A 2.5-Mb Transcript Map of a Tumor-Suppressing Subchromosomal Transferable Fragment from 11p15.5, and Isolation and Sequence Analysis of Three Novel Genes
Ren-Ju Hu,*,1 Maxwell P. Lee,*1 Timothy D. Connors, t Laura A. Johnson,* Timothy C. Burn, + Kui Su, t Gregory M. Landes, t and Andrew P. Feinberg*, 1,2
*Department of Medicine and Departments of Molecular Biology & Genetics and Oncology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, Maryland 21205; and tGenzyme Research & Development, One Mountain Road, Framingham, Massachusetts 01701
Received June 6, 1997; accepted August 15, 1997
11p15.5 is an important tumor-suppressor gene region, showing loss of heterozygosity in Wilms tumor, rhabdo- myosarcoma, adrenocortical carcinoma, and lung, ovar- ian, and breast cancer. We previously mapped directly by genetic complementation a subtransferable fragment (STF) harboring an embryonal tumor-suppressor gene and spanning about 2.5 Mb. We have now mapped the centromeric end of this STF between D11S988 and D11S12 and its telomeric end between D11S1318 and TH. We have isolated a complete contig of PAC, P1, BAC, and cosmid genomic clones spanning the entire 2.5-Mb region defined by this STF, as well as more than 200 exons from these genomic clones using exon trapping. We have isolated genes in this region by directly screen- ing cDNA libraries as well as by database searching for ESTs. Nine of these genes have been reported previously by us and by others. However, the initial mapping of most of those genes was based on FISH or somatic cell hybrid analysis, and here we precisely define their phys- ical location. These genes include RRM1, GOK (D11S4896E), Nup98, CARS, hNAP2 (NAP1L4), p57KIP2 (CDKN1C), KyLQT1 (KCNA9), TAPA-1, and ASCL2. In ad- dition, we have identified several novel genes in this region, three of which, termed TSSC1, TSSC2, and TSSC3, are reported here. TSSC1 shows homology to Rb- associated protein p48 and chromatin assembly factor CAF1, and it is located between GOK and Nup98. TSSC2 is homologous to Caenorhabditis elegans ß-mannosyl transferase, and it lies between Nup98 and CARS. TSSC3 shows homology to mouse TDAG51, which is implicated in FasL-mediated apoptosis, and it is located between hNAP2 and p57KIP2. Thus, these genes may play a role in malignancies that involve this region. @ 1997 Academic Press
INTRODUCTION
We and others previously mapped an embryonal tu- mor suppressor gene (WT2) to 11p15.5 by loss of hetero-
1 Equal contribution from these authors.
2 To whom correspondence should be addressed. Telephone: (410) 614-3489. Fax: (410) 614-9819.
zygosity (LOH) in Wilms tumor (Reeve et al., 1989; Koufos et al., 1989). Many other cancers also show fre- quent LOH of 11p15.5, including rhabdomyosarcoma, adrenocortical carcinoma, breast cancer, non-small-cell lung cancer, and hepatocellular, bladder, ovarian, and testicular cancer (Winqvist et al., 1995; Besnard-Gúe- rin et al., 1996; Shaw and Knowles, 1995; Bepler and Garcia-Blanco, 1994; Lothe et al., 1993; Weitzel et al., 1994; Byrne et al., 1993). Polymorphic markers in the minimal overlapping region of LOH include D11S12, D11S860, D11S1318, and TH. We also previously dem- onstrated directly the presence of a tumor-suppressor gene by suppressing growth of rhabdomyosarcoma cells using a subchromosomal transferable fragment (STF) isolated from 11p15.5 (Koi et al., 1993). The smallest tumor-suppressing STF is about 2.5 Mb, and its bound- aries were defined previously by a 2-Mb distance be- tween the 0-globin gene and D11S12 and on the te- lomeric side by a 500-kb interval defined by D11S724 and IGF2. Therefore, both the functional complementa- tion assay using STFs and physical mapping of dele- tions in tumors by LOH define one or more tumor- suppressor gene(s) within a region between ß-globin and IGF2. We also previously mapped five chromosome rearrangement breakpoints of the Beckwith-Wiede- mann syndrome (BWS) cluster BWSCR1 to a more lim- ited domain of the region (350 kb of ~2.5 Mb total), between D11S648 and D11S551 (Hoovers et al., 1995). We also found previously that the BWSCR1 break- points interrupt the coding sequence of KyLQT1 (Lee et al., 1997a). BWSCR1 may not contain the tumor suppressor gene, however, as other genetic complemen- tation experiments suggest a more centromeric location still within the STF (Reid et al., 1996).
We have now isolated a complete genomic contig spanning the entire 2.5-Mb tumor-suppressing STF, defining the boundaries more precisely at the centro- meric end, between D11S988 and D11S12, and the te- lomeric end, between D11S1318 and TH. Furthermore, we have isolated 200 exons within the STF and identi-
fied and localized precisely nine previously known genes. In addition, three novel genes within the STF are reported here.
