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B in human brain tumors
Gene 255 (2000) 105–116 www.elsevier.com/locate/gene
Genomic organization, chromosomal localization and regulation of expression of the neuronal nuclear matrix protein NRP/B in human brain tumors k Tae-Aug Kim 1,a, Setsuo Ota 1,a, Shuxian Jiang a, Linda M. Pasztor b, Robert A. White b, Shalom Avraham a, * a Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115, USA b Section of Medical Genetics and Molecular Medicine, Children’s Mercy Hospital, UMKC School of Medicine, Kansas City, MO 64108, USA Received 3 April 2000; received in revised form 6 June 2000; accepted 29 June 2000 Received by J.A. Engler
Abstract The nuclear matrix and its role in cell physiology are largely unknown, and the discovery of any matrix constituent whose expression is tissue- and/or cell-specific offers a new avenue of exploration. Studies of the novel neuronal nuclear matrix protein, NRP/B, reveal that it is an early and highly specific marker of neuronal induction and development in vertebrates, since its expression is restricted mainly to the developing and mature nervous system. These studies also show that NRP/B is involved in neuronal differentiation. To further examine the structure–function of NRP/B, we have cloned and characterized the murine Nrp/b gene. The murine gene consists of four exons interrupted by three introns that span 7.6 kb of DNA. The complete open reading frame is localized in exon 3, suggesting that NRP/B is highly conserved during evolution. Chromosomal analysis shows that NRP/B is localized to chromosome 13 in mouse and chromosome 5q12–13 in human. Since our previous studies demonstrated that NRP/B is expressed in primary hippocampal neurons but not in primary astrocytes, we have characterized NRP/B mRNA and protein expression in various brain cell lines and in human brain tumors. Abundant expression of NRP/B mRNA and protein was observed in human neuroblastoma cell lines (IMR32, SKN-MC, SKN-SH ), in glioblastoma cell lines (A172, T98G, U87-MG, U118-MG, U138-MG, and U373-MG), in neuroglioma (H4) and astrocytoma cell lines (CCF-STTG1 and SW1088). Confocal analysis of NRP/B in U87-MG glioblastoma cells indicated nuclear localization of NRP/B. NRP/B expression was also observed in human primary brain tumors including glioblastoma multiformae and astrocytomas (total of five cases). These results suggest that NRP/B expression is upregulated in human brain tumors including glioblastomas and astrocytomas, while under normal conditions NRP/B expression is restricted to neurons. This study implicates a role for NRP/B in brain tumor development. © 2000 Elsevier Science B.V. All rights reserved. Keywords: BTB/POZ domain; Chromosome 5q; Human brain tumor; Kelch motif; Nuclear matrix protein
Abbreviations: BTB, broad-complex tramtrack and bric-a-brac; CLSM, confocal laser scanning microscope; DMEM, Dulbecco’s modified Eagle’s medium; ENC-1, ectoderm-neural cortex-1; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC1, histone deacetylase-1; lapls 2-6, intracisternal A particle proviral element 2-6; LAZ3/BCL6, lymphoma-associated zinc finger 3/B cell; MGD, mouse genome database; N-COR, nuclear receptor co-repressor; NRP/B, nuclear restricted protein/brain; ORFs, open reading frames; POZ, poxvirus/zinc finger; REC, recombination frequency; RFLP, restriction fragment length polymorphism; RT–PCR, reverse transcriptase–polymerase chain reaction; NRP/B, nuclear restricted protein/brain; ORFs, open reading frames; POZ, poxvirus/zinc finger; REC, recombination frequency; RFLP, restriction fragment length polymorphism; RT–PCR, reverse transcriptase–polymerase chain reaction; SMRT, silencing mediator of retinoid and thyroid receptor. k This paper is dedicated to Raphael Recanati and his family for their friendship and support for our research program. * Corresponding author. Tel.: +1-617-667-0073; fax: +1-617-975-6373. E-mail address: [email protected] (S. Avraham) 1 Both authors contributed equally to this work. 0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 0 0 ) 0 0 29 7 - 3
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1. Introduction The nuclear matrix constitutes the three-dimensional filamentous protein network that maintains the domain organization of the nucleus. This component of the nuclear architecture provides the internal scaffold of the nucleus. It is formed by an ordered and highly compartmentalized protein structure consisting of a nuclear lamina, a residual nucleolus, and an internal matrix composed of a non-chromatin fibrogranular network associated with DNA (Berezney et al., 1996; Stein and Berezney, 1996). The nuclear matrix has been implicated in transcription, regulation of gene expression, the cell cycle, primary transcription processing and in linkages to intermediate filaments of the cytoskeleton (He et al., 1995; Loidl and Eberharter, 1995). A cellular hallmark of the transformed phenotype is an altered nuclear shape and the presence of abnormal nucleoli. Structural alterations of the nucleus are prevalent in cancer cells and are commonly used as pathological markers of transformation in many types of cancer. Nuclear shape is thought to reflect the internal nuclear structure and is determined, at least in part, by the nuclear matrix (Peinta et al., 1989; Dworetzky et al., 1990). The nuclear matrix contains a number of associated proteins that have been found to be involved in malignant transformation (Fey and Penman, 1984; Keese et al., 1994; Getzenberg et al., 1996). It has been directly or indirectly implicated in most of the changes considered to be pathologic hallmarks of malignant transformation, such as alterations in DNA ploidy, DNA content, nuclear shape and proliferative states (Berezney et al., 1995). Ataxin-1 (the protein encoded by the SCA1 gene), which is involved in the neurodegenerative disorder spinocerebellar ataxia, alters nuclear-matrix-associated structures (Skinner et al., 1997). Despite the apparent importance of the nuclear matrix in the regulation of many biological processes, its roles in cell physiology and in neuronal differentiation are largely unknown. Recently, we have discovered and characterized a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain-like structure in the predicted N-terminus, and a ‘kelch motif ’ in the predicted C-terminal domain ( Kim et al., 1998). NRP/B mRNA (5.5 kb) is expressed predominantly in human fetal and adult brain, with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. ENC-1, a mammalian kelch-related gene that is specifically expressed in the nervous system, was also reported to be the murine homolog of NRP/B (Hernandez et al., 1997; Kim et al., 1998). NRP/B/ENC-1 is expressed in the prospective neuroectodermal region of the epiblast during early gastrulation and throughout the nervous system
later in development. NRP/B/ENC-1 expression is highly dynamic and, after neurulation, preferentially defines prospective cortical areas. Expression of NRP/B/ENC-1 was detected at the preneurulation stage of the mouse embryo (E 6.5) in the prospective neuroectodermal region of the epiblast that later differentiates predominantly into neuroectodermal cells (Hernandez et al., 1997; Kim et al., 1998). No expression of NRP/B/ENC-1 was detected in any extraembryonic tissue. At E 8.0, its expression was detected in ectodermal derivatives and continued to be strongly expressed in the hippocampus and neocortex (Hernandez et al., 1997; Kim et al., 1998). The BTB/POZ domain at the N-terminus of NRP/B consists of approx. 115 amino acids and is expressed in several members of the kelch family ( Xue and Cooley, 1993). This domain, found primarily in zinc finger proteins, defines a newly characterized protein–protein interaction interface (Bardwell and Treisman, 1994), and also mediates both dimer and heterodimer formation in vitro (Albagli et al., 1995). NRP/B also shares significant homology to the ‘kelch’ repeats found in several kelch-related genes ( Xue and Cooley, 1993; von Bulow et al., 1995; Way et al., 1995) and in a large number of open reading frames (ORFs) within the genome of poxviruses (von Bulow et al., 1995; Way et al., 1995). NRP/B contains six repeats of kelch in the C-terminal half of the protein, while each repeat consists of about 50 amino acids. These motifs may have functional significance in binding actin, protein folding or in protein–protein interactions. We have shown previously that NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes, and that its expression is upregulated during the differentiation of rat PC12 cells, murine Neuro-2A and human SH-SY5Y neuroblastoma cells ( Kim et al., 1998). Overexpression of NRP/B in these cells augmented neuronal process formation, while treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as neuronal process formation during differentiation of PC12 cells. In this study, we aimed to characterize the genomic organization, chromosomal localization and expression of NRP/B in brain tumor cell lines and human primary brain tumors. We have determined the chromosomal assignment of the human NRP/B gene by analysis of somatic cell hybrids, and we have mapped the mouse NRP/B gene by backcross analysis. Our results suggest that NRP/B expression is upregulated in brain tumors.
2. Materials and methods 2.1. Materials Chemical reagents were purchased from Sigma (St. Louis, MO). The murine fetal l-gt10 cDNA library was
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obtained from Dr. Kunkle, Children’s Hospital, Boston, MA. Restriction endonucleases, modifying enzymes, terminal deoxynucleotidyl transferase, random priming kits, and Sephadex G-25 quickspin columns were purchased from Pharmacia-LKB (Piscataway, NJ ) and New England BioLabs (Beverly, MA). The primers for polymerase chain reaction (PCR), RT–PCR and sequencing were synthesized using an automated DNA synthesizer (Applied Biosystems, model 394). The PCR and RNA-PCR kits were obtained from Perkin-Elmer Cetus (Norwalk, CT ). Sequenase and random priming kits were obtained from US Biochemical Corp. (Cleveland, OH ) and RNA isolation kits were from Stratagene (La Jolla, CA). Human brain tumor tissues were obtained from Cooperative Human Tissue Network (CHTN ) (Philadelphia, PA). 2.2. Cell culture Primary neurons were prepared from the hippocampal regions of Sprague–Dawley rats at gestational day 18, and primary astrocytes from the cerebral cortex were prepared from postnatal day 1 rats as described ( Kim et al., 1998). Primary neurons were cultured in neurobasal medium (GIBCO) containing B-27 supplement without antibiotics. Culture of primary astrocytes was in DMEM supplemented with 10% heat-inactivated horse serum, glucose (6 mg/ml ) and 2 mM glutamine. After cultures reached confluence, the medium was changed to DMEM containing 10% FBS. Both types of primary cells were grown in poly--lysine-coated culture plates at a density of 1×105–1×106 cells per cm2. Neuroblastomas (IMR32, SKN-MC, SKN-SH ), neurogliomas (H4), glioblastomas (A172, T98G, U87-MG, U118-MG, U138-MG, U373-MG) and astrocytomas (CCF-STTG1, SW1088) were grown as described in the ATCC manual.
