A physical map of a 1.3-Mb region on the long arm of chromosome 12, spanning the GLI and LRP loci

GENOMICS 14, 117-120 (1992)

A Physical Map of a 1.3-Mb Region on the Long Arm of Chromosome 1 2, Spanning the GLI and LRP Loci ANNE FORUS A N D OLA MYKLEBOST1 Department of Tumor Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Os/o, Norway Received December 2, 1991; revised May 5, 1992

W e h a v e u s e d p u l s e d - f i e l d g e l e l e c t r o p h o r e s i s to c o n struct a long-range restriction map spanning more t h a n 1 . 3 m i l l i o n bp o f t h e q 1 3 - q 1 4 s e g m e n t o f c h r o m o some 12. Within this region lie the genes coding for the glioncogene a n d t h e l o w - d e n s i t y l i p o p r o t e i n r e c e p t o r related protein (LRP). The distance between the genes is a b o u t 2 0 0 - 3 0 0 k b . W e a l s o o b s e r v e a m e t h y l a t i o n f r e e i s l a n d 3' to t h e L R P g e n e . © 1992 AcademicPress, Inc.

INTRODUCTION Aberrations of the segment q13-q15 on chromosome 12 have been found in several tumor types. Notable are benign lipomas (Mandahl et al., 1987) and (malignant) myxoid liposarcomas (Turc-Carel et al., 1986), as well as uterine leiomyomas (Helm et al., 1988; Turc-Carel et al., 1988a) and pleomorphic adenomas of the salivary gland (Bullerdiek et al., 1987). The most specific of these rearrangements are those found in the myxoid liposarcomas, where the distal part of chromosome 12 (ql3-qter) is reciprocally translocated to 16p11. In the benign lipomas, on the other hand, the distal part of the long arm of chromosome 12 is translocated to various other chromosomes. Several putative oncogenes have been localized to the 12q13-q14 region and thus could be candidate target genes for the rearrangements. The G L I gene, situated in this region and coding for a zinc finger protein, was initially cloned because of its amplification in a glioma (Kinzler et al., 1987; Ruppert et al., 1988). Such amplification of G L I seems to be a rare event, but it has been infrequently observed also in childhood sarcomas (Roberts et al., 1989). The importance of these amplifications for cancer development or progression is at present unclear. Similar to the ras oncogene, gli has been shown to transform cells in cooperation with adenovirus E I A (Ruppert et al., 1991). It has been reported that the chromosomal region surrounding the G L I gene contains methylation sites specific for myxoid liposarcomas and that the translocation breakpoint in liposarcomas may be close to G L I (Paulien et al., 1990). 1 To whom correspondence should be addressed.

Another interesting gene found in this region is that coding for the "low-density lipoprotein receptor-related protein" or L R P (Herz et al., 1988; Myklebost et al., 1989). L R P has strong structural homologies to the LDL receptor and resembles four copies of the LDL receptor joined head to tail. The different structural motifs of L R P (and the LDL receptor) are homologous to motifs found in complement proteins, various growth factors, and proteins of the extracellular matrix (Herz et al., 1988; Bevilacqua et al., 1989; Noonan et al., 1991; Kallunki and Tryggvason, 1992). One of the segments with homology to epidermal growth factor (EGF) and other growth factors is located at the same distance from the cell surface as EGF in its precursor molecule (Herz et al., 1988). Because L R P is processed proteolytically (Lund et al., 1989; Herz et al., 1990), it is conceivable that a growth factor could be released in certain circumstances, in a way similar to that for EGF. There is good evidence that L R P is an apolipoprotein E receptor, although its physiological role in lipid metabolism is at present unclear (for review, see Brown et al., 1991). L R P seems to be bifunctional, as it is also responsible for the internalization and subsequent degradation of complexes of the protease inhibitor a2-macroglobulin and inactivated proteases (Kristensen et al., 1990; Strickland et al., 1990). MATERIALS AND METHODS D N A preparation. White blood cells, prepared after osmotic lysis of erythrocytes (Rogne et al., 1987), or tumor cells, recovered from cell cultures by trypsinization, were suspended in SE (isotonic saline with 25 m M EDTA) at a concentration of 3.3 X 107 ml, diluted rapidly 14 with 1% low-melt agarose (Bio-Rad) in SE (42°C), and cast into rectangular agarose blocks. After brief hardening of the agarose at +4°C, the blocks were ejected into lysis buffer (250 m M EDTA, pH 8, 1% Sarkosyl) and after complete cell lysis (clearing of the blocks), excess lysis buffer was removed and the blocks were incubated for at least 4 h at 42°C with 0.5 mg/ml proteinase K (Boehringer-Mannheim). Subsequently, the blocks were washed extensively with T E / S D S (10 m M Tris, pH 7.6, 1 m M EDTA, 0.1% SDS) and finally in TE (10 m M Tris, pH 7.6, 1 m M EDTA). Blocks of DNA from sperm cells were prepared as above for somatic cells, except that the proteinase K digestion was carried out in the presence of 20 m M ~-mercaptoethanol. Marker yeast chromosomes were prepared from the Saccharomuces cerevisiae strain YPH148 kindly provided by Dr. P. Hieter. In this i17