MATERIALS AND METHODS
Isolation of genomic clones and physical mapping. STSs pre- viously isolated and/or mapped to 11p15.5 (Hoovers et al., 1995) were more precisely localized within STF 74-1-6 by PCR amplification and, as a negative control, absence of amplification from its parental cell A9. BAC (Genetic Research, Inc.) and PAC (Genome Systems, Inc.) libraries were screened for the presence of STSs by PCR (described in detail under Results and in the legend to Fig. 1). P1 clones (Genome Systems, Inc.) were screened by hybridization of a high-density filter using genomic fragments as probes. Cosmids derived from YACs B40E4, D122D10, and E42F4 were described previously (Hoovers et al., 1995). Cosmids derived from YACs B176E9 and B215A11 were isolated by construction of cosmid libraries using total YAC DNA. Assembly of genomic contigs using these BAC, PAC, and P1 clones was achieved by PCR using STSs, end clones, and exons.
Exon trapping. Exon trapping was performed using the pSPL3B or pSPL3B-CAM vectors (Paul Nisson, BRL, pers. comm.). Individual BAC clones were double-digested with BamHI and Bg/II and sub- cloned into the BamHI site of splicing vector pSPL3B. PAC, P1, or pools of four cosmids were subcloned into pSPL3B-CAM. Plasmid DNAs were isolated from 20 randomly chosen transformants and analyzed by restriction digestion for the presence of inserts. If more than 50% of transformants contained inserts of differing sizes and the total number of transformants exceeded 1000, LB medium was added to the plates containing transformants and colonies were pooled for isolation of DNA. Aliquots of the transformation mixtures were saved as bacterial stocks. DNA was transfected into Cos 7 cells by lipofection. Cells were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Confluent cells were split 1:3 1 day prior to transfection. RNA was isolated 24 h posttransfection using RNAzol B (Tel-Test, Inc.) according to the manufacturer’s pro- tocol. RT-PCR was used to amplify exons using primer set SD2-SA4 provided in the exon trapping kit and subcloned in the pAMP10 vector (Life Technologies, Inc.). The pAMP10 subclones were screened by PCR using primer set SD2-SA4. Transformants with PCR products larger than 250 bp were selected, and they corre- sponded to exons with sizes above 73 bp. DNAs were isolated from these clones containing exons using the Wizard Miniprep kit (Pro- mega), and exons were sequenced using the primer SD2.
cDNA isolation and sequence analysis. cDNAs were isolated by one of three methods. First, we have developed a novel PCR-based cDNA screening method to isolate cDNAs directly and systematically con- taining the exons (M. P. Lee et al., manuscript in preparation). Second, the GenBank dbEST and TIGR THC databases were searched for ESTs using exon sequences. Third, cDNA libraries were screened by hybrid- ization using probes prepared by random priming (Feinberg and Vo- gelstein, 1983). Sequence analysis of both exons and cDNAs was carried out using Sequencher (GeneCode, Inc.), GCG (Genetics Computer Group), and BLAST (Altshul et al., 1990) software.
RESULTS
Physical Mapping of Tumor-Suppressing Subchromosomal Transferable Fragment (STF 74-1-6)
The STF 74-1-6 spans 2.5 Mb and defines at least one tumor-suppressor gene locus (Koi et al., 1993). To refine the boundary of 74-1-6, we tested a panel of STSs for their presence or absence in 74-1-6. Thus the centro- meric end of the STF was found to lie between D11S988
and D11S12 (Fig. 1). Similarly, the telomeric end of the STF was located between D11S1318 and TH (Fig. 1). The order of STSs was also consistent with mapping of somatic hybrid cells. We found that D11S988 and D11S12 were proximal to the J1-7 somatic cell hybrid (Glaser et al., 1989) breakpoint, while D11S860 and telomeric STSs were within J1-7. This mapping of D11S988 is at variance with Bepler and Garcia-Blanco (1994), who assigned STSs in the order of D11S12- D11S860-D11S988-TH, as well as with their recent mapping order of D11S12-D11S988-D11S860-TH (O’Brian and Bepler, 1997). However, we assigned D11S988 proximal to D11S12, in contrast to the previ- ous studies, because the centromeric end of the STF included D11S12 but not D11S988. Thus, the order is D11S988-D11S12-D11S860-D11S1318-TH. We also found that D11S679 and other more centromeric STSs were proximal to the J1-9 breakpoint, while D11S551 and other more telomeric STSs were within J1-9. The order of additional STSs was further established by isolation of a physical contig within the STF as de- scribed below.