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2.4. Chromosomal localization of human NRP/B Genomic DNAs from NIGMS Hybrid Mapping Panel No. 2 as well as DNA from somatic cell hybrids NA11434, NA11437, and NA11443 were obtained from the NIGMS Genetic Mutant Cell Repository (Coriel Cell Institute for Medical Research, Camden, NJ ). Mapping Panel No. 2 consisted of DNA isolated from 24 human/rodent cell hybrids retaining one or two human chromosomes. All but two of the hybrids retained a single intact human chromosome. In addition, the mapping panel included DNA samples isolated from the human and rodent parental cell lines (mouse and Chinese hamster). Human/rodent cell hybrids NA11434, NA11437 and NA11443 contained Chinese hamster chromosomes and segments of Chromosome 5 generated by somatic cell fusion with cells containing translocations or deletions involving Chromosome 5. The DNA sample NA11437 contained a der(5)t(5;13) (5qter 5p13::13q13 13qter) and thus lacked the segment 5p14 5pter. NA11434 contained 5 qter 5q13.3::5q11.2 5pter and thus was missing 5q12 and 5q13. NA11443 contained del(5) (q15 q21.3), and thus 5q21 was absent. Approx. 5 mg of DNA from human, hamster and mouse genomic DNAs were digested with BamHI, HindIII and PstI to find a suitable RFLP or unique genomic fragment for use in mapping. Subsequently, genomic DNAs from each panel were cut with PstI. Southern blots were probed with a human 1.8 kb NRP/B cDNA, and hybridizations were carried out as previously described ( White et al., 1992). Hybrids were scored on the autoradiographs for the appropriate human-specific restriction endonuclease fragment. The results were compared with the chromosome contents of the hybrid cell lines. Concordance between restriction fragments and specific chromosomes or portions of Chromosome 5 was used to establish the chromosomal localization of NRP/B.
2.3. Chromosomal localization of Nrp/b in mouse 2.5. Western blot analysis Genomic DNAs from C57BL/6J, Mus spretus, and interspecific backcross panel (C57BL/6J M. spretus) F ×C57BL/6J (BSS panel ) were obtained from The 1 Jackson Laboratory (Rowe et al., 1994). Southern blots and hybridizations were carried out as previously described ( White et al., 1992). Approximately 5 mg of genomic DNAs from the C57BL/6J and M. spretus progenitors were digested with 28 different restriction enzymes to find a suitable restriction fragment length polymorphism (RFLP) for mapping. Southern blots were probed with a human 1.8 kb NRP/B cDNA. Approx. 2 mg of DNA from the BSS type backcross panel were digested for each sample with XbaI overnight. Segregation of Nrp/b alleles was compared with that of alleles at other loci from a mouse genome database (MGD) by the Jackson Laboratory Backcross DNA Map Panel Service (Rowe et al., 1994).
Total cell lysates were analyzed by SDS–PAGE and transferred to membranes. The blots were incubated at 4°C overnight in 5% milk in PBS containing 0.1% Tween 20, followed by incubation with NRP/B specific antibodies. The blots were washed and reacted with horseradish peroxidase-conjugated anti-rabbit IgG for 1 h. Immunoreactive bands were visualized using ECL reagents. 2.6. Immunolocalization of NRP/B protein by confocal microscopy Standard immunohistochemical methods were used as described ( Kim et al., 1998). Briefly, U87-MG astrocytoma/glioblastoma cells were seeded onto sterile 18-mm glass coverslips ( Fisher Scientific, Pittsburgh,
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PA) in 12-well plates at different densities. Cells were washed at room temperature in PBS for 2–3 min, fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.2% Triton-X 100 in PBS for 5 min, and blocked in 0.1% BSA and 3% normal goat serum in PBS for 1 h at room temperature or overnight at 4°C. Cells were incubated for 1 h with NRP/B in PBS containing 0.1% BSA and 3% normal goat serum, washed, and then incubated with 1:100 dilution of Texas Red-conjugated goat anti-rabbit IgG ( Vector Laboratories, Burlingame, CA). Immunostained cells were washed, mounted with ProLong antifade reagent (Molecular Probes, Eugene, OR), and examined using a Sarastro 2000 confocal laser scanning microscope (CLSM ) (Molecular Dynamics, Sunnyvale, CA) and Leica TCS-NT (Leica, Wetzlar, Germany) confocal laser-scanning microscope optimized for simultaneous dual fluorescent imaging. 2.7. Expression of NRP/B in human brain tumors (by reverse transcription polymerase chain reaction (RT–PCR) and Northern blot analysis) RNA extracted from human primary brain tumors and from the cultured cells (1×105 cells) was reverse transcribed at 42°C for 40 min in a final volume of 50 ml as described ( Kim et al., 1998). The 5∞- and 3∞-specific primers were added in final concentrations of 5 ng/50 ml each. The mixture was subjected to 30 amplification cycles using the Perkin-Elmer thermal cycler set as follows: denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min, extension at 72°C for 2 min. The sequence of the NRP/B upstream primer was 5∞-TCGAGGATCCATGCTCTTCCTTCTGGGAGGACAG-3∞ (corresponding to position +877 to +911 bp). The nucleotide sequence of the downstream primer was 5∞-ACGTCAAGCTTACGGTACAGTCCACCTGTTTTCAC-3∞ (corresponding to position +1789 to +1824 bp). Primers for the b-actin message were: upstream primer 5∞-ATG GAT GAT GAT ATC GCC GCG-3∞ and downstream primer 5∞-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG GCC-3∞. The amplification products and poly A mRNA obtained from the brain tumor cell lines were detected by overnight hybridization with an NRP/B, b-actin, or GAPDH probe. The PCR products and NRP/B mRNA level were analyzed as previously described ( Kim et al., 1998).
3. Results 3.1. Cloning, sequence analysis, and genomic organization of the murine Nrp/b gene We previously observed that Nrp/b is expressed in rat primary hippocampal neurons but not in astrocytes,
and that its level of expression is upregulated during neuronal differentiation ( Kim et al., 1998). To determine the exon–intron organization of the murine Nrp/b gene and to identify its potential tissue-specific response elements, we screened approx. 6×105 total recombinants, from a murine liver genomic library (c-EMBL-3), for genomic clones under conditions of high stringency with a 260 bp 32P-labeled 3∞ gene-specific fragment (A3∞) of the mouse Nrp/b cDNA ( Fig. 1). The human NRP/B/ENC-1 cDNA and the mouse Nrp/b cDNA have highly conserved sequences of more than 99% ( Kim et al., 1998; Hernandez et al., 1997). We isolated a 17 kb genomic DNA clone, termed l Dash-MG-Nrp/b-1. In addition, using probes derived from both the 5∞ and 3∞-ends of the mouse Nrp/b cDNA, we isolated an additional genomic DNA clone of 21 kb (termed l Dash-MG-Nrp/b-2) (Fig. 1A). A restriction map of each genomic clone was constructed by digesting the phage DNA with a panel of restriction enzymes either separately or in various combinations: SacI, BamHI, HindIII, XbaI, and EcoRI. The DNA blots were probed under conditions of high stringency with either the 403 bp 5∞A gene-specific fragment, or the 344 bp A3∞ gene-specific fragment of the mouse Nrp/b cDNA. In parallel, a blot was prepared of mouse liver DNA that had been digested with the same panel of restriction enzymes. When this DNA blot was probed with the 344 bp A3∞ gene-specific fragment or the 403 bp 5∞A gene-specific fragment of the mouse Nrp/b cDNA, the pattern of hybridization was identical to that obtained with l Dash-MG-Nrp/b-2 (data not shown), indicating that this clone probably contained the major part of the gene that encodes the mouse Nrp/b. The restriction enzyme map of the mouse Nrp/b gene was constructed and the nucleotide sequences of l DashMG-Nrp/b-1 and l Dash-MG-Nrp/b-2 were determined according to the strategy depicted in Fig. 1. Based on the nucleotide sequences of the genomic fragments analyzed, two oligonucleotides of 21 nucleotides in length were synthesized and used as primers to determine the contiguous nucleotide sequence of the next 200–250 nucleotides (in each direction) of the double-stranded DNA. No mismatches were found between the genomic sequences and the cDNA. The exon–intron organization of the mouse Nrp/b gene was determined by this approach. Based on the nucleotide sequences of its subcloned fragments of approx. 17 kb and approx. 21 kb, we determined that the mouse Nrp/b gene comprises four exons that span about 7.6 kb of DNA ( Fig. 1). The gene is approx. 1.7 kb from the putative transcription initiation site to the end of exon 2. Our genomic l Dash-MG-Nrp/b-2 clone contained an additional 3.3 kb of 5∞-flanking sequences and 7 kb of sequences downstream of exon 3. Exon 1 contained the 5∞-untranslated sequence (5∞-UT ) and exon 2 contained the putative
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Fig. 1. (A) Genomic organization of the murine Nrp/b gene. The l Dash-MG-Nrp/b-1 and l Dash-MG-Nrp/b-2 genomic clones were isolated from the mouse liver DNA library. The exons are shown in boxes and the dotted area shown in exon 2 contains the full-length coding region of the NRP/B sequences. The restriction enzymes are shown. (B) Southern blot of the genomic Nrp/b gene. Murine genomic DNA was digested with the indicated restriction enzymes and was analyzed by Southern blot analysis using a 3∞ gene-specific NRP/B probe.
translation initiation site. The exon–intron splice junctions were sequenced for each exon, and are in accordance with the Gt…At consensus sequence (Avraham et al., 1995). An additional 500 bp of 5∞-flanking sequence was determined. Southern blot analysis of mouse genomic DNA digested with several restriction enzymes and hybridized with an Nrp/b 3∞ gene-specific probe showed a single band, indicating the existence of Nrp/b as a single gene ( Fig. 1B).