0888-7543/92 $5.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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strain, chromosome XV is broken into two fragments of 92 and 1025 kb, each containing sequences hybridizing to pBR322 and therefore providing convenient hybridization signals for alignment of autoradiograms and molecular weight determination (Fig. 2). Yeast cells were grown in YEP medium (1% yeast extract, 2 % peptone, 4% glucose, pH 5.0-5.5) at 30°C for about 24 h, washed in CPE buffer (120 m M N a 2 H P Q , 40 m M citrate, 20 m M EDTA), and resuspended in CPES (CPE + 1.2 m M sorbitol) at 1 X 109 cells/ml. The yeast suspension was mixed 1:1 with 1% low-melt agarose/CPE and cast into blocks. After hardening, the blocks were incubated overnight at 30°C with 100 U/ml zymolyase (Kyrin Breweries). Concatemers of bacteriophage X DNA were prepared from intact X particles. Bacteriophages from crude lysates of the temperature-sensirive Escherichia coli lysogen HB129X were mixed with polyethylene glycol, pelleted by standard techniques (Maniatis et aI., 1982), resuspended to a concentration corresponding to 100 #g/ml, mixed with L M T agarose, cast into blocks, and digested with proteinase as described above. Annealing to concatemers was carried out for several days in 2X SSC, 10 m M EDTA, 0.1% SDS, at room temperature. Pulsed-field gel analysis. Agarose blocks containing h u m a n genomic DNA were preequilibrated with 500 ttl of 1X restriction enzyme buffer for 30 min at 37°C and for another 15 min in a minimal volume (about 100 ~1 + block volume) of the same buffer with 100 ~g of nuclease-free bovine serum albumin before 20-30 U of the appropriate restriction enzyme (Promega or New England Biolabs) was added. Digestion was carried out for 5 h to overnight in a 37°C incubator (or 42°C for thermophilic enzymes because the blocks are made of lowmelt agarose). DNA-containing blocks were inserted into the slots of 1% agarose gels in 0.25x T B E (22.5 m M Tris-borate, 0.5 m M EDTA) and electrophoresed under various conditions in a Biometra Rothaphor programmable electrophoresis apparatus. Typical conditions giving good separation of fragments from a few kilobases to about 1 Mb are ramped pulse times from 5 to 90 s, voltage ramped from 120 to 180 V, and 110-125 ° angle between electrode positions for 37 h at 13°C. Gels were stained for 30 min with ethidium bromide (0.5 #g/ml) in H20, photographed, illuminated for 2 min on a 302-nm UV transilluminator, denatured for 4-5 h in 0.5 M NaOH/1.5 M NaC1, and blotted overnight by capillary action onto nylon filters (BioTrace, Bio-Rad, or Amersham) in the same solution. Blots were neutralized in 50 m M phosphate, pH 7.2, and baked for 2 h at 80°C before UV irradiation for 5 rain under a germicidal lamp (260 nm) at about 50 # W / c m 2. Probes. The genomic probe for GLI, pKK36P1 (which hybridizes to gli mRNA), and flanking probe pKK380 were kindly provided by Drs. Kinzler and Vogelstein (Kinzler et al., 1987). As probes for L R P either some of the partial cDNA clones (Herz et al., 1988) or a complete 15-kb cDNA insert, joined together from the original partial clones, were used.

