A novel human homologue of yeast nucleosome assembly protein, 65 kb centromeric to the p57KIP2 gene, is biallelically expressed in fetal and adult tissues
A novel human homologue of yeast nucleosome assembly protein, 65 kb centromeric to the p57 KIP2 gene, is biallelically expressed in fetal and adult tissuesRen-Ju Hu1, Maxwell P. Lee1, Laura A. Johnson1 and Andrew P. Feinberg1,2,*
Departments of 1Medicine, 2Oncology, and Molecular Biology & Genetics, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205, USA
Received May 23, 1996;Revised and Accepted August 19, 1996
Three genes on 11p15.5 are known to undergo genomic imprinting. The gene for insulin-like growth factor II (IGF2) is normally expressed from the paternal allele, while H19 and p57KIP2, a cyclin-dependent kinase inhibitor, are expressed from the maternal allele. Five germline balanced chromosomal rearrangement breakpoints from patients with Beckwith-Wiedemann syndrome (BWS) have been mapped to 11p15.5 between p57KIP2 and IGF2, and all are derived from the maternal chromosome. By positional cloning from BWS breakpoints, we have isolated a gene 100 kb and 65 kb centromeric to the proximal end of this BWS breakpoint cluster and p57KIP2, respectively. This gene is homologous to yeast nucleosome assembly protein (NAP1) and to a human homologue of NAP1, and we designate it hNAP2 (human nucleosome assembly protein 2). hNAP2 diverges in its expression pattern from IGF2, H19, and p57KIP2, and it shows biallelic expression in all tissues tested. Thus, hNAP2 is functionally insulated from the imprinting domain of 11p15.
Genomic imprinting is a modification of a specific parental chromosome in the gamete or zygote, leading to monoallelic or differential allelic expression in somatic tissues of the offspring. We and others have identified genomic imprinting of three human genes, IGF2, H19, and p57KIP2 (1 -4 ), on chromosomal band 11p15, in the order cen-p57KIP2-IGF2-H19-tel (5 ). In addition to imprinting, these genes, which lie within 800-1000 kb of each other (5 ; unpublished data), also show a similar pattern of developmental and tissue-specific expression (1 -8 ). These observations suggest that these genes are coordinately regulated by chromosome modifications on a scale much larger than that of a single gene, as has been hypothesized for imprinted domains (9 -11 ).
A first step in determining the extent and continuity of such a domain is to identify boundaries between imprinted and nonimprinted genes. Recently, Tsang et al. identified the gene L23MRP, an L23 (mitochondrial) related protein, within 40 kb of H19 (12 ). L23MRP is insulated from the effects of imprinting on the telomeric side of the domain (12 ). In this study, we similarly sought to identify genes centromeric to p57KIP2 and the BWS translocation breakpoints, and to determine their pattern of developmental expression and imprinting. We report here the isolation of a gene, which is homologous to yeast NAP1 and is located 65 kb centromeric to p57KIP2. We also present the characterization of the newly isolated gene with respect to its developmental expression and imprinting status.
We previously isolated a 500 kb cosmid contig from 11p15.5 (5 ). The contig contains a cluster of five germline chromosomal rearrangement breakpoints from BWS patients spanning 350 kb, and we have mapped p57KIP2 40 kb centromeric to this cluster (5 ). Cosmids extending 100 kb centromeric to p57KIP2 (Fig. 1 ) were screened for species-conserved sequences that detected transcripts in fetal and adult tissues. One of these, a 4 kb EcoRI fragment termed 18-4 detected unique bands in Southern blots of various mammals, amphibians and annelids (5 ). Northern blot hybridization revealed a 2.5 kb transcript in all tissues examined (5 ).
18-4 was used to screen a human placental cDNA library. From 2 * 106 plaques, four were identified as positive in primary screening. These positive plaques were further purified to yield a single homogeneous phage stock, and the inserts in recombinant phage were in vivo excised and subcloned into pBluescript and sequenced. All had identical 3' sequences and contained a polyadenylation site. The longest clone, 18-4.4, contained a long open reading frame (375 amino acid) including an ATG start codon, TAA stop codon, and polyadenylation site (Fig. 2 ). Thus, 18-4.4 appeared to contain the entire coding sequence and 3'-untranslated region.