3.2. Chromosomal mapping of mouse and human genes encoding NRP/B We determined the chromosomal location of the gene encoding NRP/B in mouse by analyzing the segregation of RFLP in DNAs derived from the offspring of The Jackson Laboratory (M. spretus×C57BL/6J ) F ×M. 1 spretus backcross. An XbaI RFLP for Nrp/b was identified by the presence of a 1.8 kb genomic DNA fragment
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Fig. 2. Mapping of mouse Nrp/b to Chromosome 13. XbaI restriction enzyme pattern for M. spretus (S) genomic DNAs and (C57BL/6J×M. spretus) F heterozygous (BS ) genomic DNAs probed with the 1.8 kb Nrp/b cDNA. The molecular size (in kb) of the Nrp/b RFLP fragment is 1 indicated. (B) Haplotype analysis of Chromosome 13 genetic markers in (C57BL/6J×M. spretus) F ×M. spretus (BSS type) backcross mice 1 showing linkage and the relative position of Nrp/b. Closed boxes indicate inheritance of the C57BL/6J (B) allele and open boxes indicate the inheritance of the M. spretus (S ) allele from the (C57BL/6J×M. spretus) F parent. Gene names and references to these loci can be found in 1 GBASE. The first two columns indicate the number of backcross progeny with no recombinations. The following columns indicate recombinational events between adjacent loci (signified by a change from open box to closed box). The number of recombinants are listed below each column and the recombination frequency (REC%) between adjacent loci is indicated.
in C57BL/6J or the absence of this fragment in M. spretus (Fig. 2A). This allele was characterized in 94 DNAs from the (C57BL/6J×M. spretus) F ×M. spretus 1 backcross panel. Haplotype analysis of these mapping
data ( Fig. 2B) indicated that the Nrp/b locus is closely linked to lapls 2-6 (intracisternal A particle proviral element 2-6) Chromosome 13 in mouse. Allelic segregation patterns for lapls 2-6 and Nrp/b were identical
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(no recombinants), indicating a distance of less than 1 centimorgan between these two genes. The calculated map distances between Nrp/b and adjacent loci D13 Bir 19 (DNA fragment BIR 19) and D13 Bir 17 (DNA fragment BIR 17), including 95% confidence limits, were determined: D13 Bir 17-4.3±2.1 cM-Nrp/b 1.1±1.1 cM-D13 Bir 19. This chromosomal localization places the mouse Nrp/b gene at the distal end of Chromosome 13 in a region homologous to human Chromosome 5q. The human NRP/B gene was mapped using DNAs from the Coriel Institute somatic cell hybrid panel 2. This panel consisted of DNA isolated from 24 human/rodent cell hybrids retaining one or two human chromosomes. All but two of the hybrids retained a single human chromosome. Hamster, human and mouse DNAs were digested with BamHI, HindIII and PstI to identify a specific RFLP pattern for the NRP/B gene in each species. Southern blots were probed with a human 1.8 kb NRP/B cDNA. Human specific 1.5 kb and 1.3 kb fragments were found on Southern blots of PstI digested genomic DNAs from the parental cell lines (hamster, human and mouse) (Fig. 3B). DNAs from the parental and the somatic hybrid cell lines were digested with PstI, Southern blotted, and then probed. Analysis of the mapping panel indicated that the human-specific PstI pattern was observed in cell line 5 which contains human Chromosome 5 ( Fig. 3A), and thus indicates localization of human NRP/B to Chromosome 5. To further localize the NRP/B gene on Chromosome 5, a Coriel Institute panel of somatic cell hybrids containing various portions of human Chromosome 5 was hybridized with the human 1.8 kb NRP/B cDNA (Fig. 3C ). The human chromosome components of the human/rodent hybrids are illustrated in Fig. 3A. Also indicated are the Chromosome 5 segments, lacking in the three somatic cell hybrids, that we selected for the regional mapping of human NRP/B. These somatic cell hybrids contained different Chromosome 5 segments which were derived from translocations and deletions. The DNA sample NA11434 was missing 5q12 and 5q13, NA11437 lacked the segment 5p14 5pter, and in NA11443, 5q21 was absent. Genomic DNA samples from Chinese hamster, human and our regional panel were cut with PstI and hybridized to the 1.8 kb NRP/B cDNA. The 1.5 kb and 1.3 kb human-specific NRP/B bands were observed in the lanes containing total human DNA, NA11437, and NA11443, but were absent in NA11434. Therefore, when chromosomal segments 5q12 and 5q13 were present in the DNA from samples containing derivatives of Chromosome 5, a signal was obtained. However, the DNA sample from cell line NA11434, in which the 5q12 and 5q13 segments were absent, lacked the 1.5 and 1.3 human-specific NRP/B
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bands. Therefore, these data indicate that NRP/B is located in either band 5q12 or 5q13. 3.3. Expression of NRP/B in human brain tumor cell lines and human primary brain tumors To analyze NRP/B expression in human brain tumors and to determine whether its expression is restricted to neuronal cells or is found in other brain cells under pathological conditions, we screened various brain tumor cell lines. As shown in our previous report, NRP/B is localized in the nucleus of primary hippocampal neurons and was biochemically purified in a nuclear matrix fraction ( Kim et al., 1998). Expression of NRP/B mRNA was analyzed in various brain tumor cell lines including neuroglioma, neuroblastoma, astrocytoma and glioblastoma, using a 3∞-gene specific probe. All of these brain tumor cells expressed a single mRNA band of 5.5 kb ( Fig. 4A), as previously described ( Kim et al. 1998). Furthermore, total lysates of these cell lines were prepared and analyzed for the expression of NRP/B protein by Western blot analysis using VD2-specific NRP/B monoclonal antibody. Interestingly, as shown in Fig. 4B, NRP/B protein expression was observed in all brain tumor cell lines tested but not in primary astrocytes as expected. Confocal analysis by immunostaining of NRP/B also showed its nuclear localization in U87-MG human glioblastoma cells and demonstrated its abundant expression in the nucleus ( Fig. 4C ). To further elucidate NRP/B expression in primary brain tumors, RNA was extracted from human brain tumors (glioblastoma multiformae, astrocytoma) and NRP/B expression was analyzed by RT–PCR using specific primers for NRP/B. Expression of NRP/B was observed in glioblastoma multiformae and astrocytic tumors, while no expression was observed in primary astrocytes (Fig. 5). These data indicate that NRP/B expression is upregulated in brain tumor cell lines as well as in human primary brain tumor tissues.
4. Discussion In this study, we describe the genomic organization of the Nrp/b gene, and the chromosomal localization and expression of NRP/B protein in human primary brain tumors. NRP/B is a novel nuclear matrix protein ( Kim et al., 1998), which is expressed abundantly in the brain and appears to be localized in primary neuronal cells. NRP/B contains a BTB/POZ domain in the N-terminus and ‘kelch’ repeats at the C-terminus, and appears to play an important role in neuronal differentiation. Based on our results, NRP/B expression is restricted to primary neurons but not to astrocytes under normal conditions. However, in brain tumors, we have observed NRP/B expression not only in neuronal cells
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but also in astrocytes and glial cells derived from tumors (Figs. 4 and 5). This upregulation of NRP/B expression in brain tumors suggests that NRP/B might be involved in brain tumorigenesis. Interestingly, it has been shown (Dhordain et al., 1997) that the LAZ3/BCL6 ( lymphoma-associated zinc finger 3/B cell ) lymphoma gene is altered in non-Hodgkin’s lymphoma, and encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal BTB/POZ domain. This protein through its BTB/POZ domain is associated with the corepressor SMRT, indicating the involvement of this domain with a complex of proteins that regulates transcriptional repression (Dhordain et al., 1997). Furthermore, it has been shown that the transcriptional corepressors SMRT and N-Cor function as silencing mediators in the transcriptional suppression mechanism involving the corepressors mSin3A and HDAC1 in multiprotein complexes. These studies support our proposed hypothesis that the BTB/POZ domain of NRP/B may be involved in the transcriptional suppression mechanism in brain tumors, similar to its role in other tumors. Several groups have reported that certain tumors have nuclear matrix proteins that are not present in the corresponding normal tissues. For example, human prostate tumors have a nuclear matrix protein that is not present in normal prostate tissue or in benign prostatic hyperplasia. Several malignancy-associated nuclear matrix proteins that are not present in normal breast tissues have been identified in human infiltrating ductal carcinomas of the breast ( Khanuja et al., 1993). Six nuclear matrix proteins that are absent in normal colon tissues have been reported in human colon adenocarcinomas ( Keese et al., 1994). These observations indicate that changes in nuclear matrix structure and protein composition might be related to important changes in the growth and differentiation of cells. Thus, these changes might be used to predict or detect neoplastic transformation. According to the ‘Tensegrity’ model postulated by Ingber (1993), cytoskeletal structure altered by mechanical forces generated through cell– extracellular matrix ( ECM ) interactions can change nuclear morphology and ultimately the pattern of gene expression. Reorganization of the nuclear matrix by extracellular signals may permit tissue-specific gene expression. Analysis of the complete cDNA sequences of the human NRP/B protein indicated a protein of 589 amino acids (aa) with a predicted M of 67 730. Interestingly, r
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a potential second translation initiation codon, ATG, at nucleotide number 220–222 has a Kozak consensus sequence. The resulting product translated from the second initiation codon is predicted to have 516 aa and a molecular mass of 57 kDa. Two-dimensional SDS– PAGE analysis of NRP/B in primary neurons revealed one dominant form of NRP/B protein of 67 kDa, indicating that the first ATG is being utilized in primary neurons and neuroblastoma cell lines. Since NRP/B expression is altered in brain tumors, the possibility that NRP/B utilizes a different ATG in other cell types such as tumors derived from brain glial cells, thereby leading to a different size of NRP/B protein, will be tested in future studies. The genomic organization of NRP/B was determined in this study (Fig. 1). This is the first nuclear neuronal matrix protein whose genomic organization has been determined. Interestingly, the coding regions are localized to one exon, suggesting that NRP/B is highly conserved during evolution. The untranslated region of NRP/B is spread within 3.7 kb and is found primarily in exons 1, 2 and 4. We have mapped the mouse Nrp/b gene to Chromosome 13 using a (C57BL/6J×M. spretus) F ×M. spretus backcross. The localization of 1 the mouse Nrp/b gene to the distal end of Chromosome 13 in a region homologous to the long arm of human Chromosome 5 is consistent with our mapping of NRP/B to Chromosome 5q12–13. The chromosomal localization of the human Enc-1 employing the fluorescence in situ hybridization method, and its mRNA expression in several cell lines, were recently reported ( Hernandez et al., 1998, 1999). Interestingly, it was reported that a defect in the region of human Chromosome 5q12–13 is associated with several types of cancer, including various leukemias, non-Hodgkin’s lymphoma, several skin cancers and spinal muscular atrophy (NCBI, Genome analysis). Spinal muscular atrophy is the second most common lethal, autosomal recessive disease in Caucasians. This disorder is characterized by hypotonia, muscle fasciculation, and decreased spontaneous activity. In addition, frequent mutations were found in the LAZ3/BCL6 lymphoma gene (that encodes a BTB/POZ domain which functions as a DNA-binding transcription repressor) (Dhordain et al., 1997), and are expressed in nonHodgkin’s lymphoma. This gene is also localized to Chromosome 5q12–13, the same chromosomal localization as the NRP/B protein.
