Isolation and characterisation of the NBR2 gene which lies head to head with the human BRCA1 gene
Isolation and characterisation of the NBR2 gene which lies head to head with the human BRCA1 geneChun-Fang Xu1,2,*, Melissa A. Brown1,2, Hans Nicolai1,2, Julie A. Chambers1,2, Beatrice L. Griffiths1,2 and Ellen Solomon1,2
1Somatic Cell Genetics Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK and 2Division of Medical and Molecular Genetics, UMDS, 8th Floor, Guy's Tower, Guy's Hospital, London SE1 9RT, UK
Received January 8, 1997;Revised and Accepted April 14, 1997
To study the regulation of BRCA1 gene expression and the potential importance of dysregulation of this gene in breast and ovarian cancer, we have examined the 5' region of the human BRCA1 gene in detail. We have identified a new gene, NBR2, which is partially related to the NBR1 gene (formerly known as 1A1-3B and mapping directly adjacent to the pseudo-BRCA1 gene) and which lies head to head with the BRCA1 gene. The physical distance between the transcription start sites of the NBR2 and BRCA1 genes is 218 bp, suggesting that regulation of the expression of both genes may be co-ordinated through a bi-directional promoter. The NBR2 gene contains five exons spanning a genomic region of ~30 kb between the BRCA1 and pseudo-BRCA1 genes. Northern analysis showed that the NBR2 gene is expressed in all the tissues examined. The NBR2 cDNA contains an open reading frame of 112 amino acids and is predicted to encode a protein of ~12 kDa. Single-strand conformation polymorphism (SSCP) analysis of the NBR2 gene failed to identify any mutations in either breast or ovarian cancer, suggesting that if the NBR2 gene is involved in the development of these cancers, other mechanisms for tumorigenesis may exist. Hybridisation of NBR2 probes to zoo blots showed that the NBR2 gene is present in human and other primates. No hybridisation to DNA from other species was observed, suggesting that genomic elements controlling BRCA1 expression may differ between species.
It is estimated that ~45% of the families with breast cancer and almost all of the families with both breast and ovarian cancer are accounted for by defects in the breast cancer susceptibility gene BRCA1. Since its isolation in 1994 (1 ), intensive analysis for mutations in this gene has been carried out world-wide and, so far, >100 distinct germline mutations, scattered over the entire coding region of the BRCA1 gene, have been identified (2 ). However, unlike other tumour suppressor genes where both germline and somatic mutations have been found, no somatic mutations in breast cancer and only a handful of somatic mutations in ovarian cancer have so far been identified (3 -5 ). This suggests that either BRCA1 is not involved in the development of sporadic cancer, or that alternative inactivating mechanisms, other than mutation in the coding region, may be important. It is conceivable that such disruption of BRCA1 function could occur both at the transcriptional and the post-transcriptional levels, by mechanisms such as promoter mutation, DNA hypermethylation, alternative splicing or antisense blocking of RNA splicing or translation. In the process of examining such alternative mechanisms, we have been studying the 5' region of the BRCA1 gene and have previously reported identification of the transcription start sites for both the BRCA1 and the NBR1 genes (6 -7 ). We have also shown that the genomic region housing the 5' ends of these genes is duplicated, with a partial copy of the genomic region containing exons 1A, 1B and 2 of the BRCA1 gene lying head to head with the NBR1 gene, while a partial copy of the NBR1 gene, containing exons 1A, 1B and 3, resides <300 bp distal to the transcription start site of the BRCA1 gene (8 ). In this study, we show that this partial copy of the 5' end of the NBR1 gene is, in fact, part of a new gene named NBR2 (Next to BRCA1 gene 2), which is situated in the genomic region between the BRCA1 and pseudo-BRCA1 genes and lies head to head with the BRCA1 gene.
We had found previously that the duplicated genomic fragment containing partial copies of exons 1A and 1B of the NBR1 gene resides in the 5'-flanking region of the BRCA1 gene (8 ). To determine whether these exons were expressed, we used a probe generated from exon 1A of the NBR1 gene to screen a human breast cDNA sublibrary selected with YAC12H4. Four positives were identified, all with an insert of ~600 bp. Sequence analysis showed the four subclones were identical to one another, and subclone 15g2e1 was studied further. The first 164 bp of 15g2e1 sequence was 91% identical with exon 1A of the NBR1 gene (7 ), and the next 54 bp was 96% identical with exon 3 of the NBR1 gene, while no significant homology was identified between the remaining sequences. However, a 100% match was found between 15g2e1 and the genomic sequence at the 5' end of the BRCA1 gene (GenBank accession no. U37574) (6 ) over a stretch of 161 bp, suggesting a new gene might be present in this region. We therefore designated it NBR2.