RESULTS

Figure la shows the hybridization patterns obtained with probes for LRP and GLI when genomic samples were cut incompletely with the rare-cutting restriction enzyme Csp45I (an isoschizomer of SfuI, recognition sequence TTCGAA). In completely digested samples both probes hybridize to a Csp45I fragment of about 340 kb, corresponding to the strongest band in Fig. la, lane 1. As can be seen (although weakly in Fig. la), identical additional bands of partially cut DNA fragments could be obtained with both probes when leukocyte DNA was cut with this enzyme. It is therefore very unlikely that the probes hybridize to different Csp45I fragments that by chance migrate at identical positions during electrophoresis. Similarly, incomplete digestion with NruI gives a partially cut band of 800 kb with both probes (Fig. lb).

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F I G . 1. Hybridization of genomic DNA samples separated by pulsed-field gel electrophoresis to probes for GLI and GRP. (a) Identical samples cut partially with Csp45I (lane 1) or completely with MluI (lane 2), blotted, and hybridized to the probes for GLI (left) and subsequently L R P (right). The molecular weights of the completely cut fragments hybridizing to the probes were 340 and 100 kb for GLI and 340 and 150 kb for LRP. (b) The sample was cut incompletely with the rare-cutting enzyme NruI, blotted, and hybridized to the GLI probe (left) and subsequently to the L R P probe (right). The molecular weights of the completely cut fragments were 300 and 500 kb, and for the common, partially cut fragment 800 kb. For both gels the pulse times were increased linearly from 5 to 90 s, and the gels were run for 37 h, giving separation from 5 to 1000 kb below the compression zone.

Most restriction enzymes used in pulsed-field gradient electrophoresis (PFGE) analysis are inhibited by methylation of their recognition sequence. This complicates analysis in different cell or tissue types, but can also be used to confirm the identity of restriction fragments detected by different probes. In DNA from sperm cells, probes for GLI and LRP hybridize to a 500-kb fragment (Fig. 2). Presumably this is because one or both sites defining the 340-kb Csp45I fragment in leukocytes are methylated. We similarly find that in some tumor cell lines in which the LRP probe hybridizes to Csp45I fragments varying between 430 and 600 kb, the GLI probe always hybridizes to Csp45I fragments of the same size (not shown). A long-range restriction map can be constructed from the fragment patterns obtained by sequential hybridization of our pulsed-field blots with probes for the GLI locus (pKK36P1 or the flanking genomic probe pKK380) and various parts of the LRP cDNA. Fig. 3 shows this map. DISCUSSION

By using PFGE and Southern blotting techniques, we have constructed long-range restriction maps covering parts of the q13-q14 segment of chromosome 12. This segment is involved in genomic rearrangements in several types of cancer and is of great interest in our effort to characterize the biology of liposarcomas.

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M A P OF C H R O M O S O M E 12q13-q14

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We have found the L R P gene to be amplified together with GLI in a rhabdomyosarcoma cell line (Forus et al., 1991). However, lack of amplification of L R P in another tumor cell line with GLI amplification suggested that L R P is not critical for the possible oncogenic consequences of the amplifications. On the other hand, the coamplification indicated that the genes might be localized close to each other. Of the many rare-cutting restriction enzymes used to construct long-range maps surrounding the two genes, only Csp45I gave genomic fragments of exactly the same size with probes from both loci. Because this is insufficient as proof for the two genes being colocalized on the same fragment, we followed two strategies to unequivocally connect the two restriction maps. The map presented in Fig. 3 was confirmed both by the partial digestion patterns of two different enzymes and by consistent variation in the size of the Csp45I fragments observed for probes for both loci in different cell types, probably because of variation of the methylation patterns. The evidence discussed above convincingly proves that the GLI and L R P genes are localized on the same 340-kb Csp45I fragment in leukocyte DNA, and thus the long-range restriction maps surrounding the two genes can be combined as shown in Fig. 3. The distance between the genes can be deduced from the map as between 220 and 330 kb. By PFGE mapping one cannot detect restriction fragments not hybridizing to any of the probes. The map will therefore be incomplete, as there may be additional restriction sites, e.g., for NotI, between the loci. A partial map of the region surrounding the GLI gene in adipocytes has been presented previously (Paulien et al., 1990). Although there are methylation differences, the similar sizes and consistent asymmetric orientation of NruI, SalI, and NotI fragments in both maps suggest that GLI has the same orientation as LRP. Interestingly, there seems to be a CpG-rich, methylation-free island about 100 kb 3' from the start of the L R P gene, resulting in frequent cuts by the rare-cutting

