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FKHL15,a New Human Member of the Forkhead Gene Family Located on Chromosome 9q22
GENOMICS
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FKHL15, a New Human Member of the Forkhead Gene Family Located on Chromosome 9q22 Brian Paul Chadwick,* Franz Obermayr,*,1 and Anna-Maria Frischauf*,2 *Molecular Analysis of Mammalian Mutation, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom Received November 18, 1996; accepted February 6, 1997
FKHL15 was isolated from a cDNA library enriched for transcripts from 9q22. Isolation and sequencing of a 3.5-kb cDNA clone identified a putative 376-aminoacid protein with greater than 80% homology over a 100-amino-acid stretch to the forkhead DNA-binding domain. The FKHL15 gene contains a region rich in alanine residues, frequently associated with transcriptional repression. The forkhead genes are believed to play important roles in development and differentiation in many different organisms and have also been implicated in the development of some tumors. The map position of FKHL15 on 9q22 places the gene within the candidate regions for the cancer predisposition syndrome multiple self-healing squamous epitheliomata and the degenerative neurological disorder hereditary sensory neuropathy type I. This is a region frequently lost in squamous cell cancer. q 1997 Academic Press
INTRODUCTION
The forkhead domain was first identified as a region of homology between the rat hepatocyte nuclear factor 3 (HNF3) and the Drosophila homeotic gene forkhead (Lai et al., 1991). The forkhead gene (fkh) obtained its name through the incapability to form the normal terminal structures of the anterior and posterior gut, instead forming two spiked structures (Weigel et al., 1989). The HNF3b gene is required for notochord development in mouse (Ang et al., 1994; Weinstein et al., 1994). The two proteins show 90% identity at the amino acid level over a 100-amino-acid stretch since called the ‘‘forkhead domain.’’ This forkhead domain is a variant of the DNA-binding helix–turn–helix (HTH) motif, binding DNA as a monomer with two loops, or wings, on the C-terminal side of the HTH, and is often referred to as the ‘‘winged-helix’’ motif (Brennen, 1993; Clark et al., 1993; Lai et al., 1993). Many other members of 1
Current address: Department of Developmental Biology, MPI for Immunology, Freiberg, Germany. 2 To whom correspondence should be addressed. Telephone: (0171) 269 2319. Fax: (0171) 269 3479.
0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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this eukaryotic transcription factor family have been identified through association with pattern formation during embryogenesis including the Drosophila segmental genes sloppy paired 1 and 2 (slp1 and slp2) (Grossniklaus et al., 1992; Hacker et al., 1992), the Caenorhabditis elegans vulval development gene lin31 (Miller et al., 1993), the Axial gene in zebrafish (ax1) (Strahle et al., 1993), the axis formation gene XFKH1 in Xenopus (Dirksen and Jamrich, 1992), and the head patterning gene crocodile in Drosophila (Hacker et al., 1995). In mouse, disruptions in the forkhead-containing gene whn give rise to the nude mutation (Nehls et al., 1994; Segre et al., 1995). Other members of the group have been identified by degenerate PCR and lowstringency hybridization of the forkhead domain (Bork et al., 1992; Clevidence et al., 1993; Hacker et al., 1992; Kaestner et al., 1993; Larsson et al., 1995; Murphy et al., 1994; Pierrou et al., 1994). Some members of the gene family have been shown to be involved directly in the process of tumorigenesis, such as the retroviral oncogene qin (Li and Vogt, 1993) and the PAX3 gene through the translocation and fusion of the PAX3 activation domain from 2q35 to the forkhead domain of a novel gene, FKHR, on 13q14 (Galili et al., 1993; Shapiro et al., 1993). The forkhead gene family has been divided into five classes (class I–V), and members of each class have greater than 75% homology at the amino acid level over the forkhead domain, whereas homology between groups is less than 70% (Murphy et al., 1994; Sasaki and Hogan, 1994). Variation in the amino acids in the DNA-binding domain alter the binding specificity of the proteins (Lai et al., 1991; Oettinger et al., 1991). Twelve human gene members of the forkhead gene family have been described: the forkhead-related activators, FREAC1–8 (Larsson et al., 1995; Pierrou et al., 1994); the human brain factor I, HBF-1 (Murphy et al., 1994); the interleukin-binding factor, ILF (Li et al., 1991); the human T-cell leukemia virus factor, HTLF (Li et al., 1992); and the FKHR gene (Galili et al., 1993; Shapiro et al., 1993). Here we describe the cloning of a new member of the human forkhead gene family, FKHL15 (forkhead-like
