU) were able to enhance GLI2 induced transcription. These results are qualitatively similar to those previously reported in C3H/10T½ cells . L-FU and 2HFU were only able to enhance GLI2 activity 2 to 3 fold in 293 cells, whereas 5 to 8 fold inductions are seen in C3H/10T½ cells. This may reflect the fact that the latter cell line expresses additional components of the Hedgehog signaling pathway, which are required for full activity of FU. Unlike the previous investigations  it was not possible to see an effect of L-FU on SUFU. Again this difference may be explained by the various properties of the cell lines used. Understanding the signaling events downstream of SMO may reveal functional differences of the proteins involved, as compared to their fruit fly counterparts. Although SUFU inhibits GLI transcription factors and su(fu) inhibits Ci, there are still striking differences. As yet there have been no reports of a cos2 counterpart in vertebrates. Instead it has been observed that FU interacts with all GLI proteins and SUFU , even though fu does not bind to Ci . It has also been observed that both L-FU and SUFU can be found in the nucleus [5–8], which has not been observed for fu or su(fu). It is likely that both FU and SUFU are shuttled in and out of the nucleus by binding to GLI proteins [5, 8]. Though basic activities of both FU and SUFU in regulation of GLI have been conserved, it also appears that significant differences from their fruit fly counterparts exist. Clearly, FU is not having an effect on GLI1 similar to the one seen on GLI2. Additional investigations are needed in order to establish the role of FU in hedgehog signaling and GLI control. The role of the different isoforms also remains to be elucidated. These have to be tested individually for their regulation of all GLI proteins and proteolytic products. Fu is known to have at least two separate physiological functions in the fly, one of which is dependent upon the kinase domain . Likewise, FU may well have two or more distinct functions in signaling, represented by different domains, isoforms and protein interactions.
FU is localized on chromosome 2q35 very close to IHH. Though SD1 has been mapped to this region, we have not identified a causative role for FU in this disorder. FU consists of 29 exons of which 1 and 2 encode 5'UTRs and 3 to 9 encode a kinase domain. For the first time it is shown that transcripts of different sizes are expressed and alternative splicing takes place, probably leading to the generation of different protein isoforms. FU protein is likely to be involved in the Hedgehog signaling pathway since it can enhance the activity of GLI2. In contrast, it has no effect on GLI1 and an effect on SUFU cannot be observed in 293 cells.
The FU cDNA clones
Two almost full-length human FU clones were identified in the Incyte database. Both 1HFU and 2HFU were cloned in the vector pINCY. A third clone was available from Kazusa DNA Research Institute (Chiba, Japan) and termed Ngo3689 (Gene name KIAA1278). This clone was in the vector pBluescript II SK+. The human BAC clone AC009974 was obtained from Research Genetics (Huntsville, AL). The human GLI, human SUFU, 12GLI-RE-luciferase reporter and β-galactosidase vectors have been described previously .
FU cDNA subcloning
Expression constructs for different isoforms of FU was obtained by direct PCR or extension overlap PCR, using end-primers having specific restriction sites and the high fidelity VentR DNA polymerase (New England Biolabs, Beverly, MA). The cDNA for the long form of FU (L-FU) was subcloned into pCDNA3.1-HisB using the NotI and XbaI sites. 2HFU and the short FU (S-FU) cDNAs were subcloned into pCDNA3.1-HisC using the KpnI and XbaI sites.
DNA sequencing and analyses
All PCR generated products were analyzed by DNA sequencing. The Big-Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) was used according to instructions. Sequencing was performed at CyberGene AB (Huddinge, Sweden). Sequence alignments were done using the DIALIGN program  available at the BiBiServ from University of Bielefeld, Germany. Sequence information of proteins, clones and chromosomes were obtained from the Swiss-Pro , Entrez  and Ensembl  databases.
Analyses of genomic DNA from family members with SD1
After informed consent was obtained, blood was taken from affected and unaffected family members and DNA extracted from peripheral blood leukocytes according to standard methods. Intronic primers were designed to amplify exons 3–29 of FU either as single exons with flanking intronic sequences or as products containing two exons with flanking intronic sequence and the complete intervening intron. The primer sequences can be obtained upon request. PCR was performed in a standard fashion and products were sequenced using either the Thermosequenase CyTM5.5 Dye Terminator or DYEnamic ET Dye Terminator Cycle Sequencing kits (Amersham Biosciences, Piscataway, NJ). Electrophoresis and analysis were performed on either an Automated Laser Fluorescence (ALF) DNA sequencer or MegaBACE DNA sequencer (Amersham Biosciences) after purification with Autoseq columns (Amersham Biosciences). For exon 27, the PCR product was purified using the enzymatic ExoI-SAP purification method, sequenced using the Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech) and analysed on an ABI 3100 genetic analyzer (Applied Biosystems). PCR products containing exon 11 or exons 15/16 were digested with BstNI or AciI, respectively, and the bands resolved on 3–4% agarose gels to confirm sequence changes in the patient with SD1 and to determine their frequency in a panel of 44 CEPH individuals.
Northern blot analysis
Commercially available Human MTN 12-lane Blot 2, Human Fetal MTN Blot II and Human Endocrine System MTN Blot Northern blots (Clontech, Paolo Alto, CA) were obtained and used with PCR generated hybridization probes. DNA probes were made by direct PCR, amplifying the sequences corresponding to exon 13 and 28. The generated fragments were then labeled with 32P-ATP using the High Prime DNA labeling kit (Boehringer Mannheim, Mannheim, Germany) according to instructions. Hybridization of Northern blots was done with labeled DNA probes in ExpressHyp (Clontech) at 68°C according to instructions. The blots were then analyzed with a Fujix Bas 2000 phosphoimager (Fuji Photo Film, Tokyo, Japan).
Expression analysis by nested PCR
Primers for nested PCR analyses
Sequence from exon
Outer PCR primer pairs
Inner PCR primer pairs
Reporter gene assays
The cDNA clones were used in transfections of HEK293 cells in 24 well culture plates. Basically this was done as previously described . In short, the 293 cells were transfected using Superfect Transfection Reagent (Clontech), with 100 ng of the luciferase reporter and β-galactosidase as well as different amounts and combinations of GLI, FU and SUFU constructs. For every assay there was a corresponding control with an equal amount of empty vector. The cells were harvested 24 hours after transfection with 50 μl of lysis buffer from the Galacto-Light kit (Applied Biosystems). Of this was 10 μl used for β-galactosidase assay and the rest for luciferase assay using the Luciferase Assay kit (BioThema, Dalarö, Sweden). Analyses were done on a Microplate Luminometer (Berthold Detection System, Pforzheim, Germany).
We thank Kristin Bosse for helping recruiting the family and Chad T. Morgan for excellent technical assistance. This work was supported by the Swedish Cancer Society with a postdoctoral position to TØ and research grant to RT and PGZ, by a grant from Pharmacia to RT and by a grant from the South Carolina Department of Disabilities and Special Needs (SCDDSN) to CES. RCB was supported by a postdoctoral fellowship from the Fund for Scientific Research-Flanders (FWO).
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