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~21~ F I G . 2. Hybridization p a t t e r n s of t h e GLI a n d L R P probes to genomic D N A samples from leukocytes and s p e r m cells. Samples from leukocytes (lane 2) a n d s p e r m cells (lane 3) from t h e same individual were cut with Csp45I, separated by P F G E , blotted, a n d hybridized to t h e GLI (left) and L R P probes ( r i g h t ) . T h e molecular weights of t h e hybridizing b a n d s were in b o t h cases 340 kb for leukocyte D N A (lane 2) and 500 kb for s p e r m D N A (lane 3). Lane 1 contains yeast m a r k e r c h r o m o s o m e s a n d shows background hybridization to t h e two p a r t s of t h e b r o k e n c h r o m o s o m e X V (molecular weights 92 a n d 1025 kb), hybridizing weakly to p B R - c o n t a i n i n g probes. T h e pulse time was increased linearly from 5 to 90 s, and t h e gel was run for 37 h, giving separation from 5 to 1000 kb below the compression zone.

LRP is closely related to the LDL receptor, but also contains structural motifs similar to those found in growth factors, extracellular matrix proteins, and complement components. Because L R P contains many repeated segments related to other proteins, the gene might be susceptible to unbalanced crossing over events resulting in translocations. Interestingly, in one lipoma, the distal part of chromosome 12q was found to be translocated to the p13.1 segment of chromosome 19 (TurcCarel et al., 1988b), where the gene for the closely related LDL receptor is located.

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F I G . 3. Long-range restriction map of t h e region surrounding t h e GLI and L R P genes. Horizontal arrows indicate the restriction fragments recognized by the probes. Vertical arrows and d a s h e d boxes indicate double-digestion fragments, a n d n u m b e r s indicate molecular weights in kilobases. T h e shaded areas indicate the m a x i m u m length of each gene.

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e n z y m e s . T h i s s u g g e s t s t h a t a t l e a s t o n e o t h e r g e n e is l o c a t e d b e t w e e n L R P a n d G L I , a n d we h o p e t o be able t o c h a r a c t e r i z e t h i s g e n e b y c l o n i n g p a r t s of t h e i n t e r g e n i c region. O u r m a p covers a g e n o m i c a r e a of m o r e t h a n 1.3 m i l l i o n b p a n d s h o u l d b e of v a l u e i n t h e f u r t h e r m a p p i n g of g e n e s localized to t h i s c h r o m o s o m a l s e g m e n t . H o w e v e r , as t h e size of a c h r o m o s o m a l b a n d p r o b a b l y is o n t h e o r d e r of t e n s of m e g a b a s e s , a c o n s i d e r a b l e effort is still r e q u i r e d for t h e c o n s t r u c t i o n of a c o m p l e t e p h y s i c a l m a p of t h e q 1 3 - q 1 4 s e g m e n t of c h r o m o s o m e 12.

ACKNOWLEDGMENTS This work was supported by the Norwegian Cancer Research Fund, the Norwegian Research Council for Science and the Humanities, and the University of Oslo. We are grateful to Dr. J. Herz for making his full-length construction of the LRP cDNA available to us.