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gene-15), close to the genetic marker D9S180 on chromosome 9q22. The position of FKHL15 locates it within the overlapping candidate regions for the genes for hereditary sensory neuropathy type I, HSN-1 (Nicholson et al., 1996), and the skin cancer predisposition disorder multiple self-healing squamous epitheliomata, ESS1 (Goudie et al., 1993). HSN-1 is a dominant disorder that causes loss of sensation, especially to pain and temperature, and is the most common of a group of degenerative disorders of sensory neurons. ESS1 is a recessive disorder and is characterized by a predisposition of multiple squamous cell cancers of the skin that resolve spontaneously. Loss of heterozygosity in squamous cell cancer frequently shows informative loss of D9S180, suggesting the presence of a tumor supressor gene involved in squamous cell cancer on 9q22 (Holmberg et al., 1996). The predicted FKHL15 protein contains a second domain rich in alanine residues close to the forkhead DNA-binding domain. Alanine-rich domains have been found in other developmental DNA-binding proteins, including the homeobox protein EVX1 (Briata et al., 1995), the Msx-1 homeotic gene (Catron et al., 1995), and the Drosophila homeotic genes kruppel (Licht et al., 1994) and engrailed (Han and Manley, 1993), and are believed to be responsible for transcriptional repression activity. MATERIALS AND METHODS cDNA selection. cDNA selection was performed essentially as described (Korn et al., 1992). In brief, first-strand cDNA was prepared from fibroblast poly(A)/ RNA with random hexamer primers. After second-strand synthesis adapters were ligated to the blunt ends, and the cDNA population was amplified by 25 cycles of PCR with adapterspecific primers. Six cosmids and two PACs (F. Obermayr, unpublished data) from the 9q22 region were nick-translated in the presence of biotin–dUTP (Boehringer). Cosmid and PAC clones were prehybridized with 150-fold weight excess of human Cot-1 DNA (Gibco BRL) and hybridized at 427C for 16 h to uncloned PCR-amplified cDNA. Biotinylated hybridization products were immobilized by streptavidin-coated magnetic beads on MPC-E (Dynal) and washed at 657C for 10 min each with 21 SSC, 11 SSC, 0.31 SCC, 0.21 SSC, and 0.11 SSC before elution in water at 857C for 10 min. Eluted cDNA was sized on chromaspin-400 columns (Clontech), and PCR amplification was carried out as described above. Hybridization, immobilization, and elution were carried out once more before a final amplification by PCR for 16 cycles using primers containing uracil. PCR products were sized and cloned into the pAMP10 vector (Gibco BRL). Fibroblast cDNA clones (n Å 1440) were picked into 96-well microtiter plates and transferred to 384-well microtiter plates before spotting by robot onto nylon membranes in a 3 1 3 duplicate highdensity array. Isolation and sequencing of cDNA clones. HFF12G12, a random clone from the selected cDNA library (nucleotides 2093 – 2632 of 3* UTR), was hybridized to a spotted filter of the library and identified ú50 other cohybridizing cDNA clones in the library. This group of cDNA clones was called FKHL15 (previously called GRC-3). HFF12G12 was used for the further analysis of the group. A human keratinocyte stem cell poly(T)-primed cDNA library was hybridized with HFF12G12 (Phil Jones and Fiona Watt, unpublished results), and 6 hybridizing cDNA clones were picked, and cDNA preparations made using QiaTip-100 (Qiagen). Each clone was 3.5-kb in
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FIG. 1. Genomic DNA digested with HindIII. (Lane 1) Human lymphoblastoid cell line GM1416B (ATCC). (Lane 2) Somatic cell hybrid 640-63a12 (Jones and Kao, 1984), containing the long arm of human chromosome 9 on a hamster background. (Lane 3) Hamster cell line A23. (Lane 4) Somatic cell hybrid CV340/BW containing human chromosome fragments 9pter–q22.3 and 6pter–p23 and a fragment of X on a mouse background (Mike Dixon, Manchester, UK). (Lane 5) Mouse lymphoblastoid cell line BW5147. Hybridization was with the FKHL15 cDNA clone HFF12G12. size. The clones were sequenced by primer walking using a fluorescently labeled dye-terminator cycle sequencing kit according to the manufacturer’s instructions (Applied Biosystems) and electrophoresed on an ABI373 (Applied Biosystems). DNA sequences were analyzed by searches using the programs BLAST-N, BLAST-X, and FASTA. Northern analysis. The single-copy cDNA clone HFF12G12 was hybridized to Northern blots (Clontech; human multiple-tissue Northern blots Cat. Nos. 7760-1 and 7759-1). Hybridization was performed according to the manufacturer’s instructions.