DNA sequencing was performed on both strands, revealing a single large open reading frame containing an ATG start codon at nucleotide 150 (Fig. 2 ). We designated this the translation start site as there was no open reading frame upstream of this codon, and it included a Kozak consensus sequence for translational initiation (13 ). There was a long 1200 nucleotide 3' untranslated region of the gene containing a polyadenylation site. The entire gene encoded a highly acidic predicted protein of 375 amino acids. Interestingly, one of the four cDNA clones had an alternative splicing variant form, which removed the last valine and the stop codon. Consequently, the last valine was replaced by an additional 12 amino acids (KEPSQPAECKQQ).
A search of GenBank and EMBL databases revealed that 18-4.4 showed 58% amino acid conservation with yeast nucleosome assembly protein (NAP1) (14 ). Yeast NAP1 was isolated from S.cerevisiae using antibodies directed against a protein purified from a HeLa cell extract, based on its activity to promote nucleosome assembly (14 ). 18-4.4 also showed 70% nucleic acid homology and 81% amino acid conservation with a second human gene, previously termed hNRP (15 ). We designate 18-4.4 as hNAP2 (human nucleosome assembly protein 2) and rename hNRP as hNAP1, a designation with which K. Siminovitch (15 ) concurs.
Northern blot analysis, quantified on a PhosphoImager, revealed abundant expression of a 2.5 kb transcript at comparable levels in all adult and fetal tissues examined, although the testis showed a threefold higher level of expression than in other tissues (Fig. 4 a,b). These results are in marked contrast to p57KIP2, IGF2 and H19 expression, which show marked tissue and developmental specific expression (1 -8 ).
The pattern of expression of hNAP2 suggested that it is not regulated similarly to the known imprinted genes on 11p15. To test directly for the presence or absence of imprinting of hNAP2, we developed an assay for allele-specific expression. RNA was extracted from 22 fibroblast cell cultures, reverse transcribed, and the cDNA products were PCR-amplified, using six overlapping primer sets spanning the cDNA sequence (Table 1 ). The entire coding region was contained within this set of PCR products, ranging from 220 to 370 bp. The PCR products were then examined by single strand conformational polymorphism (SSCP) analysis (20 ).
Primer pairs B and C (Table 1 ) both detected a transcribed polymorphism, informative in approximately 10% of tested individuals. Subcloning and sequencing of the variant PCR products indicated a sequence change from A to G at nucleotide 443, with no change in the encoded amino acid leucine (Fig. 2 ). To facilitate screening for polymorphic samples using genomic DNA, we sequenced the 3' flanking intron. An intron-specific primer was then synthesized, and it was paired with the forward primer C (Table 1 , primer pair G), for SSCP analysis of DNA (legend to Fig. 5 ). Thus, primer set G could be used for DNA analysis, and primer set C could be used for RT-PCR (legend to Fig. 5 ). Furthermore, as the latter primers were derived from distinct exons separated by 2 kb, amplification of two alleles of the cDNA product could easily be distinguished from inadvertent DNA contamination (Fig. 5 ).
Nine fetal genomic DNA samples were screened for heterozygosity at nucleotide 443, of which one was heterozygous for the polymorphism. RNA was then prepared from this fetal specimen, using adrenal, kidney, lung, heart, liver, limb, placenta and tongue tissue. The RNA was reverse transcribed, and the cDNA was amplified using primer pair C. SSCP analysis revealed biallelic expression of hNAP2 in all tissues tested (Fig. 5 b).