Contig Assembly and Exon Trapping of STF 74-1-6
We previously mapped five chromosome rearrange- ment breakpoints of BWS patients to a 350-kb interval within the much larger STF (Hoovers et al., 1995). At that time, we isolated a 500-kb cosmid contig containing these BWS translocation breakpoints. In addition, we had also identified five YACs, B176E9, B215A11, B40E4, D122D10, and E42F4, which mapped within the 74-1-6 interval. To extend the molecular analysis to the fivefold larger region of STF 74-1-6 while expanding our existing contig, we directed our effort toward assembling an entire genomic contig through this ~2.5-Mb region. Using a PCR-based screening method, we initially isolated 14 PAC, P1, and BAC clones that contained amplicons using D11S12, D11S860, D11S1044, D11S1193, D11S470, D11S26, or D11S1318. We then achieved closure of the contig by isolating 16 additional PAC, P1, and BAC clones using amplicons derived from the end clone sequences and trapped exons (Fig. 1 and its legend). A minimal overlapping set is shown in Fig. 1. The order of the PAC, P1, and BAC clones in the contig was also confirmed by the mapping of additional trapped exons and STSs. These genomic clones allowed us to assign the order of known STSs as follows: Cen-D11S988-D11S12-RRM1- D11S860-D11S1193-(D11S459/Z104)-D11S470- D11S26-D11S601-D11S648-D11S679-D11S724- D11S551-D11S1318-TH-INS-IGF2-H19-Tel (Fig. 1; the order of STSs within the parentheses was not determined). We also generated cosmid libraries repre- senting YACs B176E9 and B215A11, and isolated 50 cosmids (data not shown). We then systematically iso- lated exons from the entire 2.5-Mb region using these PAC, P1, BAC, and cosmid clones.
The genomic DNAs from PAC, P1, BAC, or cosmids were double-digested with BamHI and Bg/II and shotgun
J1-7
J1-9
BWSCR1
74-1-6
D11S988
D11S12
D11S860
D11S1193
D11S459
D11S26
D11S601
D11S648
D11S679
D11S724
D11S551
D11S1318
TH
IGF2
H19
Cen
Tel
B176E9
B215A11
B40E9
D122D10
B115F11 A167E5
28A6
181C14
113L6
4013
E42F4
105C10
118H17
143G7
D22
161F14
59N22
711P1
112J14
318P18
124M3
172K20
163C11
CARS
hNAP2
TSSC3
p57KIP2
| TAPA-1
ASCL2
TH
INS
IGF2
RRMI GOK TSSC1
TSSC2
KvLQT1
H19
NUP98
100 kb
cloned into pSPL3B or pSPL3B-CAM for exon trapping. The trapped exons were isolated and sequenced and com- pared among themselves and with vector to identify a set of 200 nonredundant genuine trapped exons. The genomic origins of the nonredundant collection of trapped exons were determined both by hybridization to the gridded set of genomic clones and by using DNA from the genomic clones as templates for PCR. The exon trap density of this interval was about 100 trapped exons per megabase. This density is intermediate to those determined for other ge- nomic intervals, including the 700-kb PKD1 interval (137 trapped exons/Mb) (Burn et al., 1996), the 600-kb BRCA1 region (76 trapped exons/Mb) (Brody et al., 1995), and the 2.5-Mb Down syndrome region (41 trapped exons/Mb) (Lucente et al., 1995).
These exons were further analyzed by searching the GenBank database for homology to known genes and to ESTs. Many of the exons were identical to several previously isolated cDNAs. We identified 3 exons of ribonucleotide reductase M1 (RRM1), 3 exons of GOK,3
15 exons of Nup98, 4 exons of cystenyl tRNA synthe- tase (CARS), 1 exon of hNAP2, and 3 exons of KyLQT1 (Fig. 1).
cDNA Identification, Isolation, and Mapping
We used three approaches to identify cDNAs. First, we isolated cDNAs containing the exons by directly screening cDNA libraries using a novel PCR-based strategy (M. P. Lee et al., manuscript in preparation). Second, we searched for ESTs in the GenBank and TIGR databases using exon sequences. Third, we iso- lated cDNAs from phage libraries by hybridization, us- ing either exons or genomic fragments as probes. BLAST search using exon sequences as queries identi- fied nine known genes. However, many of them were previously mapped only at the level of cytogenetic reso- lution, and their transcription orientations were also not known. We were able to determine here the precise genomic locations of these nine genes and transcription orientations of some of them, and they are as follows:
Ribonucleotide reductase M1 subunit. We isolated three exons, N227 and N2229 from PAC clone 59N22, and C1043 from PAC C10. RRM1 mapped near
3 The HGMW-approved symbols for the genes described in this paper are D11S4896E for GOK, NAP1L4 for hNAP2, CDKN1C for p57KIP2, and KCNA9 for KyLQT1.
D11S12, and the gene was transcribed from telomere to centromere (Fig. 1). N227, N2229, and C1043 matched amino acids 8-34, amino acids 218-263, and amino acids 565-589, respectively, of RRM1. RRM1 was the most proximal gene identified within STF 74-1-6.
Putative transmembrane protein GOK (D11S4896E). We initially isolated three exons, M335, M356, and N2222 from PAC clones 124M3 and 59N22. We subse- quently isolated three cDNAs containing these exons. Sequence analysis indicated that they corresponded to a single gene with an open reading frame encoding an 87-kDa protein. A database search revealed that it is identical to a recently isolated gene, GOK, a putative transmembrane protein proposed to possess kinase ac- tivity based on primary sequence analysis (Parker et al., 1996). GOK was mapped immediately telomeric to RRM1, and the orientation of transcription was from telomere to centromere. GOK was also found to be 300kb centromeric to Nup98 (Fig. 1).