RESULTS AND DISCUSSION
Isolation of FKHL15 and Chromosomal Location To isolate transcribed sequences from human chromosome 9q22 we employed the technique of cDNA selection (Korn et al., 1992). One group of selected cDNA clones, named FKHL15, hybridized to a 5.5-kb HindIII fragment of the cosmid clones LLNLc115D0635 and LLNLc115D04183 from the Lawrence Livermore chromosome 9 cosmid library (data not shown). These two cosmids contain the genetic marker D9S180 as assayed by PCR (data not shown). The cDNA clone HFF12G12, a representative member of the group, was used for the further characterization of FKHL15. To confirm the location of FKHL15 on chromosome 9q22, HFF12G12 was hybridized to a somatic cell mapping panel (Fig. 1). A hybridizing 5.5-kb HindIII fragment was observed for both somatic cell hybrids and was shown to be single-copy human specific and also showed that HFF12G12 is located on 9q. The cosmids LLNLc115D0635 (35D6) and LLNLc115C04183 (183C4) form the distal end of a 300-kb cosmid and PAC contig between D9S780 and D9S180. Other cDNA groups selected with this contig correspond to the tropomodulin gene, TMOD (Sung et al., 1996); the nuclear cap-binding protein, NCBP (Chadwick et al., 1996); the xeroderma pigmentosum complementation
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FIG. 2. Full cDNA sequence for FKHL15. The coding sequence begins at position 689 and ends at 1816. The sequence for FKHL15 is shown including the 5* and 3* untranslated regions and the ORF. The polyalanine coding sequence is underlined, as are the potential polyadenylation sites. The forkhead domain is outlined in bold. (Accession No. U89995)
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FIG. 3. Alignments of FKHL15 forkhead domain to the most closely related members of the forkhead family. Identical amino acids are represented by dots, and the amino acid identity level is shown at the end of each row as a percentage. The relative positions of the helical and basic regions of the 100-amino-acid forkhead domain are shown below the alignments. The forkhead, HNF3b, HNF3g, and XFD-1 genes are all members of class I. SLP2 is a member of group II, FD3 is a member of group III, and BF-1 is a member of group V.
group A gene, XPA (Satokata et al., 1993); and the keratin-18-like 3 gene, KRT18L3 (Romano et al., 1988) (Fig. 4). Sequence analysis of the clones in the selected cDNA library showed 100% sequence identity to KRT18. The human KRT18 gene has previously been mapped to human chromosome 12q13 (Yoon et al., 1994). There are, however, three sequences hybridizing to KRT18 on chromosomes X, 9, and 11 (Romano et al., 1988). It is very likely that KRT18L3, which maps to chromosome 9, was responsible for the isolation of KRT18 transcipts in the cDNA selection procedure. Database searching with the KRT18 sequence identified
an 88% homology to a 1.5-kb sequence entry for the characterization of the upstream region of XPA (Accession No. hsu16815) (Satokata et al., 1993). This defines the exact position of KRT18L3 with respect to XPA. When the KRT18L3 sequence was aligned with KRT18, it was found that the AUG start codon at base position 194 had been lost, and it is therefore likely that KRT18L3 is a pseudogene (data not shown). KRT18L3 is not present on mouse chromosome 4 (data not shown), which would suggest that the KRT18L3 pseudogene adjacent to XPA is of relatively recent evolutionary origin.
FIG. 4. A comprehensive physical and transcriptional map of the D9S780–D9S180 region. (A) Relative positions of the markers D9S780, XPA, and D9S180 in relation to the FKHL15 locus according to the YAC map of Morris and Reis (1994). The cosmid (B) and PAC (C) clone maps were derived by hybridization of whole cosmids and PACs and verified by restriction digests. (D) Schematic representation of the locations of the genes (not to scale).
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FIG. 5. Multiple-tissue Northern hybridizations. (A) Hybridization with the FKHL15 cDNA clone HFF12G12. (B) Hybridization with b-actin probe.