Fig. 3. Chromosomal mapping of human genes encoding NRP/B. (A) Mapping of human NRP/B to 5q12–q13. Chromosome 5 and the somatic cell hybrid panel are indicated. The Chromosome 5 content of each hybrid is shown by a solid bar. The deleted segments of Chromosome 5 are indicated to the right. The NRP/B gene localization is indicated on the left. (B) Autoradiogram showing detection of the NRP/B 1.5 and 1.3 kb human-specific bands within the mapping panel. The cell lines from which the DNAs were isolated are shown above each lane. (C ) Mapping of NRP/B in humans to Chromosome 5. PstI digested genomic DNAs from hamster (h), human (H ) and mouse (M ), as well as 24 human/rodent somatic cell hybrids ( labeled 1–22, X and Y ) probed with human NRP/B cDNA. The human-specific RFLP bands are indicated with arrowheads (1.5 and 1.3 kb in size) and are seen in the human control lane and in lane 5.
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C.
Fig. 4. Expression of NRP/B mRNA and protein in human brain tumor cell lines.(A) Expression of NRP/B mRNA in human brain tumor cell lines. The probe used in this blot was derived from the 3∞-gene specific region of NRP/B cDNA (344 bp). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) was used as an internal control for the amount of RNA loaded. (B) Western blot analysis. Total cell lysates obtained from the indicated cells (5×106) were analyzed on 8% SDS–PAGE. Blots were probed with monoclonal anti-NRP/B antibody ( VD2). NRP/B is shown as 67 kDa. Primary hippocampal neurons and astrocytes were prepared from rat brain as indicated in Materials and methods. (C ) Confocal analysis of NRP/B protein in U87-MG human glioblastoma cells. Proliferating U87-MG cells were fixed in 3% paraformaldehyde and immunostained with purified VD2 monoclonal anti-NRP/B antibody. (a) indicates control IgG and (b) immunostaining with NRP/B. Bars=5 mm.
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Fig. 5. Analysis of NRP/B expression in human brain tumors by RT– PCR. Total mRNA was prepared from human brain tumors and NRP/B expression was determined by RT–PCR as described in Materials and methods. The PCR products were electrophoresed on a 2% agarose gel and hybridized with gene-specific probes for NRP/B and actin. Actin was used as an internal control in this set of experiments.
NRP/B sequences contain two domains: the BTB/POZ domain in the N-terminus and the ‘kelch’ repeats in the C-terminus. Kelch protein is a component of intercellular cytoplasmic bridges (ring canals) in Drosophila egg chambers which connect nurse cells and the developing oocyte ( Xue and Cooley, 1993; Robinson et al., 1994). In the ring canals, kelch is co-localized and associates with actin filaments ( Xue and Cooley, 1993; Robinson et al., 1994). Using structural analysis, it was suggested that the ‘kelch’ motifs form a ‘superbarrel’ structure of b sheets (Sondek et al., 1996). Interestingly, crystallographic study of the b-subunit of the G protein (Sondek et al., 1996) revealed that its b-stranded motifs form a ‘superbarrel’ structure and play an important role in protein–protein interactions. Accumulating evidence suggests that the ‘superbarrel’ structure in such signaling proteins might play an important role in recruiting cytoskeletal proteins. By analogy, NRP/B with its six repeat structure may communicate directly with structural elements and recruit nuclear matrix proteins to form a nuclear scaffold structure. Cell-type-specific expression of nuclear matrix proteins has been observed by Fey and Penman (1984). In normal brain tissue, NRP/B appears to be restricted to primary neurons. Furthermore, its spatial and temporal distribution are strongly toward cortical areas, as indicated in developmental studies (Hernandez et al., 1997; Kim et al., 1998). This suggests that the NRP/B protein might be involved in the regulation and coordination of gene expression that control neuronal development. The subcellular localization of the NRP/B protein to the nucleus was determined by immunostaining several cell types including primary hippocampal neurons, and glioblastoma cell lines such as U87-MG. We used mouse
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monoclonal NRP/B and polyclonal NRP/B antibodies for the immunohistochemical analysis. Confocal micrographs using these antibodies indicated that NRP/B was highly expressed in the nucleus of primary neurons and in glioblastomas/astrocytomas, but not in primary astrocytes (Fig. 5). The distribution of NRP/B was condensed in the peripheral heterochromatin and in the nuclear matrix of the nucleoplasm. These results suggest that NRP/B might be involved in the regulation of gene transcription through the dynamic network of nuclear matrix proteins and the structural organization of the nucleus. In summary, the upregulation of NRP/B expression in brain tumors may indicate a potential role of NRP/B in brain tumor development. This role of NRP/B could be due to the involvement of the BTB/POZ domain in tumor regulation as shown in non-Hodgkin’s lymphoma (Chen et al., 1993), and/or to the action of the BTB/POZ as a silencer or co-suppressor of transcription (Dhordain et al., 1997; Nagy et al., 1997). This hypothesis will be tested in future studies.
Acknowledgements We thank Dr. Bijia Deng for her help in establishing rat primary hippocampal cultures. The authors wish to thank Lucy Rowe and Ed Birkenmeier of The Jackson Laboratory for supplying the DNA panel and for performing the analyses of linkage data. We thank Dr. Hava Avraham for her advice and comments on the manuscript, Janet Delahanty for her help in editing the manuscript, Dan Kelley for preparing the artwork, and Mikyung Kim for her typing assistance. This work was supported by grants from the Paul Patton Memorial Trust (R.A.W.) and from Katherine B. Richardson Associates (R.A.W.).
References Albagli, O., Dhordain, P., Deweindt, D., Lecocq, G., Leprince, D., 1995. The BTB/POZ domain: a new protein–protein interaction motif common to DNA- and actin-binding proteins. Cell Growth. Differ. 6, 1193–1198. Avraham, S., Jiang, S., Ota, S., Fu, Y., Deng, B., Dowler, L.L., White, R.A., Avraham, H., 1995. Structural and functional studies of the intracellular tyrosine kinase MATK gene and its translated product. J. Biol. Chem. 270, 1833–1842. Bardwell, V.J., Treisman, R., 1994. The POZ domain: a conserved protein–protein interaction motif. Genes Dev. 8, 1664–1677. Berezney, R., Mortillaro, M.J., Ma, H., Wei, X., Samarabandu, J., 1995. The nuclear matrix: A structural milieu for nuclear genomic function. Int. Rev. Cytol. 162A, 1–65. Berezney, R., Mortillaro, M., Ma, H., Meng, C., Samarabandu, J., Wei, X., Somanathan, S., Liou, W.S., Pan, S.J., Cheng, P.C., 1996. Connecting nuclear architecture and genomic function. J. Cell Biochem. 62, 223–226.