Oligonucleotide sequences used in PCR, marathon amplification and SSCP analysis
Name
Position
Sequence
PR1
Exon 3
5'-GGACACCGCTGCTGAGGTCCTTGC-3'
G8
Exon 4
5'-GCAGAGCTGGAGCTCGATCATGC-3'
CA8
Exon 2
5'-AAAATAAAATACCTGGATGAGG-3'
G5
Exon 4
5'-AAATGAACAGCCAGATGAAG-3'
1F
5' Flanking
5'-TCACAGTAATTGCTGTACGA-3'
1R
Intron 1
5'-TTCCGCAACGCATGCTGGAA-3'
2F
Intron 1
5'-TCTGAACACCTAGCAATAAGT-3'
2R
Intron 2
5'-CTTCTATGTACATTATAACCT-3'
3F
Intron 2
5'-TGTTCTCCTAATATTCCTTAC-3'
3R
Intron 3
5'-TCCTGAGTAGCTTGGACTACA-3'
4F
Intron 3
5'-CATTCATTTCACCCCTTTCTG-3'
4R
Intron 4
5'-TACCACTGGCTTTCAGGCTAC-3'
5F1
Intron 4
5'-TATAAGTGAGAACGTGTGGTG-3'
5R1
Exon 5
5'-CTTCCAACCATAGTTAGGTAT-3'
5F2
Exon 5
5'-CATGATAGGAGCGTCCTTTGT-3'
5R2
Exon 5
5'-GGATACAAATGAACAGCCAGA-3'
5F3
Exon 5
5'-GCCGGGAGGATGGTGCACCCC-3'
5R3
3' Flanking
5'-ACAGGACCGCCCTCACGTCAG-3'
PR1 and G8 are NBR2-specific Marathon amplification primers. CA8 and G5 are primers used to generate NBR2 cDNA probes. The rest of the primers are used in SSCP analysis.
We have previously constructed a cosmid and PAC contig covering the BRCA1 and NBR1 region (8 -10 ). To map the physical position of the NBR2 gene, probes from the 5' and 3' ends of the NBR2 cDNA were generated and hybridised to filters containing PAC103014, and cosmids A11100, D06121 and B09174 (8 ) (Fig. 3 ). The results confirmed that the NBR2 gene lies between the BRCA1 and pseudo-BRCA1 genes, spanning ~30 kb of genomic DNA. Further sequence analysis of cosmid D06121 revealed that NBR2 is transcribed in the opposite direction from BRCA1 and consists of five exons, with the last exon being alternatively used (Fig. 3 , Table 2 ). The GT-AG pairs which are essential for correct splicing were found at all the intron-exon junctions (Table 2 ). The transcription start site of the NBR2 gene is separated from that of the BRCA1 gene by 218 bp, suggesting that the intergenic region may function as a bi-directional promoter.
To look for conservation of the NBR2 gene across species, probes from the NBR2 cDNA were hybridised to zoo blots (Fig. 4 ). Under the conditions used, hybridisation signals were detected in primates but not in other species, including mouse. This is consistent with previous mapping studies of the BRCA1 region in mouse, which showed that the BRCA1 gene is not duplicated and that it lies directly adjacent to the NBR1 gene (11 ). These results suggest that the NBR2 is of recent origin.ab
We have isolated a new gene NBR2 residing in the genomic region between the breast cancer susceptibility gene BRCA1 (1 ) and NBR1 (7 ). The close proximity of the NBR2 gene to the BRCA1 gene and the potential shared use of an intergenic promoter region raises the possibility that regulation of the expression of these genes may be co-ordinated, or that the proteins encoded by these genes may be involved in the same biochemical pathway. Examples of such divergently transcribed genes include the Wilm's tumour genes WT1/Wit-1 (12 ), the ataxia telangiectasia genes ATM/E14 (13 ), the collagen genes [alpha]1/[alpha]2 (14 ) and TAP1/LMP2 genes (15 ).
BRCA1 is a tumour suppressor gene and yet functionally disrupting mutations are rarely found in sporadic cancers. This raises the possibility that other mechanisms, such as dysregulated expression of the BRCA1 gene, are important in the development of sporadic cancer, and indeed reduced levels of BRCA1 transcripts have been detected in breast tumours (16 ). In an attempt to elucidate the regulation of BRCA1 gene expression, we have analysed the cis-control elements in the promoter region of the BRCA1 gene. Our results have shown that the BRCA1 gene is under complex regulation: firstly, its transcription is under the control of dual promoters generating two distinct transcripts differing by the alternative use of the first exons (6 ) and, secondly, one of these promoters is shared with the NBR2 gene and is bi-directional (manuscript in preparation). The 218 bp intergenic sequence contains no TATA box, but there are other cis-elements that can bind transcription factors and function either uni-directionally or bi-directionally. Thus, the BRCA1 and NBR2 genes may be co-regulated in a spatial and temporal manner, depending on the relative abundance of specific transcription factors and, therefore, when the balance between the expression of the two genes is disrupted, abnormal cell growth and differentiation may occur.