Isolation and Sequencing of cDNA Clones for FKHL15 HFF12G12 was used to screen a human keratinocyte stem cell cDNA library. Six identical clones of 3.5 kb were isolated. The FKHL15 cDNA clones were sequenced and used to search against entries in GenBank. A potential open reading frame was observed for nt 689 to 1816 with the ATG at 689 having a moderate match to the initiation start site for vertebrates (CGCGCCAUGA for FKHL15 versus GCCGCCAUGG) (Kozak, 1989). The full DNA sequence for FKHL15 is shown in Fig. 2 with the amino acid sequence shown below. Three putative polyadenylation signals of AATAAA are found at positions 2249–2254, 3411–3416, and 3480–3485 (see Fig. 2). The region 830–1150 showed greater than 80% nucleic acid homology to members of the forkhead gene family. The forkhead domain spans amino acid residues 53 through 153. Figure 3 shows the alignment of the forkhead region of FKHL15 against related members of the forkhead family. The most closely related members (HFH-7 and freac-8) do not fall into any of the five classes according to Murphy et al. (1994), due to not satisfying the greater than 75% amino acid homology criteria, and may form a new sixth class. FKHL15 shares homology over the forkhead domain only with the other 12 known human members. A putative nuclear localization signal of 5 basic amino acid residues upstream of the forkhead domain is observed at residues 43–47 and a second one at the end of the forkhead domain at amino acid residues 157–161 (Robbins et al., 1991). A polyalanine tract of 19 alanine residues is found at position 64–82 downstream of the forkhead domain. A similar-length polyalanine tract is also observed in the mouse homeobox engrailed-1 DNAbinding protein (Han and Manley, 1993) (data not
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shown). Domains rich in alanine residues have also been found in other developmental DNA-binding proteins including the Drosophila kruppel gene (Licht et al., 1994), the TBP-binding protein Dr1 (Yeung et al., 1994), the human homeobox gene EVX1 (Briata et al., 1995), and the murine homeodomain gene Msx-1 (Catron et al., 1995). These alanine-rich domains are believed to be responsible for the transcriptional repression activity associated with the above genes. The alanine-rich domain in FKHL15 may serve a similar function. The last 9 alanine residues are encoded by a GCC trinucleotide repeat, while a mixture of alanine codons is used for the first 10 residues highlighted in Fig. 2. The C-terminal region of the coding sequence shows no significant homology to any entries in GenBank. Attempts were made to isolate the mouse homologue for the FKHL15 gene using a probe derived from the C-terminal region of the coding sequence. No signals were observed on mouse Northern blots, cDNA libraries, or genomic Southern blots, suggesting that the mouse DNA sequence for the non-forkhead region of FKHL15 is not highly conserved (data not shown). Expression of FKHL15 Human multiple-tissue Northerns were hybridized with HFF12G12 (Fig. 5). A single 4.5-kb message was detected in adult brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. An additional 1.5-kb message was also detected in adult heart and a 1.8-kb message in pancreas. Adult colon, small intestine, and prostate showed the presence of only the 1.5-kb message, while adult testis showed a 2.8-kb message and adult thymus gave 5.0-, 4.5-, and 3.2-kb messages. Expression was strongest in heart and pancreas. The presence of FKHL15 was not detected in adult spleen, ovary, or peripheral blood leukocyte.
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The variation in message size could be due to a combination of alternatively spliced products for FKHL15 and the potential use of different polyadenylation sites. The presence of a forkhead domain within the FKHL15 gene suggests that one of the functions of FKHL15 may be to bind DNA, with an important role in development. The alanine-rich domain located close to the C-terminal side of the forkhead region is similar to those that have been observed in other DNA-binding proteins associated with transcriptional repression. It is therefore possible that FKHL15 also possesses transcriptional repression activity. The map position for FKHL15 around D9S180 places it within the candidate regions for the genetic disorders HSN-1 (Nicholson et al., 1996) and ESS1 (Goudie et al., 1993). More recently the presence of a tumor suppressor locus around D9S180 has been implicated through allele loss studies for chromosome 9q, showing a 50% loss of the D9S180 marker in squamous cell cancer (Holmberg et al., 1996). Recently several developmental genes have been shown to cause certain cancers when mutated, such as the human tumor suppressor gene PTCH and downstream targets WNT1 and GLI (Pennisi, 1996). ACKNOWLEDGMENTS We are grateful to Carol Jones for the provision of the 640-63a12 somatic cell hybrid and to Mike Dixon for the CV/340 somatic cell hybrid. We are also grateful to Phil Jones and Fiona Watt for the provision of the human keratinocyte stem cell cDNA library. We thank Bernd Korn for the advice on and protocol for the cDNA selection. We are grateful to Pieter de Jong for the Lawrence Livermore chromosome 9-specific gene library LL09Nc01 and the PAC library. These were constructed at the Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550, under the auspices of the National Laboratory Gene Library Project sponsored by the U.S. Department of Energy. This work was supported by the Imperial Cancer Research Fund.