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Chen, Z., Brand, N.J., Chen, A., Chen, S.J., Tong, J.H., Wang, Z.Y., Waxman, S., Zelent, A., 1993. Fusion between a novel Kruppellike zinc finger gene and the retinoic acid receptor-alpha locus due to a variant t(11,17) translocation associated with acute promyelocytic leukaemia. EMBO J. 12, 1161–1167. Dhordain, P., Albagli, O., Lin, R.J., Ansieau, S., Quief, S., Leutz, A., Kerckaert, J.P., Evans, R.M., Leprince, D., 1997. Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein. Proc. Natl. Acad. Sci. USA 94, 10762–10767. Dworetzky, S.I., Fey, E.G., Penman, S., Lian, J.B., Stein, J.L., Stein, G.S., 1990. Progressive changes in the protein composition of the nuclear matrix during rat osteoblast differentiation. Proc. Natl. Acad. Sci. USA 87, 4605–4609. Fey, E.G., Penman, S., 1984. Tumor promoters induce a specific morphological signature in the nuclear matrix-intermediate filament scaffold of Madin–Darby canine kidney (MDCK ) cell colonies. Proc. Natl. Acad. Sci. USA 81, 859–866. Getzenberg, R.H., Konety, B.R., Oeler, T.A., Quigley, M.M., Hakam, A., Becich, M.J., Bahnson, R.R., 1996. Bladder cancer-associated nuclear matrix proteins. Cancer Res. 56, 1690–1694. He, D., Zeng, C., Brinkley, B., 1995. Nuclear matrix proteins as structural and functional components of the mitotic apparatus. Int. Rev. Cytol. 162B, 1–74. Hernandez, M.C., Andres-Barquin, P.J., Holt, I., Israel, M.A., 1998. Cloning of human ENC-1 and evaluation of its expression and regulation in nervous system tumors. Exp. Cell Res. 242, 470–477. Hernandez, M.C., Andres-Barquin, P.J., Kuo, W.L., Israel, M.A., 1999. Assignment of the ectodermal-neural cortex 1 gene ( ENC1) to human chromosome band 5q13 by in situ hybridization. Cytogenet. Cell Genet. 87, 89–90. Hernandez, M.-C., Andres-Barquin, P.J., Martinez, S., Bulfone, A., Rubenstein, J.L.R., Israel, M.A., 1997. ENC-1: a novel mammalian kelch-related gene specifically expressed in the nervous system encodes an actin-binding protein. J. Neurosci. 17, 3038–3051. Ingber, D., 1993. The riddle of morphogenesis: a question of solution chemistry or molecular cell engineering? Cell 75, 1249–1252. Keese, S.K., Meneghini, M.D., Szaro, R.P., Wu, Y.J., 1994. Nuclear matrix proteins in human colon cancer. Proc. Natl. Acad. Sci. USA 91, 1913–1916. Khanuja, P.S., Lehr, J.E., Soule, H.D., Gehani, S.K., Noto, A.C.,
Choudhury, S., Chen, R., Pienta, K.J., 1993. Nuclear matrix proteins in normal and breast cancer cells. Cancer Res. 53, 3394–3398. Kim, T.-A., Lim, J., Ota, S., Raja, S., Rogers, R., Rivnay, B., Avraham, H., Avraham, S., 1998. NRP/B, a novel nuclear matrix protein, associates with p110RB and is involved in neuronal differentiation. J. Cell. Biol. 141, 553–566. Loidl, P., Eberharter, A., 1995. Nuclear matrix and the cell cycle. Int. Rev. Cytol. 162B, 377–403. Nagy, L., Kao, H.Y., Chakravarti, D., Lin, R.J., Hassig, C.A., Ayer, D.E., Schreiber, S.L., Evans, R.M., 1997. Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase. Cell 89, 373–380. Peinta, K.J., Partin, A.W., Coffey, D.S., 1989. Cancer as a disease of DNA organization and dynamic cell structure. Cancer Res. 49, 2525–2532. Robinson, D., Cant, K., Cooley, L., 1994. Morphogenesis of Drosophila ovarian ring canals. Development 120, 2015–2025. Rowe, L.B., Nadeau, J.H., Turner, R., Frankel, W.N., Letts, V.A., Eppig, J.T., Ko, M.S.H., Thurston, S.J., Birkenmeier, E.H., 1994. Maps from two interspecific backcross panels available as a community genetic mapping resource. Mamm. Genome 5, 253–274. Skinner, P.J., Koshy, B.T., Cummings, C.J., Klement, I.A., Helin, K., Servadio, A., Zoghbi, H.Y., Orr, H.T., 1997. Ataxin-1 with an expanded glutamine tract alters nuclear matrix associated structures. Nature 389, 971–974. Sondek, J., Bohrn, A., Lambright, D., Hamm, H., Sigler, P., 1996. Crystal structure of a G protein bc dimer at 2.1 A resolution. Nature 379, 369–374. Stein, G.S., Berezney, R., 1996. Nuclear structure and function. J. Cell Biochem. 62, 147–148. von Bulow, M., Heid, H., Hess, H., Franke, W.W., 1995. Molecular nature of calicin, a major basic protein of the mammalian sperm head cytoskeleton. Exp. Cell Res. 219, 407–413. Way, M., Sanders, M., Chafel, M., Tu, Y.-H., Knight, A., Matsudaira, P., 1995. b-Scruin, a homolog of the actin crosslinking protein scruin, is localized to the acrosomal vesicle of Limulus sperm. J. Cell Sci. 108, 3155–3162. White, R.A., Peters, L.L., Adkison, L.R., Korsgren, C., Cohen, C.M., Lux, S.E., 1992. The murine pallid mutation is a platelet storage pool disease associated with the protein 4.2 (pallidin) gene. Nat. Genet. 2, 80–83. Xue, F., Cooley, L., 1993. Kelch encodes a component of intercellular bridges in Drosophila egg chambers. Cell 72, 681–693.
Genomic organization, chromosomal localization and regulation of expression of the neuronal nuclear matrix protein NRP/B in human brain tumors k Tae-Aug Kim 1,a, Setsuo Ota 1,a, Shuxian Jiang a, Linda M. Pasztor b, Robert A. White b, Shalom Avraham a, * a Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115, USA b Section of Medical Genetics and Molecular Medicine, Children’s Mercy Hospital, UMKC School of Medicine, Kansas City, MO 64108, USA Received 3 April 2000; received in revised form 6 June 2000; accepted 29 June 2000 Received by J.A. Engler
Abstract The nuclear matrix and its role in cell physiology are largely unknown, and the discovery of any matrix constituent whose expression is tissue- and/or cell-specific offers a new avenue of exploration. Studies of the novel neuronal nuclear matrix protein, NRP/B, reveal that it is an early and highly specific marker of neuronal induction and development in vertebrates, since its expression is restricted mainly to the developing and mature nervous system. These studies also show that NRP/B is involved in neuronal differentiation. To further examine the structure–function of NRP/B, we have cloned and characterized the murine Nrp/b gene. The murine gene consists of four exons interrupted by three introns that span 7.6 kb of DNA. The complete open reading frame is localized in exon 3, suggesting that NRP/B is highly conserved during evolution. Chromosomal analysis shows that NRP/B is localized to chromosome 13 in mouse and chromosome 5q12–13 in human. Since our previous studies demonstrated that NRP/B is expressed in primary hippocampal neurons but not in primary astrocytes, we have characterized NRP/B mRNA and protein expression in various brain cell lines and in human brain tumors. Abundant expression of NRP/B mRNA and protein was observed in human neuroblastoma cell lines (IMR32, SKN-MC, SKN-SH ), in glioblastoma cell lines (A172, T98G, U87-MG, U118-MG, U138-MG, and U373-MG), in neuroglioma (H4) and astrocytoma cell lines (CCF-STTG1 and SW1088). Confocal analysis of NRP/B in U87-MG glioblastoma cells indicated nuclear localization of NRP/B. NRP/B expression was also observed in human primary brain tumors including glioblastoma multiformae and astrocytomas (total of five cases). These results suggest that NRP/B expression is upregulated in human brain tumors including glioblastomas and astrocytomas, while under normal conditions NRP/B expression is restricted to neurons. This study implicates a role for NRP/B in brain tumor development. © 2000 Elsevier Science B.V. All rights reserved. Keywords: BTB/POZ domain; Chromosome 5q; Human brain tumor; Kelch motif; Nuclear matrix protein
Abbreviations: BTB, broad-complex tramtrack and bric-a-brac; CLSM, confocal laser scanning microscope; DMEM, Dulbecco’s modified Eagle’s medium; ENC-1, ectoderm-neural cortex-1; ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC1, histone deacetylase-1; lapls 2-6, intracisternal A particle proviral element 2-6; LAZ3/BCL6, lymphoma-associated zinc finger 3/B cell; MGD, mouse genome database; N-COR, nuclear receptor co-repressor; NRP/B, nuclear restricted protein/brain; ORFs, open reading frames; POZ, poxvirus/zinc finger; REC, recombination frequency; RFLP, restriction fragment length polymorphism; RT–PCR, reverse transcriptase–polymerase chain reaction; NRP/B, nuclear restricted protein/brain; ORFs, open reading frames; POZ, poxvirus/zinc finger; REC, recombination frequency; RFLP, restriction fragment length polymorphism; RT–PCR, reverse transcriptase–polymerase chain reaction; SMRT, silencing mediator of retinoid and thyroid receptor. k This paper is dedicated to Raphael Recanati and his family for their friendship and support for our research program. * Corresponding author. Tel.: +1-617-667-0073; fax: +1-617-975-6373. E-mail address: [email protected] (S. Avraham) 1 Both authors contributed equally to this work. 0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 0 0 ) 0 0 29 7 - 3
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1. Introduction The nuclear matrix constitutes the three-dimensional filamentous protein network that maintains the domain organization of the nucleus. This component of the nuclear architecture provides the internal scaffold of the nucleus. It is formed by an ordered and highly compartmentalized protein structure consisting of a nuclear lamina, a residual nucleolus, and an internal matrix composed of a non-chromatin fibrogranular network associated with DNA (Berezney et al., 1996; Stein and Berezney, 1996). The nuclear matrix has been implicated in transcription, regulation of gene expression, the cell cycle, primary transcription processing and in linkages to intermediate filaments of the cytoskeleton (He et al., 1995; Loidl and Eberharter, 1995). A cellular hallmark of the transformed phenotype is an altered nuclear shape and the presence of abnormal nucleoli. Structural alterations of the nucleus are prevalent in cancer cells and are commonly used as pathological markers of transformation in many types of cancer. Nuclear shape is thought to reflect the internal nuclear structure and is determined, at least in part, by the nuclear matrix (Peinta et al., 1989; Dworetzky et al., 1990). The nuclear matrix contains a number of associated proteins that have been found to be involved in malignant transformation (Fey and Penman, 1984; Keese et al., 1994; Getzenberg et al., 1996). It has been directly or indirectly implicated in most of the changes considered to be pathologic hallmarks of malignant transformation, such as alterations in DNA ploidy, DNA content, nuclear shape and proliferative states (Berezney et al., 1995). Ataxin-1 (the protein encoded by the SCA1 gene), which is involved in the neurodegenerative disorder spinocerebellar ataxia, alters nuclear-matrix-associated structures (Skinner et al., 1997). Despite the apparent importance of the nuclear matrix in the regulation of many biological processes, its roles in cell physiology and in neuronal differentiation are largely unknown. Recently, we have discovered and characterized a novel nuclear matrix protein, NRP/B (nuclear restricted protein/brain), which contains two major structural elements: a BTB domain-like structure in the predicted N-terminus, and a ‘kelch motif ’ in the predicted C-terminal domain ( Kim et al., 1998). NRP/B mRNA (5.5 kb) is expressed predominantly in human fetal and adult brain, with minor expression in kidney and pancreas. During mouse embryogenesis, NRP/B mRNA expression is upregulated in the nervous system. ENC-1, a mammalian kelch-related gene that is specifically expressed in the nervous system, was also reported to be the murine homolog of NRP/B (Hernandez et al., 1997; Kim et al., 1998). NRP/B/ENC-1 is expressed in the prospective neuroectodermal region of the epiblast during early gastrulation and throughout the nervous system
later in development. NRP/B/ENC-1 expression is highly dynamic and, after neurulation, preferentially defines prospective cortical areas. Expression of NRP/B/ENC-1 was detected at the preneurulation stage of the mouse embryo (E 6.5) in the prospective neuroectodermal region of the epiblast that later differentiates predominantly into neuroectodermal cells (Hernandez et al., 1997; Kim et al., 1998). No expression of NRP/B/ENC-1 was detected in any extraembryonic tissue. At E 8.0, its expression was detected in ectodermal derivatives and continued to be strongly expressed in the hippocampus and neocortex (Hernandez et al., 1997; Kim et al., 1998). The BTB/POZ domain at the N-terminus of NRP/B consists of approx. 115 amino acids and is expressed in several members of the kelch family ( Xue and Cooley, 1993). This domain, found primarily in zinc finger proteins, defines a newly characterized protein–protein interaction interface (Bardwell and Treisman, 1994), and also mediates both dimer and heterodimer formation in vitro (Albagli et al., 1995). NRP/B also shares significant homology to the ‘kelch’ repeats found in several kelch-related genes ( Xue and Cooley, 1993; von Bulow et al., 1995; Way et al., 1995) and in a large number of open reading frames (ORFs) within the genome of poxviruses (von Bulow et al., 1995; Way et al., 1995). NRP/B contains six repeats of kelch in the C-terminal half of the protein, while each repeat consists of about 50 amino acids. These motifs may have functional significance in binding actin, protein folding or in protein–protein interactions. We have shown previously that NRP/B protein is expressed in rat primary hippocampal neurons, but not in primary astrocytes, and that its expression is upregulated during the differentiation of rat PC12 cells, murine Neuro-2A and human SH-SY5Y neuroblastoma cells ( Kim et al., 1998). Overexpression of NRP/B in these cells augmented neuronal process formation, while treatment with antisense NRP/B oligodeoxynucleotides inhibited the neurite development of rat primary hippocampal neurons as well as neuronal process formation during differentiation of PC12 cells. In this study, we aimed to characterize the genomic organization, chromosomal localization and expression of NRP/B in brain tumor cell lines and human primary brain tumors. We have determined the chromosomal assignment of the human NRP/B gene by analysis of somatic cell hybrids, and we have mapped the mouse NRP/B gene by backcross analysis. Our results suggest that NRP/B expression is upregulated in brain tumors.
