Genomic organization, chromosomal localization and regulation of expression of the neuronal nuclear matrix protein NRP/B in human brain tumors☆
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 (Dworetzky et al., 1990, Peinta et al., 1989). The nuclear matrix contains a number of associated proteins that have been found to be involved in malignant transformation (Fey and Penman, 1984, Getzenberg et al., 1996, Keese et al., 1994). 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 and Jeon, 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 (von Bulow et al., 1995, Way et al., 1995, Xue and Cooley, 1993) 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.
Section snippets
Materials
Chemical reagents were purchased from Sigma (St. Louis, MO). The murine fetal λ-gt10 cDNA library was 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
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 (γ-EMBL-3), for genomic clones under conditions of high stringency with a 260 bp
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
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
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This paper is dedicated to Raphael Recanati and his family for their friendship and support for our research program.
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Both authors contributed equally to this work.