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ARTICLE NO.
FKHL15, a New Human Member of the Forkhead Gene Family Located on Chromosome 9q22 Brian Paul Chadwick,* Franz Obermayr,*,1 and Anna-Maria Frischauf*,2 *Molecular Analysis of Mammalian Mutation, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom Received November 18, 1996; accepted February 6, 1997
FKHL15 was isolated from a cDNA library enriched for transcripts from 9q22. Isolation and sequencing of a 3.5-kb cDNA clone identified a putative 376-aminoacid protein with greater than 80% homology over a 100-amino-acid stretch to the forkhead DNA-binding domain. The FKHL15 gene contains a region rich in alanine residues, frequently associated with transcriptional repression. The forkhead genes are believed to play important roles in development and differentiation in many different organisms and have also been implicated in the development of some tumors. The map position of FKHL15 on 9q22 places the gene within the candidate regions for the cancer predisposition syndrome multiple self-healing squamous epitheliomata and the degenerative neurological disorder hereditary sensory neuropathy type I. This is a region frequently lost in squamous cell cancer. q 1997 Academic Press
INTRODUCTION
The forkhead domain was first identified as a region of homology between the rat hepatocyte nuclear factor 3 (HNF3) and the Drosophila homeotic gene forkhead (Lai et al., 1991). The forkhead gene (fkh) obtained its name through the incapability to form the normal terminal structures of the anterior and posterior gut, instead forming two spiked structures (Weigel et al., 1989). The HNF3b gene is required for notochord development in mouse (Ang et al., 1994; Weinstein et al., 1994). The two proteins show 90% identity at the amino acid level over a 100-amino-acid stretch since called the ‘‘forkhead domain.’’ This forkhead domain is a variant of the DNA-binding helix–turn–helix (HTH) motif, binding DNA as a monomer with two loops, or wings, on the C-terminal side of the HTH, and is often referred to as the ‘‘winged-helix’’ motif (Brennen, 1993; Clark et al., 1993; Lai et al., 1993). Many other members of 1
Current address: Department of Developmental Biology, MPI for Immunology, Freiberg, Germany. 2 To whom correspondence should be addressed. Telephone: (0171) 269 2319. Fax: (0171) 269 3479.
0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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this eukaryotic transcription factor family have been identified through association with pattern formation during embryogenesis including the Drosophila segmental genes sloppy paired 1 and 2 (slp1 and slp2) (Grossniklaus et al., 1992; Hacker et al., 1992), the Caenorhabditis elegans vulval development gene lin31 (Miller et al., 1993), the Axial gene in zebrafish (ax1) (Strahle et al., 1993), the axis formation gene XFKH1 in Xenopus (Dirksen and Jamrich, 1992), and the head patterning gene crocodile in Drosophila (Hacker et al., 1995). In mouse, disruptions in the forkhead-containing gene whn give rise to the nude mutation (Nehls et al., 1994; Segre et al., 1995). Other members of the group have been identified by degenerate PCR and lowstringency hybridization of the forkhead domain (Bork et al., 1992; Clevidence et al., 1993; Hacker et al., 1992; Kaestner et al., 1993; Larsson et al., 1995; Murphy et al., 1994; Pierrou et al., 1994). Some members of the gene family have been shown to be involved directly in the process of tumorigenesis, such as the retroviral oncogene qin (Li and Vogt, 1993) and the PAX3 gene through the translocation and fusion of the PAX3 activation domain from 2q35 to the forkhead domain of a novel gene, FKHR, on 13q14 (Galili et al., 1993; Shapiro et al., 1993). The forkhead gene family has been divided into five classes (class I–V), and members of each class have greater than 75% homology at the amino acid level over the forkhead domain, whereas homology between groups is less than 70% (Murphy et al., 1994; Sasaki and Hogan, 1994). Variation in the amino acids in the DNA-binding domain alter the binding specificity of the proteins (Lai et al., 1991; Oettinger et al., 1991). Twelve human gene members of the forkhead gene family have been described: the forkhead-related activators, FREAC1–8 (Larsson et al., 1995; Pierrou et al., 1994); the human brain factor I, HBF-1 (Murphy et al., 1994); the interleukin-binding factor, ILF (Li et al., 1991); the human T-cell leukemia virus factor, HTLF (Li et al., 1992); and the FKHR gene (Galili et al., 1993; Shapiro et al., 1993). Here we describe the cloning of a new member of the human forkhead gene family, FKHL15 (forkhead-like