2. Materials and methods 2.1. Materials Chemical reagents were purchased from Sigma (St. Louis, MO). The murine fetal l-gt10 cDNA library was
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obtained from Dr. Kunkle, Children’s Hospital, Boston, MA. Restriction endonucleases, modifying enzymes, terminal deoxynucleotidyl transferase, random priming kits, and Sephadex G-25 quickspin columns were purchased from Pharmacia-LKB (Piscataway, NJ ) and New England BioLabs (Beverly, MA). The primers for polymerase chain reaction (PCR), RT–PCR and sequencing were synthesized using an automated DNA synthesizer (Applied Biosystems, model 394). The PCR and RNA-PCR kits were obtained from Perkin-Elmer Cetus (Norwalk, CT ). Sequenase and random priming kits were obtained from US Biochemical Corp. (Cleveland, OH ) and RNA isolation kits were from Stratagene (La Jolla, CA). Human brain tumor tissues were obtained from Cooperative Human Tissue Network (CHTN ) (Philadelphia, PA). 2.2. Cell culture Primary neurons were prepared from the hippocampal regions of Sprague–Dawley rats at gestational day 18, and primary astrocytes from the cerebral cortex were prepared from postnatal day 1 rats as described ( Kim et al., 1998). Primary neurons were cultured in neurobasal medium (GIBCO) containing B-27 supplement without antibiotics. Culture of primary astrocytes was in DMEM supplemented with 10% heat-inactivated horse serum, glucose (6 mg/ml ) and 2 mM glutamine. After cultures reached confluence, the medium was changed to DMEM containing 10% FBS. Both types of primary cells were grown in poly--lysine-coated culture plates at a density of 1×105–1×106 cells per cm2. Neuroblastomas (IMR32, SKN-MC, SKN-SH ), neurogliomas (H4), glioblastomas (A172, T98G, U87-MG, U118-MG, U138-MG, U373-MG) and astrocytomas (CCF-STTG1, SW1088) were grown as described in the ATCC manual.
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2.4. Chromosomal localization of human NRP/B Genomic DNAs from NIGMS Hybrid Mapping Panel No. 2 as well as DNA from somatic cell hybrids NA11434, NA11437, and NA11443 were obtained from the NIGMS Genetic Mutant Cell Repository (Coriel Cell Institute for Medical Research, Camden, NJ ). Mapping Panel No. 2 consisted of DNA isolated from 24 human/rodent cell hybrids retaining one or two human chromosomes. All but two of the hybrids retained a single intact human chromosome. In addition, the mapping panel included DNA samples isolated from the human and rodent parental cell lines (mouse and Chinese hamster). Human/rodent cell hybrids NA11434, NA11437 and NA11443 contained Chinese hamster chromosomes and segments of Chromosome 5 generated by somatic cell fusion with cells containing translocations or deletions involving Chromosome 5. The DNA sample NA11437 contained a der(5)t(5;13) (5qter 5p13::13q13 13qter) and thus lacked the segment 5p14 5pter. NA11434 contained 5 qter 5q13.3::5q11.2 5pter and thus was missing 5q12 and 5q13. NA11443 contained del(5) (q15 q21.3), and thus 5q21 was absent. Approx. 5 mg of DNA from human, hamster and mouse genomic DNAs were digested with BamHI, HindIII and PstI to find a suitable RFLP or unique genomic fragment for use in mapping. Subsequently, genomic DNAs from each panel were cut with PstI. Southern blots were probed with a human 1.8 kb NRP/B cDNA, and hybridizations were carried out as previously described ( White et al., 1992). Hybrids were scored on the autoradiographs for the appropriate human-specific restriction endonuclease fragment. The results were compared with the chromosome contents of the hybrid cell lines. Concordance between restriction fragments and specific chromosomes or portions of Chromosome 5 was used to establish the chromosomal localization of NRP/B.
2.3. Chromosomal localization of Nrp/b in mouse 2.5. Western blot analysis Genomic DNAs from C57BL/6J, Mus spretus, and interspecific backcross panel (C57BL/6J M. spretus) F ×C57BL/6J (BSS panel ) were obtained from The 1 Jackson Laboratory (Rowe et al., 1994). Southern blots and hybridizations were carried out as previously described ( White et al., 1992). Approximately 5 mg of genomic DNAs from the C57BL/6J and M. spretus progenitors were digested with 28 different restriction enzymes to find a suitable restriction fragment length polymorphism (RFLP) for mapping. Southern blots were probed with a human 1.8 kb NRP/B cDNA. Approx. 2 mg of DNA from the BSS type backcross panel were digested for each sample with XbaI overnight. Segregation of Nrp/b alleles was compared with that of alleles at other loci from a mouse genome database (MGD) by the Jackson Laboratory Backcross DNA Map Panel Service (Rowe et al., 1994).
Total cell lysates were analyzed by SDS–PAGE and transferred to membranes. The blots were incubated at 4°C overnight in 5% milk in PBS containing 0.1% Tween 20, followed by incubation with NRP/B specific antibodies. The blots were washed and reacted with horseradish peroxidase-conjugated anti-rabbit IgG for 1 h. Immunoreactive bands were visualized using ECL reagents. 2.6. Immunolocalization of NRP/B protein by confocal microscopy Standard immunohistochemical methods were used as described ( Kim et al., 1998). Briefly, U87-MG astrocytoma/glioblastoma cells were seeded onto sterile 18-mm glass coverslips ( Fisher Scientific, Pittsburgh,
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PA) in 12-well plates at different densities. Cells were washed at room temperature in PBS for 2–3 min, fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.2% Triton-X 100 in PBS for 5 min, and blocked in 0.1% BSA and 3% normal goat serum in PBS for 1 h at room temperature or overnight at 4°C. Cells were incubated for 1 h with NRP/B in PBS containing 0.1% BSA and 3% normal goat serum, washed, and then incubated with 1:100 dilution of Texas Red-conjugated goat anti-rabbit IgG ( Vector Laboratories, Burlingame, CA). Immunostained cells were washed, mounted with ProLong antifade reagent (Molecular Probes, Eugene, OR), and examined using a Sarastro 2000 confocal laser scanning microscope (CLSM ) (Molecular Dynamics, Sunnyvale, CA) and Leica TCS-NT (Leica, Wetzlar, Germany) confocal laser-scanning microscope optimized for simultaneous dual fluorescent imaging. 2.7. Expression of NRP/B in human brain tumors (by reverse transcription polymerase chain reaction (RT–PCR) and Northern blot analysis) RNA extracted from human primary brain tumors and from the cultured cells (1×105 cells) was reverse transcribed at 42°C for 40 min in a final volume of 50 ml as described ( Kim et al., 1998). The 5∞- and 3∞-specific primers were added in final concentrations of 5 ng/50 ml each. The mixture was subjected to 30 amplification cycles using the Perkin-Elmer thermal cycler set as follows: denaturation at 94°C for 1 min, primer annealing at 55°C for 1 min, extension at 72°C for 2 min. The sequence of the NRP/B upstream primer was 5∞-TCGAGGATCCATGCTCTTCCTTCTGGGAGGACAG-3∞ (corresponding to position +877 to +911 bp). The nucleotide sequence of the downstream primer was 5∞-ACGTCAAGCTTACGGTACAGTCCACCTGTTTTCAC-3∞ (corresponding to position +1789 to +1824 bp). Primers for the b-actin message were: upstream primer 5∞-ATG GAT GAT GAT ATC GCC GCG-3∞ and downstream primer 5∞-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG GCC-3∞. The amplification products and poly A mRNA obtained from the brain tumor cell lines were detected by overnight hybridization with an NRP/B, b-actin, or GAPDH probe. The PCR products and NRP/B mRNA level were analyzed as previously described ( Kim et al., 1998).