Nucleoporin Nup98. We and others previously re- ported the identification of a gene fusion, composed of Nup98 and HoxA9, in patients with acute myeloid leu- kemia and showing the translocation t(7; 11)(p15;p15) (Nakamura et al., 1996; Borrow et al., 1996). The tran- scriptional orientation of Nup98 is from centromere to telomere (Borrow et al., 1996), Northern blot analysis revealed the presence of both 4- and 7-kb transcripts, and a cDNA was isolated for the 4-kb isoform (Borrow et al., 1996). Nup98 functions as a subunit of the nu- clear pore complex for import of nuclear protein from the cytoplasm (Radu et al., 1995). In the present study, we isolated 15 trapped exons of the Nup98 gene from P1 clone 711P1, as well as cosmids derived from YAC B176E9. Using these trapped exons, we isolated three cDNAs corresponding to Nup98. Sequencing analysis indicated that two of these cDNAs correspond to the previously cloned 4-kb isoform Nup98 gene. The third cDNA clone was 6.5 kb and apparently corresponds to the 7-kb isoform of the gene. We found that the 7-kb isoform of Nup98 was generated by alternative RNA splicing. Thus, Northern blot analysis using a probe derived from the common 5’-end sequence hybridized to transcripts of two sizes, 4 and 7 kb. By comparison, hybridization using a probe derived from 3’-end se- quences of the longer isoform detected only the 7-kb transcript (data not shown). Nup98 mapped approxi- mately 300 kb telomeric to RRM1 (Fig. 1).
Cysteinyl tRNA synthetase. Four exons, ET74, ET77, ET198, and ET225, isolated from cosmids de- rived from YAC B40E4, corresponded to amino acids 1-25, 59-90, 142-217, and 537-562, respectively, of CARS. CARS mapped between D11S26 and D11S601, and its transcriptional orientation was from centro- mere to telomere (Fig. 1). The order of these exons was determined as Cen-(ET77/ET198)-ET225-ET74-Tel. The gene was located immediately centromeric to hNAP2, and the distance between CARS and hNAP2 was about 60 kb (Fig. 1).
Human nucleosome assembly protein 2 (NAP1L4). In the course of the present study, we isolated an exon of hNAP2, which we had previously cloned by screening a Lambda ZAP II cDNA library using a genomic DNA fragment as probe, and the transcriptional orientation of hNAP2 is from centromere to telomere (Hu et al., 1996). It is biallelically expressed, while two more te- lomeric genes, p57KIP2 and KyLQT1, are imprinted (Hu et al., 1996).
Cyclin-dependent kinase inhibitor p57KIP2 (CDKN1C). p57KIP2 is a CDK inhibitor closely related to p27KIPI (Matsuoka et al., 1995; Lee et al., 1995). It was origi- nally mapped to 11p15 cytogenetically (Matsuoka et al., 1995), and we more precisely localized it within YAC B40E4 between D11S648 and D11S679 (Hoovers et al., 1995) and showed that it is imprinted and ex- pressed from the maternal chromosome (Matsuoka et al., 1996). Recently, p57KIP2 has shown mutations in a small fraction of BWS patients (Hatada et al., 1996; Lee et al., 1997b). It is located 65 kb telomeric to hNAP2, and its transcriptional orientation is from cen- tromere to telomere (Hu et al., 1996).
Voltage-gated potassium channel K,LQT1 (KCNA9). KyLQT1 was isolated by positional cloning, it encodes a voltage-gated potassium channel, and mutations in the gene cause the cardiac rhythm disorder long QT syndrome (Wang et al., 1996). We previously isolated three exons of KyLQT1 and determined that the gene spans 350 kb and encompasses five BWS translocation breakpoints. We also determined that KyLQT1 is im- printed, consistent with a role in BWS (Lee et al., 1997a). The transcriptional orientation of the KyLQT1 gene is from telomere to centromere (Lee et al., 1997a). In the current study, we found that the tail to tail distance between the p57KIP2 and KyLQT1 genes is 40 kb (Fig. 1). We also identified and isolated four cDNA clones embedded within introns of KyLQT1 (data not shown).
Transmembrane protein target for anti-proliferative antibody (TAPA-1). TAPA-1 was originally isolated as a target for an antiproliferative monoclonal antibody raised against a human B cell lymphoma, and it was mapped cytogenetically to 11p15 (Oren et al., 1990). Using published sequence data, we designed PCR primers to map TAPA-1 initially to the tumor-sup- pressing STF, and we then precisely localized it to the overlapping region of two BAC clones, 161F14 and 318P18. TAPA-1 was telomeric to KyLQT1, with the distance between two genes less than 100 kb (Fig. 1).