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gene-15), close to the genetic marker D9S180 on chromosome 9q22. The position of FKHL15 locates it within the overlapping candidate regions for the genes for hereditary sensory neuropathy type I, HSN-1 (Nicholson et al., 1996), and the skin cancer predisposition disorder multiple self-healing squamous epitheliomata, ESS1 (Goudie et al., 1993). HSN-1 is a dominant disorder that causes loss of sensation, especially to pain and temperature, and is the most common of a group of degenerative disorders of sensory neurons. ESS1 is a recessive disorder and is characterized by a predisposition of multiple squamous cell cancers of the skin that resolve spontaneously. Loss of heterozygosity in squamous cell cancer frequently shows informative loss of D9S180, suggesting the presence of a tumor supressor gene involved in squamous cell cancer on 9q22 (Holmberg et al., 1996). The predicted FKHL15 protein contains a second domain rich in alanine residues close to the forkhead DNA-binding domain. Alanine-rich domains have been found in other developmental DNA-binding proteins, including the homeobox protein EVX1 (Briata et al., 1995), the Msx-1 homeotic gene (Catron et al., 1995), and the Drosophila homeotic genes kruppel (Licht et al., 1994) and engrailed (Han and Manley, 1993), and are believed to be responsible for transcriptional repression activity. MATERIALS AND METHODS cDNA selection. cDNA selection was performed essentially as described (Korn et al., 1992). In brief, first-strand cDNA was prepared from fibroblast poly(A)/ RNA with random hexamer primers. After second-strand synthesis adapters were ligated to the blunt ends, and the cDNA population was amplified by 25 cycles of PCR with adapterspecific primers. Six cosmids and two PACs (F. Obermayr, unpublished data) from the 9q22 region were nick-translated in the presence of biotin–dUTP (Boehringer). Cosmid and PAC clones were prehybridized with 150-fold weight excess of human Cot-1 DNA (Gibco BRL) and hybridized at 427C for 16 h to uncloned PCR-amplified cDNA. Biotinylated hybridization products were immobilized by streptavidin-coated magnetic beads on MPC-E (Dynal) and washed at 657C for 10 min each with 21 SSC, 11 SSC, 0.31 SCC, 0.21 SSC, and 0.11 SSC before elution in water at 857C for 10 min. Eluted cDNA was sized on chromaspin-400 columns (Clontech), and PCR amplification was carried out as described above. Hybridization, immobilization, and elution were carried out once more before a final amplification by PCR for 16 cycles using primers containing uracil. PCR products were sized and cloned into the pAMP10 vector (Gibco BRL). Fibroblast cDNA clones (n Å 1440) were picked into 96-well microtiter plates and transferred to 384-well microtiter plates before spotting by robot onto nylon membranes in a 3 1 3 duplicate highdensity array. Isolation and sequencing of cDNA clones. HFF12G12, a random clone from the selected cDNA library (nucleotides 2093 – 2632 of 3* UTR), was hybridized to a spotted filter of the library and identified ú50 other cohybridizing cDNA clones in the library. This group of cDNA clones was called FKHL15 (previously called GRC-3). HFF12G12 was used for the further analysis of the group. A human keratinocyte stem cell poly(T)-primed cDNA library was hybridized with HFF12G12 (Phil Jones and Fiona Watt, unpublished results), and 6 hybridizing cDNA clones were picked, and cDNA preparations made using QiaTip-100 (Qiagen). Each clone was 3.5-kb in
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FIG. 1. Genomic DNA digested with HindIII. (Lane 1) Human lymphoblastoid cell line GM1416B (ATCC). (Lane 2) Somatic cell hybrid 640-63a12 (Jones and Kao, 1984), containing the long arm of human chromosome 9 on a hamster background. (Lane 3) Hamster cell line A23. (Lane 4) Somatic cell hybrid CV340/BW containing human chromosome fragments 9pter–q22.3 and 6pter–p23 and a fragment of X on a mouse background (Mike Dixon, Manchester, UK). (Lane 5) Mouse lymphoblastoid cell line BW5147. Hybridization was with the FKHL15 cDNA clone HFF12G12. size. The clones were sequenced by primer walking using a fluorescently labeled dye-terminator cycle sequencing kit according to the manufacturer’s instructions (Applied Biosystems) and electrophoresed on an ABI373 (Applied Biosystems). DNA sequences were analyzed by searches using the programs BLAST-N, BLAST-X, and FASTA. Northern analysis. The single-copy cDNA clone HFF12G12 was hybridized to Northern blots (Clontech; human multiple-tissue Northern blots Cat. Nos. 7760-1 and 7759-1). Hybridization was performed according to the manufacturer’s instructions.