3. Results 3.1. Cloning, sequence analysis, and genomic organization of the murine Nrp/b gene We previously observed that Nrp/b is expressed in rat primary hippocampal neurons but not in astrocytes,
and that its level of expression is upregulated during neuronal differentiation ( Kim et al., 1998). To determine the exon–intron organization of the murine Nrp/b gene and to identify its potential tissue-specific response elements, we screened approx. 6×105 total recombinants, from a murine liver genomic library (c-EMBL-3), for genomic clones under conditions of high stringency with a 260 bp 32P-labeled 3∞ gene-specific fragment (A3∞) of the mouse Nrp/b cDNA ( Fig. 1). The human NRP/B/ENC-1 cDNA and the mouse Nrp/b cDNA have highly conserved sequences of more than 99% ( Kim et al., 1998; Hernandez et al., 1997). We isolated a 17 kb genomic DNA clone, termed l Dash-MG-Nrp/b-1. In addition, using probes derived from both the 5∞ and 3∞-ends of the mouse Nrp/b cDNA, we isolated an additional genomic DNA clone of 21 kb (termed l Dash-MG-Nrp/b-2) (Fig. 1A). A restriction map of each genomic clone was constructed by digesting the phage DNA with a panel of restriction enzymes either separately or in various combinations: SacI, BamHI, HindIII, XbaI, and EcoRI. The DNA blots were probed under conditions of high stringency with either the 403 bp 5∞A gene-specific fragment, or the 344 bp A3∞ gene-specific fragment of the mouse Nrp/b cDNA. In parallel, a blot was prepared of mouse liver DNA that had been digested with the same panel of restriction enzymes. When this DNA blot was probed with the 344 bp A3∞ gene-specific fragment or the 403 bp 5∞A gene-specific fragment of the mouse Nrp/b cDNA, the pattern of hybridization was identical to that obtained with l Dash-MG-Nrp/b-2 (data not shown), indicating that this clone probably contained the major part of the gene that encodes the mouse Nrp/b. The restriction enzyme map of the mouse Nrp/b gene was constructed and the nucleotide sequences of l DashMG-Nrp/b-1 and l Dash-MG-Nrp/b-2 were determined according to the strategy depicted in Fig. 1. Based on the nucleotide sequences of the genomic fragments analyzed, two oligonucleotides of 21 nucleotides in length were synthesized and used as primers to determine the contiguous nucleotide sequence of the next 200–250 nucleotides (in each direction) of the double-stranded DNA. No mismatches were found between the genomic sequences and the cDNA. The exon–intron organization of the mouse Nrp/b gene was determined by this approach. Based on the nucleotide sequences of its subcloned fragments of approx. 17 kb and approx. 21 kb, we determined that the mouse Nrp/b gene comprises four exons that span about 7.6 kb of DNA ( Fig. 1). The gene is approx. 1.7 kb from the putative transcription initiation site to the end of exon 2. Our genomic l Dash-MG-Nrp/b-2 clone contained an additional 3.3 kb of 5∞-flanking sequences and 7 kb of sequences downstream of exon 3. Exon 1 contained the 5∞-untranslated sequence (5∞-UT ) and exon 2 contained the putative
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Fig. 1. (A) Genomic organization of the murine Nrp/b gene. The l Dash-MG-Nrp/b-1 and l Dash-MG-Nrp/b-2 genomic clones were isolated from the mouse liver DNA library. The exons are shown in boxes and the dotted area shown in exon 2 contains the full-length coding region of the NRP/B sequences. The restriction enzymes are shown. (B) Southern blot of the genomic Nrp/b gene. Murine genomic DNA was digested with the indicated restriction enzymes and was analyzed by Southern blot analysis using a 3∞ gene-specific NRP/B probe.
translation initiation site. The exon–intron splice junctions were sequenced for each exon, and are in accordance with the Gt…At consensus sequence (Avraham et al., 1995). An additional 500 bp of 5∞-flanking sequence was determined. Southern blot analysis of mouse genomic DNA digested with several restriction enzymes and hybridized with an Nrp/b 3∞ gene-specific probe showed a single band, indicating the existence of Nrp/b as a single gene ( Fig. 1B).
3.2. Chromosomal mapping of mouse and human genes encoding NRP/B We determined the chromosomal location of the gene encoding NRP/B in mouse by analyzing the segregation of RFLP in DNAs derived from the offspring of The Jackson Laboratory (M. spretus×C57BL/6J ) F ×M. 1 spretus backcross. An XbaI RFLP for Nrp/b was identified by the presence of a 1.8 kb genomic DNA fragment
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Fig. 2. Mapping of mouse Nrp/b to Chromosome 13. XbaI restriction enzyme pattern for M. spretus (S) genomic DNAs and (C57BL/6J×M. spretus) F heterozygous (BS ) genomic DNAs probed with the 1.8 kb Nrp/b cDNA. The molecular size (in kb) of the Nrp/b RFLP fragment is 1 indicated. (B) Haplotype analysis of Chromosome 13 genetic markers in (C57BL/6J×M. spretus) F ×M. spretus (BSS type) backcross mice 1 showing linkage and the relative position of Nrp/b. Closed boxes indicate inheritance of the C57BL/6J (B) allele and open boxes indicate the inheritance of the M. spretus (S ) allele from the (C57BL/6J×M. spretus) F parent. Gene names and references to these loci can be found in 1 GBASE. The first two columns indicate the number of backcross progeny with no recombinations. The following columns indicate recombinational events between adjacent loci (signified by a change from open box to closed box). The number of recombinants are listed below each column and the recombination frequency (REC%) between adjacent loci is indicated.
in C57BL/6J or the absence of this fragment in M. spretus (Fig. 2A). This allele was characterized in 94 DNAs from the (C57BL/6J×M. spretus) F ×M. spretus 1 backcross panel. Haplotype analysis of these mapping
data ( Fig. 2B) indicated that the Nrp/b locus is closely linked to lapls 2-6 (intracisternal A particle proviral element 2-6) Chromosome 13 in mouse. Allelic segregation patterns for lapls 2-6 and Nrp/b were identical
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(no recombinants), indicating a distance of less than 1 centimorgan between these two genes. The calculated map distances between Nrp/b and adjacent loci D13 Bir 19 (DNA fragment BIR 19) and D13 Bir 17 (DNA fragment BIR 17), including 95% confidence limits, were determined: D13 Bir 17-4.3±2.1 cM-Nrp/b 1.1±1.1 cM-D13 Bir 19. This chromosomal localization places the mouse Nrp/b gene at the distal end of Chromosome 13 in a region homologous to human Chromosome 5q. The human NRP/B gene was mapped using DNAs from the Coriel Institute somatic cell hybrid panel 2. This panel consisted of DNA isolated from 24 human/rodent cell hybrids retaining one or two human chromosomes. All but two of the hybrids retained a single human chromosome. Hamster, human and mouse DNAs were digested with BamHI, HindIII and PstI to identify a specific RFLP pattern for the NRP/B gene in each species. Southern blots were probed with a human 1.8 kb NRP/B cDNA. Human specific 1.5 kb and 1.3 kb fragments were found on Southern blots of PstI digested genomic DNAs from the parental cell lines (hamster, human and mouse) (Fig. 3B). DNAs from the parental and the somatic hybrid cell lines were digested with PstI, Southern blotted, and then probed. Analysis of the mapping panel indicated that the human-specific PstI pattern was observed in cell line 5 which contains human Chromosome 5 ( Fig. 3A), and thus indicates localization of human NRP/B to Chromosome 5. To further localize the NRP/B gene on Chromosome 5, a Coriel Institute panel of somatic cell hybrids containing various portions of human Chromosome 5 was hybridized with the human 1.8 kb NRP/B cDNA (Fig. 3C ). The human chromosome components of the human/rodent hybrids are illustrated in Fig. 3A. Also indicated are the Chromosome 5 segments, lacking in the three somatic cell hybrids, that we selected for the regional mapping of human NRP/B. These somatic cell hybrids contained different Chromosome 5 segments which were derived from translocations and deletions. The DNA sample NA11434 was missing 5q12 and 5q13, NA11437 lacked the segment 5p14 5pter, and in NA11443, 5q21 was absent. Genomic DNA samples from Chinese hamster, human and our regional panel were cut with PstI and hybridized to the 1.8 kb NRP/B cDNA. The 1.5 kb and 1.3 kb human-specific NRP/B bands were observed in the lanes containing total human DNA, NA11437, and NA11443, but were absent in NA11434. Therefore, when chromosomal segments 5q12 and 5q13 were present in the DNA from samples containing derivatives of Chromosome 5, a signal was obtained. However, the DNA sample from cell line NA11434, in which the 5q12 and 5q13 segments were absent, lacked the 1.5 and 1.3 human-specific NRP/B
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bands. Therefore, these data indicate that NRP/B is located in either band 5q12 or 5q13. 3.3. Expression of NRP/B in human brain tumor cell lines and human primary brain tumors To analyze NRP/B expression in human brain tumors and to determine whether its expression is restricted to neuronal cells or is found in other brain cells under pathological conditions, we screened various brain tumor cell lines. As shown in our previous report, NRP/B is localized in the nucleus of primary hippocampal neurons and was biochemically purified in a nuclear matrix fraction ( Kim et al., 1998). Expression of NRP/B mRNA was analyzed in various brain tumor cell lines including neuroglioma, neuroblastoma, astrocytoma and glioblastoma, using a 3∞-gene specific probe. All of these brain tumor cells expressed a single mRNA band of 5.5 kb ( Fig. 4A), as previously described ( Kim et al. 1998). Furthermore, total lysates of these cell lines were prepared and analyzed for the expression of NRP/B protein by Western blot analysis using VD2-specific NRP/B monoclonal antibody. Interestingly, as shown in Fig. 4B, NRP/B protein expression was observed in all brain tumor cell lines tested but not in primary astrocytes as expected. Confocal analysis by immunostaining of NRP/B also showed its nuclear localization in U87-MG human glioblastoma cells and demonstrated its abundant expression in the nucleus ( Fig. 4C ). To further elucidate NRP/B expression in primary brain tumors, RNA was extracted from human brain tumors (glioblastoma multiformae, astrocytoma) and NRP/B expression was analyzed by RT–PCR using specific primers for NRP/B. Expression of NRP/B was observed in glioblastoma multiformae and astrocytic tumors, while no expression was observed in primary astrocytes (Fig. 5). These data indicate that NRP/B expression is upregulated in brain tumor cell lines as well as in human primary brain tumor tissues.