Human achaete-scute homolog 2 (ASCL2). A hu- man homologue of mouse achaete-scute homologue 2 (MASH2) was mapped earlier cytogenetically to 11p15 (Miyamoto et al., 1996). We isolated cosmid 23e5 from a flow-sorted human chromosome 11 li- brary (Heding et al., 1992), using the mouse gene MASH2 (Johnson et al., 1990) as a probe. We isolated from cosmid 23e5 three trapped exons, which were further mapped within 318P18, but not 161F14, indi-
A
AATTCGGCACGAGAAGACTTCCAGTTTGGAGTCGTTTGCTGCGGGGAGGGAATGAATGGG CGCTGGGAACACGCCCGCGAGGTGGGGACGCGCCGGCCGTAGCGAGGTCCTTAGCGTGTG AGTGGCCGGGGTCGGGTCGCTTCCCCGCAGCATGGAGGACGATGCACCAGTGATCTACGG
| MEDDAPVIYG | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| GCTGGAGTTCCAGGCACGTGCCTTAACACCTCAAACTGCAGAAACAGATGCCATTCGGTT | |||||||||||||
| LEFQARALTPQTAETDAIRF | |||||||||||||
| TTTGGTTGGGACGCAGTCTCTTAAATATGATAATCAGATCCATATCATAGATTTTGACGA | |||||||||||||
| LVGTQSLKY | DNQIHI | IDF DD | |||||||||||
| TGAAAACAACATTATAAATAAAAATGTCCTCCTCCATCAAGCGGGTGAAATCTGGCATAT | |||||||||||||
| ENNIINKNVLLHQAGEI | WHI | ||||||||||||
| TAGCGCTAGCCCTGCAGACAGAGGTGTGCTGACGACCTGCTACAACAGAACTTCAGACAG | |||||||||||||
| SASPADRGVLTTCYNRTSDS | |||||||||||||
| CAAAGTCCTGACATGTGCAGCCGTGTGGAGGATGCCGAAGGAATTGGAATCAGGCAGCCA | |||||||||||||
| KVLTCAAV | WRMPKELESGSH | ||||||||||||
| CGAGTCCCCTGATGATTCATCCAGCACTGCACAGACCCTGGAGCTGCTCTGTCACCTTGA | |||||||||||||
| ESPDDSSSTAQTLELL | CHLD | ||||||||||||
| CAACACAGCCCATGGCAACATGGCCTGTGTCGTGTGGGAGCCAATGGGAGATGGGAAGAA | |||||||||||||
| T | M | ||||||||||||
| AATCATTTCCTTGGCTGATAACCATATCCTGCTGTGGGATTTACAGGAAAGCTCGAGCCA | |||||||||||||
| I ISLA | DNHILL | WDL | Q | ESSS Q | |||||||||
| GGCTGTGCTGGCCAGCTCAGCGTCCCTGGAAGGGAAGGGACAACTGAAGTTCACCTCAGG | |||||||||||||
| AVLASSASLEGKG | QL | KFTSG | |||||||||||
| ACGGTGGAGCCCACATCATAACTGCACCCAGGTGGCCACAGCGAACGACACCACCCTCCG | |||||||||||||
| RWSPHHNCTQVATANDTTLR | |||||||||||||
| TGGCTGGGACACCCGGAGCATGAGCCAGATCTACTGCATAGAGAATGCCCACGGACAGCT | |||||||||||||
| GWDTR | SMS | Q | IY | CIENAHGQL | |||||||||
| GGTGCGGGACCTTGACTTTAATCCCAATAAGCAGTACTACTTGGCCAGCTGCGGAGACGA | |||||||||||||
| VRDLDF | NPNKQYY | LAS | CGDD | ||||||||||
| CTGTAAGGTGAAGTTCTGGGACACCCGAAATGTCACCGAACCCGTGAAGACCCTGGAGGA | |||||||||||||
| CKVKFWDTRNVTEPVKTL | |||||||||||||
| GCACTCCCACTGGGTGTGGAACGTCCGCTACAACCACTCTCATGACCAGCTGGTCCTCAC | |||||||||||||
| H | |||||||||||||
| SHWVWNVRYNHSHDQLVL GGGCAGCAGTGACAGCAGAGTCATCCTTTCCAACATGGTGTCCATCTCGTCGGAGCCCTT | |||||||||||||
| SSDSRVILSNMVSISSEPF CGGCCACTTGGTAGACGACGATGACATCAGTGACCAGGAGGACCACCGTTCTGAAGAGAA | |||||||||||||
| GHLVDD | DDIS | D | QEDHRSEEK | ||||||||||
| GAGCAAGGAGCCCCTGCAGGACAACGTGATCGCCACCTACGAGGAGCACGAGGACAGCGT | |||||||||||||
| SKEPLQDNVIATYEEHEDS V | |||||||||||||