Our data show that the emergence of the NBR2 gene in the BRCA1 neighbourhood is a recent evolutionary event, which probably occurred after the mammalian radiation into different orders ~80-100 million years ago. Due to the absence of the NBR2 gene in species other than primates, the chromatin structure and immediate cis-control elements in the BRCA1 promoter are likely to be significantly different across species. Indeed, sequence comparison between the 5'-flanking region of the human and mouse BRCA1 gene has revealed that only a CCAAT box and an SP1-binding site were conserved between the two species and that other potential binding sites for transcription factors, such as PEA3 and AP1 present in the human BRCA1 promoter (6 ), are absent in the mouse (11 ). Thus, it is very conceivable that the temporal and spatial tissue-specific expression and therefore possibly the function of the BRCA1 gene may be significantly different between the two species. The significance of newly acquired genomic sequence in the control of gene expression in primates has been observed previously in other genes. In particular, it has been very elegantly demonstrated in the apo E-CI-CI'gene cluster, where in the human the 5' end of the recently evolved apo CI'gene controls the tissue-specific expression pattern not only for apo CI but also for the more distantly located apo E (17 ,18 ), suggesting that in mouse without the apo CI'gene the tissue expression pattern of these genes are unlikely to be the same.
Differences in the regulation of human and mouse BRCA1 have implications for the interpretation of animal model experiments and suggest that caution should be exercised when inference of BRCA1 function in humans is made based on experiments performed using mice. Indeed, different phenotypes were observed between a naturally occurring human BRCA1 `knockout' and several experimentally engineered mice knockouts. In the former, the individual is developmentally normal and had breast cancer in her early thirties (19 ) while in the latter the BRCA1-deleted (-/-) mice are embryonic lethal with no evidence of tumour development in heterozygous mice (+/-) up to 1 year of age (20 -22 ). Therefore, when a murine model is used to study the function of a human gene, it must be remembered, as Kinzler and Vogelstein (23 ) recently put it, that human and mice `are not the same beasts'.
YAC clone 12H4 (9 ) was used in the construction of a cDNA sublibrary by hybrid selection (24 ) using a commercially available human breast cDNA library (Clontech). The sublibrary was enriched during two rounds of enrichment-amplification and cloned with EcoRI-XbaI into pBluescript. Approximately 800 clones were obtained and picked into microtitre plates and spotted on filters.
Marathon cDNA amplification was performed following the manufacturer's protocol (Clontech) using 1 [mu]g of poly(A)+ RNA from mammary gland (Clontech) as template. PR1 and G8 (Table 1 ) were used as NBR2 gene-specific primers, in combination with the AP1 primer (provided in the kit), for the 5' and 3' RACE long distance PCR respectively (Boehringer- Mannheim expandT long template PCR system). The PCR products were separated on an agarose gel, purified using a Geneclean II kit (BIO 101), and subsequently cloned into the pGEMT vector (Promega). Upon transformation, white colonies were picked and cultured in 500 [mu]l of L-Broth/Amp for 1 h. Fifty [mu]l of the culture were boiled for 10 min and 1 [mu]l of the supernatant was used as template in PCR screening, with T7 and SP6 primers. The PCR products were column purified (Pharmacia Biotech, MicrospinTM S-400HR) and subjected to automatic sequencing.
Plasmid and cosmid DNA was purified with QIAGEN columns, while PCR products were purified with a MicrospinT S-400HR column (Pharmacia Biotech). Sequencing was performed using a Taq DyeDeoxy Terminator Cycle Sequencing Kit (ABI), and analysed on an ABI 373A sequencer. Sequence analysis was carried out using the GCG programme.
Northern analysis was performed as described previously (11 ). Briefly, poly(A)+ RNA from different tissues [on commercially available MTN blots (Clontech)] were hybridised with an [[alpha]-32P]dCTP-labelled probe generated by PCR using CA8 and G5 primers (Table 1 ) from the NBR2 gene, at 42oC for 18-24 h. Filters were washed twice in 2* SSC, 0.05% SDS at room temperature for 20 min and then twice in 0.1* SSC, 0.1% SDS at 50oC for 20 min.
Zoo blots were prepared as described before (11 ), and hybridised with the above-described NBR2 probe in 5* SSPE, 5* Denhardt's solution, 0.5% SDS, and 0.01% yeast RNA. Filters were washed to either 1* SSC or 0.5* SSC at 65oC for 15 min and exposed to X-ray film for 1-4 days.
Single strand conformation polymorphism (SSCP) analysis was performed as previously described (25 ). Briefly, PCR was performed on genomic DNA with intron-based primers (Table 1 ) flanking each of the exons with [[alpha]-32P]dCTP. Amplified PCR products were diluted 1:4 with loading buffer, denatured at 95oC for 5 min and separated on an MDE gel (J. T. Baker Inc.) for 12-16 h at room temperature. After electrophoresis, the gel was dried and exposed to X-ray film at -70oC overnight.
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*To whom correspondence should be addressed. Tel: +44 171 955 5000 ext. 5581; Fax: +44 171 955 4644; Email: xu@icrf.icnet.uk
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