4. Discussion In this study, we describe the genomic organization of the Nrp/b gene, and the chromosomal localization and expression of NRP/B protein in human primary brain tumors. NRP/B is a novel nuclear matrix protein ( Kim et al., 1998), which is expressed abundantly in the brain and appears to be localized in primary neuronal cells. NRP/B contains a BTB/POZ domain in the N-terminus and ‘kelch’ repeats at the C-terminus, and appears to play an important role in neuronal differentiation. Based on our results, NRP/B expression is restricted to primary neurons but not to astrocytes under normal conditions. However, in brain tumors, we have observed NRP/B expression not only in neuronal cells
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but also in astrocytes and glial cells derived from tumors (Figs. 4 and 5). This upregulation of NRP/B expression in brain tumors suggests that NRP/B might be involved in brain tumorigenesis. Interestingly, it has been shown (Dhordain et al., 1997) that the LAZ3/BCL6 ( lymphoma-associated zinc finger 3/B cell ) lymphoma gene is altered in non-Hodgkin’s lymphoma, and encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal BTB/POZ domain. This protein through its BTB/POZ domain is associated with the corepressor SMRT, indicating the involvement of this domain with a complex of proteins that regulates transcriptional repression (Dhordain et al., 1997). Furthermore, it has been shown that the transcriptional corepressors SMRT and N-Cor function as silencing mediators in the transcriptional suppression mechanism involving the corepressors mSin3A and HDAC1 in multiprotein complexes. These studies support our proposed hypothesis that the BTB/POZ domain of NRP/B may be involved in the transcriptional suppression mechanism in brain tumors, similar to its role in other tumors. Several groups have reported that certain tumors have nuclear matrix proteins that are not present in the corresponding normal tissues. For example, human prostate tumors have a nuclear matrix protein that is not present in normal prostate tissue or in benign prostatic hyperplasia. Several malignancy-associated nuclear matrix proteins that are not present in normal breast tissues have been identified in human infiltrating ductal carcinomas of the breast ( Khanuja et al., 1993). Six nuclear matrix proteins that are absent in normal colon tissues have been reported in human colon adenocarcinomas ( Keese et al., 1994). These observations indicate that changes in nuclear matrix structure and protein composition might be related to important changes in the growth and differentiation of cells. Thus, these changes might be used to predict or detect neoplastic transformation. According to the ‘Tensegrity’ model postulated by Ingber (1993), cytoskeletal structure altered by mechanical forces generated through cell– extracellular matrix ( ECM ) interactions can change nuclear morphology and ultimately the pattern of gene expression. Reorganization of the nuclear matrix by extracellular signals may permit tissue-specific gene expression. Analysis of the complete cDNA sequences of the human NRP/B protein indicated a protein of 589 amino acids (aa) with a predicted M of 67 730. Interestingly, r
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a potential second translation initiation codon, ATG, at nucleotide number 220–222 has a Kozak consensus sequence. The resulting product translated from the second initiation codon is predicted to have 516 aa and a molecular mass of 57 kDa. Two-dimensional SDS– PAGE analysis of NRP/B in primary neurons revealed one dominant form of NRP/B protein of 67 kDa, indicating that the first ATG is being utilized in primary neurons and neuroblastoma cell lines. Since NRP/B expression is altered in brain tumors, the possibility that NRP/B utilizes a different ATG in other cell types such as tumors derived from brain glial cells, thereby leading to a different size of NRP/B protein, will be tested in future studies. The genomic organization of NRP/B was determined in this study (Fig. 1). This is the first nuclear neuronal matrix protein whose genomic organization has been determined. Interestingly, the coding regions are localized to one exon, suggesting that NRP/B is highly conserved during evolution. The untranslated region of NRP/B is spread within 3.7 kb and is found primarily in exons 1, 2 and 4. We have mapped the mouse Nrp/b gene to Chromosome 13 using a (C57BL/6J×M. spretus) F ×M. spretus backcross. The localization of 1 the mouse Nrp/b gene to the distal end of Chromosome 13 in a region homologous to the long arm of human Chromosome 5 is consistent with our mapping of NRP/B to Chromosome 5q12–13. The chromosomal localization of the human Enc-1 employing the fluorescence in situ hybridization method, and its mRNA expression in several cell lines, were recently reported ( Hernandez et al., 1998, 1999). Interestingly, it was reported that a defect in the region of human Chromosome 5q12–13 is associated with several types of cancer, including various leukemias, non-Hodgkin’s lymphoma, several skin cancers and spinal muscular atrophy (NCBI, Genome analysis). Spinal muscular atrophy is the second most common lethal, autosomal recessive disease in Caucasians. This disorder is characterized by hypotonia, muscle fasciculation, and decreased spontaneous activity. In addition, frequent mutations were found in the LAZ3/BCL6 lymphoma gene (that encodes a BTB/POZ domain which functions as a DNA-binding transcription repressor) (Dhordain et al., 1997), and are expressed in nonHodgkin’s lymphoma. This gene is also localized to Chromosome 5q12–13, the same chromosomal localization as the NRP/B protein.
Fig. 3. Chromosomal mapping of human genes encoding NRP/B. (A) Mapping of human NRP/B to 5q12–q13. Chromosome 5 and the somatic cell hybrid panel are indicated. The Chromosome 5 content of each hybrid is shown by a solid bar. The deleted segments of Chromosome 5 are indicated to the right. The NRP/B gene localization is indicated on the left. (B) Autoradiogram showing detection of the NRP/B 1.5 and 1.3 kb human-specific bands within the mapping panel. The cell lines from which the DNAs were isolated are shown above each lane. (C ) Mapping of NRP/B in humans to Chromosome 5. PstI digested genomic DNAs from hamster (h), human (H ) and mouse (M ), as well as 24 human/rodent somatic cell hybrids ( labeled 1–22, X and Y ) probed with human NRP/B cDNA. The human-specific RFLP bands are indicated with arrowheads (1.5 and 1.3 kb in size) and are seen in the human control lane and in lane 5.
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Fig. 4. Expression of NRP/B mRNA and protein in human brain tumor cell lines.(A) Expression of NRP/B mRNA in human brain tumor cell lines. The probe used in this blot was derived from the 3∞-gene specific region of NRP/B cDNA (344 bp). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) was used as an internal control for the amount of RNA loaded. (B) Western blot analysis. Total cell lysates obtained from the indicated cells (5×106) were analyzed on 8% SDS–PAGE. Blots were probed with monoclonal anti-NRP/B antibody ( VD2). NRP/B is shown as 67 kDa. Primary hippocampal neurons and astrocytes were prepared from rat brain as indicated in Materials and methods. (C ) Confocal analysis of NRP/B protein in U87-MG human glioblastoma cells. Proliferating U87-MG cells were fixed in 3% paraformaldehyde and immunostained with purified VD2 monoclonal anti-NRP/B antibody. (a) indicates control IgG and (b) immunostaining with NRP/B. Bars=5 mm.
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Fig. 5. Analysis of NRP/B expression in human brain tumors by RT– PCR. Total mRNA was prepared from human brain tumors and NRP/B expression was determined by RT–PCR as described in Materials and methods. The PCR products were electrophoresed on a 2% agarose gel and hybridized with gene-specific probes for NRP/B and actin. Actin was used as an internal control in this set of experiments.
NRP/B sequences contain two domains: the BTB/POZ domain in the N-terminus and the ‘kelch’ repeats in the C-terminus. Kelch protein is a component of intercellular cytoplasmic bridges (ring canals) in Drosophila egg chambers which connect nurse cells and the developing oocyte ( Xue and Cooley, 1993; Robinson et al., 1994). In the ring canals, kelch is co-localized and associates with actin filaments ( Xue and Cooley, 1993; Robinson et al., 1994). Using structural analysis, it was suggested that the ‘kelch’ motifs form a ‘superbarrel’ structure of b sheets (Sondek et al., 1996). Interestingly, crystallographic study of the b-subunit of the G protein (Sondek et al., 1996) revealed that its b-stranded motifs form a ‘superbarrel’ structure and play an important role in protein–protein interactions. Accumulating evidence suggests that the ‘superbarrel’ structure in such signaling proteins might play an important role in recruiting cytoskeletal proteins. By analogy, NRP/B with its six repeat structure may communicate directly with structural elements and recruit nuclear matrix proteins to form a nuclear scaffold structure. Cell-type-specific expression of nuclear matrix proteins has been observed by Fey and Penman (1984). In normal brain tissue, NRP/B appears to be restricted to primary neurons. Furthermore, its spatial and temporal distribution are strongly toward cortical areas, as indicated in developmental studies (Hernandez et al., 1997; Kim et al., 1998). This suggests that the NRP/B protein might be involved in the regulation and coordination of gene expression that control neuronal development. The subcellular localization of the NRP/B protein to the nucleus was determined by immunostaining several cell types including primary hippocampal neurons, and glioblastoma cell lines such as U87-MG. We used mouse
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monoclonal NRP/B and polyclonal NRP/B antibodies for the immunohistochemical analysis. Confocal micrographs using these antibodies indicated that NRP/B was highly expressed in the nucleus of primary neurons and in glioblastomas/astrocytomas, but not in primary astrocytes (Fig. 5). The distribution of NRP/B was condensed in the peripheral heterochromatin and in the nuclear matrix of the nucleoplasm. These results suggest that NRP/B might be involved in the regulation of gene transcription through the dynamic network of nuclear matrix proteins and the structural organization of the nucleus. In summary, the upregulation of NRP/B expression in brain tumors may indicate a potential role of NRP/B in brain tumor development. This role of NRP/B could be due to the involvement of the BTB/POZ domain in tumor regulation as shown in non-Hodgkin’s lymphoma (Chen et al., 1993), and/or to the action of the BTB/POZ as a silencer or co-suppressor of transcription (Dhordain et al., 1997; Nagy et al., 1997). This hypothesis will be tested in future studies.
Acknowledgements We thank Dr. Bijia Deng for her help in establishing rat primary hippocampal cultures. The authors wish to thank Lucy Rowe and Ed Birkenmeier of The Jackson Laboratory for supplying the DNA panel and for performing the analyses of linkage data. We thank Dr. Hava Avraham for her advice and comments on the manuscript, Janet Delahanty for her help in editing the manuscript, Dan Kelley for preparing the artwork, and Mikyung Kim for her typing assistance. This work was supported by grants from the Paul Patton Memorial Trust (R.A.W.) and from Katherine B. Richardson Associates (R.A.W.).
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