RESULTS AND DISCUSSION
Isolation of FKHL15 and Chromosomal Location To isolate transcribed sequences from human chromosome 9q22 we employed the technique of cDNA selection (Korn et al., 1992). One group of selected cDNA clones, named FKHL15, hybridized to a 5.5-kb HindIII fragment of the cosmid clones LLNLc115D0635 and LLNLc115D04183 from the Lawrence Livermore chromosome 9 cosmid library (data not shown). These two cosmids contain the genetic marker D9S180 as assayed by PCR (data not shown). The cDNA clone HFF12G12, a representative member of the group, was used for the further characterization of FKHL15. To confirm the location of FKHL15 on chromosome 9q22, HFF12G12 was hybridized to a somatic cell mapping panel (Fig. 1). A hybridizing 5.5-kb HindIII fragment was observed for both somatic cell hybrids and was shown to be single-copy human specific and also showed that HFF12G12 is located on 9q. The cosmids LLNLc115D0635 (35D6) and LLNLc115C04183 (183C4) form the distal end of a 300-kb cosmid and PAC contig between D9S780 and D9S180. Other cDNA groups selected with this contig correspond to the tropomodulin gene, TMOD (Sung et al., 1996); the nuclear cap-binding protein, NCBP (Chadwick et al., 1996); the xeroderma pigmentosum complementation
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FIG. 2. Full cDNA sequence for FKHL15. The coding sequence begins at position 689 and ends at 1816. The sequence for FKHL15 is shown including the 5* and 3* untranslated regions and the ORF. The polyalanine coding sequence is underlined, as are the potential polyadenylation sites. The forkhead domain is outlined in bold. (Accession No. U89995)
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FIG. 3. Alignments of FKHL15 forkhead domain to the most closely related members of the forkhead family. Identical amino acids are represented by dots, and the amino acid identity level is shown at the end of each row as a percentage. The relative positions of the helical and basic regions of the 100-amino-acid forkhead domain are shown below the alignments. The forkhead, HNF3b, HNF3g, and XFD-1 genes are all members of class I. SLP2 is a member of group II, FD3 is a member of group III, and BF-1 is a member of group V.
group A gene, XPA (Satokata et al., 1993); and the keratin-18-like 3 gene, KRT18L3 (Romano et al., 1988) (Fig. 4). Sequence analysis of the clones in the selected cDNA library showed 100% sequence identity to KRT18. The human KRT18 gene has previously been mapped to human chromosome 12q13 (Yoon et al., 1994). There are, however, three sequences hybridizing to KRT18 on chromosomes X, 9, and 11 (Romano et al., 1988). It is very likely that KRT18L3, which maps to chromosome 9, was responsible for the isolation of KRT18 transcipts in the cDNA selection procedure. Database searching with the KRT18 sequence identified
an 88% homology to a 1.5-kb sequence entry for the characterization of the upstream region of XPA (Accession No. hsu16815) (Satokata et al., 1993). This defines the exact position of KRT18L3 with respect to XPA. When the KRT18L3 sequence was aligned with KRT18, it was found that the AUG start codon at base position 194 had been lost, and it is therefore likely that KRT18L3 is a pseudogene (data not shown). KRT18L3 is not present on mouse chromosome 4 (data not shown), which would suggest that the KRT18L3 pseudogene adjacent to XPA is of relatively recent evolutionary origin.
FIG. 4. A comprehensive physical and transcriptional map of the D9S780–D9S180 region. (A) Relative positions of the markers D9S780, XPA, and D9S180 in relation to the FKHL15 locus according to the YAC map of Morris and Reis (1994). The cosmid (B) and PAC (C) clone maps were derived by hybridization of whole cosmids and PACs and verified by restriction digests. (D) Schematic representation of the locations of the genes (not to scale).
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FIG. 5. Multiple-tissue Northern hybridizations. (A) Hybridization with the FKHL15 cDNA clone HFF12G12. (B) Hybridization with b-actin probe.