| CTATGCCGTGGACTGGTCCTCGGCTGACCCGTGGCTGTTTGCCTCCCTGAGCTATGACGG | |||||||||||||
| YAVDWS | SADPWLFASLSYDG | ||||||||||||
| GAGGCTCGTGATCAACAGGGTGCCCAGGGCCCTGAAGTACCACATCCTGCTATGACTCCC | |||||||||||||
| RLVINRVPRALKYHILL* | |||||||||||||
| GGGCCTGGGTTATCCAGGTCCCATTGAGTGGTTTTCCTCTTGGCAGATTCTCAAACAGTC GCAGCTCTTTGGAGGTGACTCGTGTTCCAGGTGGATCCCTCTCTGGGAGAGCCGCTGTTC CCTTCCTGTAGCAGCAGCATTTATGAATGGGGTGAATGGGGCTATTGTCGACGGCACAGC TAATGCCCGAACCCAGCCCCTGTCGGCAGAGACAGAGCCCCACATTATTATGTGAATAAC AATGTTTTCTGTTTTAAGGGTGTCAGGAGTTTCGCTTTTTAAAAAAATGTCTGTTCCTGC AGTAGTAACTCTTCTTTCTCTTGAGAGTAAAAAATGAAATAAAATAAATCCACGCTGACA | |||||||||||||
| AAAAAAAAAAAAAAAAAAAAAAAAA | |||||||||||||
| B | ||
| TSSC1 | 142 | WEPMGDGKKIISLADNHILLWD 163 W P G + + D+ I LWD |
| RbAp48 | 185 | WNPNLSGHLLSASDDHTICLWD 206 |
| TSSC1 | 192 | WSPHHNCTQVATANDTTLRGWDTRS 216 W H + A+D L WDTRS |
| RbAp48 | 235 | WHLLHESLFGSVADDQKLMIWDTRS 259 |
| TSSC1 | ||
| RbAp48 | ||
| TSSC1 | 346 | HEDSVYAVDWSSADPWLFASLSYD 369 H + W+ +PW+ S+S D |
| RbAp48 | 373 | HTAKISDFSWNPNEPWVICSVSED 396 |
225 NAHGQLVRDLDFNPNKQYYLASCGDDCKVKFWDTRNVTEPVKTLEEHSHWVWNVRYNHSHDQLVLTGSSDSRVILSNMVSISSE 308 +AH V L FNP ++ LA+ D V WD RN+ + + E H ++ V+++ ++ ++ + +D R+ + ++ I E 270 DAHTAEVNCLSFNPYSEFILATGSADKTVALWDLRNLKLKLHSFESHKDEIFQVQWSPHNETILASSGTDRRLNVWDLSKIGEE 353
FIG. 2. Sequence of TSSC1 and homology to RbAp48. (A) Nucleotide and predicted amino acid sequences of TSSC1. The cDNA of TSSC1 contained 1705 bp. The initiation methionine is at nucleotide 152, and the stop codon is at nucleotide 1315. The open reading frame contains 387 amino acids. Underlined sequences indicate three exon-trapped sequences. One exon is from 808 to 972, and the other exon is from 973 to 1140. (B) Comparison of amino acid sequences of TSSC1 and RbAp48 (shown) and RbAp46 (CAF1, not shown). + denotes conservative amino acid substitutions. TSSC1 shows 30% identity to RbAp48, and the homology is more extensive in the C-terminal region. Comparison was done using BLASTP.
| TSSC2 | 2 NAMREDLADNWHIRAVTVYDKP 67 NAMR DL D W IRA T YD+P | |
| ß-mannosyl transferase | 194 NAMRRDLMDRWGIRASTFYDRP 215 | |
| TSSC2 | 262 TSWTEDEDFSILLAALEKFEQ 324 TSWT DE F ILL AL +++ | |
| ß-mannosyl transferase | 271 TSWTPDERFEILLDALVAYDK 291 | |
| TSSC2 | 346 LPSLVCVITGKGPLREYYSRLIHQKHFQHIQVCTPWLEARTTP 474 LP ++ +ITGKGPL+ Y + IH+K+ +++ V TPWLEA P | |
| ß-mannosyl transferase | 295 LPRVLMIITGKGPLKAKYLQEIHEKNLKNVDVLTPWLEAEDYP 337 | |
| TSSC2 | 494 LGVCLHTSSSGLDLPMKVVDMFGCCLPVCAVNFKCLHELVKHEENGLVFEDSEELA 661 LG+ LHTS+SGLDLPMKVVDMFG +P A+ FKC+ ELV+ + NG +F+DSE+L+ | |
| ß-mannosyl transferase | 345 | LGISLHTSTSGLDLPMKVVDMFGAKVPALALKFKCIDELVEEKTNGYLFDDSEQLS 400 |
cating that the gene is telomeric to TAPA-1. The esti- mated distance between TAPA-1 and ASCL2 is less than 100 kb (Fig. 1).