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Kristensen, T., Moestrup, S. K., Gliemann, J., Bendtsen, L., Sand, O., and Sottrup-Jensen, L. (1990). Evidence that the newly cloned lowdensity-lipoproteinreceptor related protein (LRP) is the a2-macroglobulin receptor. F E B S Lett. 276: 151-155. Lund, H., Takahashi, K., Hamilton, R. L., and Havel, R. J. (1989). Lipoprotein binding and endosomal itinerary of the low density lipoprotein receptor-related protein in rat liver. Proc. Natl. Acad. Sci. USA 86: 9318-9322. Mandahl, N., Heim, S., Johansson, B., Bennet, K., Mertens, F., Olsson, G., R55ser, B., Rydholm, A., Will,n, H., and Mitelman, F. (1987). Lipomas have characteristic structural chromosomal rearrangements of 12q13-q14. Int. J. Cancer 39: 685-688. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Myklebost, O., Arheden, K., Rogne, S., Geurts van Kessel, A. H. M., Mandahl, N., Herz, J., Stanley, K. K., Heim, S., and Mitelman, F. (1989). The gene for the human putative apoE receptor is on chromosome 12 in the segment q13-q14. Genomics 5: 65-69. Noonan, D. M., Fulle, A., Valente, P., Cai, S., Horigan, E., Sasaki, M., Yamada, Y., and Hassell, J. R. (1991). The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor and the neural cell adhesion molecule. Genomics 11: 1170. Paulien, S., Turc-Carel, C., Cin, P. D., Jani-Sait, S., Sreekantaiah, C., Leong, S. P. L., Vogelstein, B., Kinzler, K. W., Sandberg, A. A., and Gemmill, R. M. (1990). Myxoid liposarcoma with t(12;16) (q13;p11) contains site-specific differences in methylation patterns surrounding a zinc-finger gene mapped to the breakpoint region on chromosome 12. Cancer Res. 50: 7902-7907. Roberts, W. M., Douglass, E. C., Peiper, S. C., Houghton, P. J., and Look, A. T. (1989). Amplification of the gli gene in childhood sarcomas. Cancer Res. 49: 5407-5413. Rogne, S., Skretting, G., Larsen, F., Myklebost, O., Mev~g, B., Carlson, L. A., Holmquist, L., Gjone, E., and Prydz, H. (1987). The isolation of a cDNA clone for human lecithin:cholesterol acyl transferase and its use to analyse the genes in patients with LCAT deficiency and fish eye disease. Biochem. Biophys. Res. Commun. 148: 161-169. Ruppert, J. M., Kinzler, K. W., Wong, A. J., Bigner, S. H., Kao, F-T., Law, M. L., Seuanez, H. N., O'Brien, S. J., and Vogelstein, B. (1988). The GLI-Kruppel family of human genes. Mol. Cell. Biol. 8: 3104-3113. Ruppert, J. M., Vogelstein, B., and Kinzler, K. W. (1991). The zinc finger protein GLI transforms primary cells in cooperation with adenovirus E1A. Mol. Cell. Biol. 11: 1724-1728. Strickland, D. K., Ashcom, J. D., Williams, S., Burgess, W. H., Migliorini, M., and Argraves, W. S. (1990). Sequence identity between the a2-macroglobulin receptor and low density lipoprotein receptorrelated protein suggests that this molecule is a multifunctional receptor. J. Biol. Chem. 265: 17401-17404. Turc-Carel, C., Limon, J., Dal Cin, P., Rao, U., Karakousis, C., and Sandberg, A. A. (1986). Cytogenetic studies of adipose tissue tumors. II. Recurrent reciprocal translocation t(12;16)(q13;pll) in myxoid liposarcomas. Cancer Genet. Cytogenet. 23: 291-299. Turc-Carel, C., Dal Cin, P., Boghosian, L., Terk-Zakarian, J., and Sandberg, A. A. (1988a). Consistent breakpoints in the region 14q22-q24 in uterine leiomyoma. Cancer Genet. Cytogenet. 32: 2531. Turc-Carel, C., Dal Cin, P., Boghosian, L., Leong, S. P. L., and Sandberg, A. A. (1988b). Breakpoints in benign lipoma may be at 12q13 or 12q14. Cancer Genet. Cytogenet. 36: 131-135.