Isolation and Sequencing of cDNA Clones for FKHL15 HFF12G12 was used to screen a human keratinocyte stem cell cDNA library. Six identical clones of 3.5 kb were isolated. The FKHL15 cDNA clones were sequenced and used to search against entries in GenBank. A potential open reading frame was observed for nt 689 to 1816 with the ATG at 689 having a moderate match to the initiation start site for vertebrates (CGCGCCAUGA for FKHL15 versus GCCGCCAUGG) (Kozak, 1989). The full DNA sequence for FKHL15 is shown in Fig. 2 with the amino acid sequence shown below. Three putative polyadenylation signals of AATAAA are found at positions 2249–2254, 3411–3416, and 3480–3485 (see Fig. 2). The region 830–1150 showed greater than 80% nucleic acid homology to members of the forkhead gene family. The forkhead domain spans amino acid residues 53 through 153. Figure 3 shows the alignment of the forkhead region of FKHL15 against related members of the forkhead family. The most closely related members (HFH-7 and freac-8) do not fall into any of the five classes according to Murphy et al. (1994), due to not satisfying the greater than 75% amino acid homology criteria, and may form a new sixth class. FKHL15 shares homology over the forkhead domain only with the other 12 known human members. A putative nuclear localization signal of 5 basic amino acid residues upstream of the forkhead domain is observed at residues 43–47 and a second one at the end of the forkhead domain at amino acid residues 157–161 (Robbins et al., 1991). A polyalanine tract of 19 alanine residues is found at position 64–82 downstream of the forkhead domain. A similar-length polyalanine tract is also observed in the mouse homeobox engrailed-1 DNAbinding protein (Han and Manley, 1993) (data not
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shown). Domains rich in alanine residues have also been found in other developmental DNA-binding proteins including the Drosophila kruppel gene (Licht et al., 1994), the TBP-binding protein Dr1 (Yeung et al., 1994), the human homeobox gene EVX1 (Briata et al., 1995), and the murine homeodomain gene Msx-1 (Catron et al., 1995). These alanine-rich domains are believed to be responsible for the transcriptional repression activity associated with the above genes. The alanine-rich domain in FKHL15 may serve a similar function. The last 9 alanine residues are encoded by a GCC trinucleotide repeat, while a mixture of alanine codons is used for the first 10 residues highlighted in Fig. 2. The C-terminal region of the coding sequence shows no significant homology to any entries in GenBank. Attempts were made to isolate the mouse homologue for the FKHL15 gene using a probe derived from the C-terminal region of the coding sequence. No signals were observed on mouse Northern blots, cDNA libraries, or genomic Southern blots, suggesting that the mouse DNA sequence for the non-forkhead region of FKHL15 is not highly conserved (data not shown). Expression of FKHL15 Human multiple-tissue Northerns were hybridized with HFF12G12 (Fig. 5). A single 4.5-kb message was detected in adult brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. An additional 1.5-kb message was also detected in adult heart and a 1.8-kb message in pancreas. Adult colon, small intestine, and prostate showed the presence of only the 1.5-kb message, while adult testis showed a 2.8-kb message and adult thymus gave 5.0-, 4.5-, and 3.2-kb messages. Expression was strongest in heart and pancreas. The presence of FKHL15 was not detected in adult spleen, ovary, or peripheral blood leukocyte.
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The variation in message size could be due to a combination of alternatively spliced products for FKHL15 and the potential use of different polyadenylation sites. The presence of a forkhead domain within the FKHL15 gene suggests that one of the functions of FKHL15 may be to bind DNA, with an important role in development. The alanine-rich domain located close to the C-terminal side of the forkhead region is similar to those that have been observed in other DNA-binding proteins associated with transcriptional repression. It is therefore possible that FKHL15 also possesses transcriptional repression activity. The map position for FKHL15 around D9S180 places it within the candidate regions for the genetic disorders HSN-1 (Nicholson et al., 1996) and ESS1 (Goudie et al., 1993). More recently the presence of a tumor suppressor locus around D9S180 has been implicated through allele loss studies for chromosome 9q, showing a 50% loss of the D9S180 marker in squamous cell cancer (Holmberg et al., 1996). Recently several developmental genes have been shown to cause certain cancers when mutated, such as the human tumor suppressor gene PTCH and downstream targets WNT1 and GLI (Pennisi, 1996). ACKNOWLEDGMENTS We are grateful to Carol Jones for the provision of the 640-63a12 somatic cell hybrid and to Mike Dixon for the CV/340 somatic cell hybrid. We are also grateful to Phil Jones and Fiona Watt for the provision of the human keratinocyte stem cell cDNA library. We thank Bernd Korn for the advice on and protocol for the cDNA selection. We are grateful to Pieter de Jong for the Lawrence Livermore chromosome 9-specific gene library LL09Nc01 and the PAC library. These were constructed at the Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550, under the auspices of the National Laboratory Gene Library Project sponsored by the U.S. Department of Energy. This work was supported by the Imperial Cancer Research Fund.
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