Novel Genes
In addition to the nine known genes described pre- viously, we have identified and isolated more than 20 cDNAs from this region, and we have obtained full- length sequence of the coding region of 2 of them and partial sequence for 1 cDNA, and these three novel genes are described below:
Tumor-suppressing STF cDNA 1 (TSSC1). We ini- tially identified two ESTs, HIBBC84 and 307785, con- taining exons N176B9 and N176B45, respectively, by exon trapping from cosmids derived from YAC B176E9. We completely sequenced these two cDNA clones, which indicated that the two cDNAs overlapped and that HIBBC84 contained the polyadenylation site. A third exon, 176A, was also found within 307785. Addi- tional BLAST search of dbEST identified a third EST, 53159, which extended the sequence toward the 5’ end (Fig. 2A). The initiation methionine is located at nucle- otide 152, and the flanking sequences match the Kozak consensus sequence. An in-frame stop codon was found in the 5’ untranslated region, indicating that the cod- ing sequence begins at nucleotide 152. The complete sequence of this gene revealed that it encodes a pre- dicted protein of 387 amino acids (Fig. 2A) and that it shows homology to tumor-suppressor Rb-associated protein p48 (RbAp48) (Qian et al., 1993) (Fig. 2B, P = 10-10) and Drosophila chromatin assembly factor 1 (CAF1, P = 10-1º) (Tyler et al., 1996). TSSC1 was lo- cated between GOK and Nup98 (Fig. 1).
Tumor-suppressing STF cDNA2 (TSSC2). We iso- lated three exons from cosmids derived from YAC B215A11. BLAST search identified the THC 195102, which showed homology to Caenorhabditis elegans ß- mannosyl transferase (P = 10-52) (Fig. 3). TSSC2 was located between Nup98 and CARS (Fig. 1). We are in the process of isolating a full-length cDNA for this gene.
Tumor-suppressing STF cDNA 3 (TSSC3). We identified an EST, 131507, containing exon 3952 trapped from cosmid 395, located between hNAP2 and p57KIP2. Sequencing of 131507 indicated the presence of a polyadenylation site. Additional BLAST search identified a second EST, 212115, which extended the cDNA sequence toward the 5’ end. Sequence analysis revealed that TSSC3 encodes a predicted protein of 152 amino acids (Fig. 4A) and that it shows strong homol- ogy to mouse TDAG51 (Fig. 4B, P = 10-32). TDAG51 upregulates Fas and FasL and thereby causes apoptosis of T cell hybridoma cells (Park et al., 1996). TSSC3 was located about 15 kb telomeric to hNAP2, with its transcriptional orientation from centromere to telomere (Fig. 1).
DISCUSSION
We have established a complete 2.5-Mb genomic con- tig consisting of PAC, P1, BAC, and cosmid clones that encompass the entire region spanning an embryonal tumor-suppressor gene, the location of which was de- fined by functional complementation with a STF. We then isolated more than 200 unique exons from these genomic clones, and these reagents enabled us to iden- tify and isolate known and novel genes. These include nine previously identified genes that we have now mapped precisely within this interval. The nine known genes identified within the STF are RRM1, GOK, Nup98, CARS, hNAP2, p57KIP2, KyLQT1, TAPA-1, and ASCL2. Of these genes, six were not previously known to lie within the interval defined by the tumor-sup- pressing STF. We have now physically mapped these genes. Nup98 was mapped to within 300 kb telomeric to RRM1. CARS was mapped 40 kb centromeric to hNAP2. TAPA-1 was mapped within 100 kb telomeric to KyLQT1. ASCL2 was mapped between TAPA-1 and TH, which lie within 200 kb of each other. We were also able to determine the transcriptional orientation of CARS (Cen to Tel), hNAP2 (Cen to Tel), p57KIP2 (Cen to Tel), and KyLQT1 (Tel to Cen).
A CCCGCGCTCGGCACGACATGAAATCCCCCGACGAGGTGCTACGCGAGGGCGAGTTGGAGA MKSPDEVLREGELE
AGCGCAGCGACAGCCTCTTCCAGCTATGGAAGAAGAAGCGCGGGGTGCTCACCTCCGACC KRSDSLF QL WKKKRGVLTSD GCCTGAGCCTGTTCCCCGCCAGCCCCCGCGCGCGCCCCAAGGAGCTGCGCTTCCACTCCA RLSLF PASPRAR PKELRF HS TCCTCAAGGTGGACTGCGTGGAGCGCACGGGCAAGTACGTGTACTTCACCATCGTCACCA ILKVDCVERTGKY VYFTIVT CCGACCACAAGGAGATCGACTTCCGCTGCGCGGGCGAGAGCTGCTGGAACGCGGCCATCG
TDHKEIDFR CAGES CWNAAI CGCTGGCGCTCATCGATTTCCAGAACCGCCGCGCCCTGCAGGACTTTCGCAGCCGCCAGG ALALI DF Q NR RAL QD FR SRQ AACGCACCGCACCCGCCGCACCCGCCGAGGACGCCGTGGCTGCCGCGGCCGCCGCACCCT ER TAPA A PAE DAVAAAAAAP CCGAGCCCTCGGAGCCCTCCAGGCCATCCCCGCAGCCCAAACCCCGCACGCCATGAGCCC SEP SE PS R PS PQ PKPRTP *
GCCGCGGGCCATACGCTGGACGAGTCGGACCGAGGCTAGGACGTGGCCGGCGCTCTCCAG CCCTGCAGCAGAAGAACTTCCCGTGCGCGCGGATCCTCGCTCCGTTGCACGGGCGCCTTA AGTTATTGGACTATCTAATATCTATGTATTTATTTCGCTGGTTCTTTGTAGTCACATATT TTATAGTCTTAATATCTTGTTTTTGCATCACTGTGCCCATTGCAAATAAATCACTTGGCC AGTTTGCTTTTCTAAAAAAAAAAAAAAAAAAAAA
| B TSSC3 | 6 EVLREGELEKRSDSLFQLWKKKRGVLTSDRLSLFP 40 + L+EG LEKRSD L QLWKKK +LT + L L P |
|---|---|
| TDAG51 | 8 KALKEGVLEKRSDGLLQLWKKKCCILTEEGLLLIP 42 |
| TSSC3 | 43 PRARPKELRFHSILKVDCVERTGKYVYFTIVTTDHKEIDFRCAGESCWNAAIALALIDFQNRRAL 107 P + KEL F ++ VDCVER GKY+YFT+V T+ KEIDFRC + WNA I L ++ ++NR+A+ |
| TDAG51 | 75 PPVKLKELHFSNMKTVDCVERKGKYMYFTVVMTEGKEIDFRCPQDQGWNAEITLQMVQYKNRQAI 139 |
FIG. 4. Sequence of TSSC3 and homology to mouse TDAG51. (A) Nucleotide and predicted amino acid sequences of TSSC3. The cDNA of TSSC3 contains 754 bp. The initiation methionine is at nucleotide 476, and the stop codon is at nucleotide 476. The open reading frame encodes 152 amino acids. Underlined nucleotides indicate the trapped exon 3952 sequence. (B) Comparison of amino acid sequences of TSSC3 and mouse TDAG51. + denotes conservative amino acid substitutions. TSSC3 shows 55% identity to mouse TDAG51. Comparison was done using BLASTP.
We have also isolated more than 20 novel cDNAs within this 2.5-Mb genomic contig. Of these, we have obtained the full-length coding sequence of two novel genes and partial sequence of a third, which are pre- sented here. Two of the novel genes, TSSC1 and TSSC3, are particularly interesting, as sequence analy- sis suggests a potential role for both genes in tumori- genesis. TSSC1 shows homology to RbAp48 (Fig. 2B), which is one of the major proteins associated with the tumor-suppressor Rb (Qian et al., 1993). RbAp48 was also recently found to form a tight complex with histone deacetylase (Taunton et al., 1996). Acetylation and deacetylation of lysine residues of histones regulates transcription through interaction between positive charges of histones and negatively charged DNA (Pazin and Kadonaga, 1997). TSSC1 also shares significant homology with chromatin assembly factor 1 (CAF1) (Tyler et al., 1996), which is identical to RbAp46, a protein closely related to RbAp48 (Verreault et al., 1996), and CAF1 stimulates DNA replication-depen- dent nucleosome assembly (Stillman, 1986). Given the homology of TSSC1 to RbAp48 and CAF1, TSSC1 likely also plays a role in gene silencing, and TSSC1 may serve as a growth suppressor of one or more tumor types that undergo LOH of 11p15.
TSSC3 is also particularly interesting, as it shares extensive homology to mouse TDAG51 (Fig. 4B), which
is known to induce Fas expression and Fas-mediated apoptosis of T cell hybridoma cells (Park et al., 1996). Breast cancer lines are defective in Fas-mediated apoptosis and Fas expression (Keane et al., 1996). Thus, TSSC3 is a candidate tumor-suppressor gene in this region, particularly for breast cancer, which shows LOH of 11p15.5 in half of advanced tumors (Winqvist et al., 1995).
In summary, identification of individual genes and/ or mapping of already known genes to the general re- gion of 11p15 has attracted considerable attention be- cause of the biological importance of this region to hu- man disease (Parker et al., 1996, Hu et al., 1996; Ma- tsuoka et al., 1995; Lee et al., 1995; Oren et al., 1990; Alders et al., 1997; Hannigan et al., 1997). The work described here should contribute significantly to these efforts, as we have precisely localized nine known genes within the critical region involved in at least one type of human tumor, and we also report three novel genes within the critical region, two of which are promising candidates for further investigation.
ACKNOWLEDGMENTS
This work was supported by NIH Grant CA54358. We thank Paul Nisson (BRL) for providing the pSPL3B and pSPL3B-CAM exon trap- ping vectors to the Feinberg laboratory, David Law for providing YAC B176E9, Jason Ravenel and Shoshana Levy for helpful discus-
sion, and Jolene Patey for preparing the manuscript. TSSC1 and TSSC3 have been deposited with GenBank under accession numbers AF019952 and AF019953, respectively.
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