In order to rule out the possibility that imprinting might arise postnatally, we also screened 18 normal kidney specimens from children with Wilms' tumor and identified three samples heterozygous for this polymorphism. RT-PCR again showed biallelic expression in these three normal kidneys (Fig. 5 b, samples 9,12,13). One of the tumors had undergone loss of heterozygosity (LOH) of chromosome 11 (Fig. 5 b lane 9; Fig. 5 c lanes 2-3). LOH was confirmed using the flanking microsatellite markers D11S860 and D11S1318, which are located centromeric and telomeric to hNAP2, respectively (data not shown). Thus, both DNA-PCR and RT-PCR showed only one allele present in the DNA of the tumor (Fig. 5 c), again confirming that the two bands observed on SSCP-PCR of normal tissue correspond to the two transcribed alleles of the gene, and that hNAP2 exhibits biallelic expression.
One of the most intriguing aspects of the study of genomic imprinting is that it may involve a level of cis-acting gene regulation larger in scope than that of a single gene. At least two human chromosomes are now known to harbor multiple imprinted genes spanning hundreds of kilobases to several megabases. These include, on chromosome 15: SNRPN (21 ,22 ), PAR1 and PAR5 (11 ), IPW (23 ), ZNF127 (24 ), and the as yet unidentified Angelman syndrome gene; and on chromosome 11: IGF2, H19, and p57KIP2 (1 -4 ).
Little is known about the organization of these imprinted chromosomal domains or the cis-acting sequences that regulate them. On chromosome 15, several imprinted genes lie within 300 kb of each other, although the imprinted Angelman syndrome gene is separated from SNRPN by the presumably nonimprinted P albinism gene (25 ). Deletion of several kilobases upstream of SNRPN affects imprinting over 2 Mb (10 ,11 ), suggesting that loss of an imprinting control center may have long-range cis-acting effects on gene regulation.
On chromosome 11p15.5, IGF2 and H19 are separated from p57KIP2 by 800-1000 kb (5 ) (unpublished data). All three genes show tissue and developmental specific expression (1 -8 ). All three genes are imprinted, although the expressed parental allele differs among them (IGF2 paternal; H19 and p57KIP2 maternal) (1 -4 ). In addition, all three genes escape imprinting in some brain tissues (1 -4 ). Thus, it would appear that these genes are part of a domain of imprinted genes, similar to the Prader-Willi/Angelman syndrome gene region of chromosome 15. In addition, we have isolated between p57KIP2 and IGF2 five balanced germline chromosomal rearrangement breakpoints from BWS patients (5 ). All five breakpoints are derived from the maternal chromosome, consistent with imprinting in this region.
A unique genomic DNA fragment, 18-4, was gel purified from cosmid B40 (5 ), labeled by random priming (28 ), and used to screen a human placenta [lambda]ZAPII cDNA library (Stratagene). Hybridization and washing conditions were as described (29 ). Positive primary phage were further purified through secondary and tertiary screening. The inserts in [lambda]ZAPII were in vivo excised and subcloned into pBluescript (Stratagene) as recommended by the manufacturer. cDNA clones were sequenced bidirectionally using an ABI 50 automated sequencer. Sequence comparison and analysis of cDNAs were performed using the GCG (Genetics Computer Group) and BLAST (30 ) software packages. Tissue-specific expression was analyzed using multiple tissue Northern blots (Clontech) following standard methods (31 ). The relative expression levels of hNAP2 vs. GAPDH in Northern blots were quantitated by using a PhosphoImager 445SI (Molecular Dynamics) and densitometer (PDI). Mapping of the cDNA was performed using a somatic cell hybrid chromosomal panel (Coriell Cell Repository), using either mouse or Chinese hamster hybrids containing each human monochromosome. Fine mapping was performed using Southern blots to DNA from YAC and cosmid contigs in 11p15.5 (5 ).
Normal fetal tissue was obtained from the University of Washington Fetal Tissue Bank. Frozen Wilms' tumor and matched normal kidney samples were obtained from the Cooperative Human Tissue Network, Children's Cancer Group, and Pediatric Oncology Group. Specimens were maintained at -135oC until use. RNAzol B reagent (Tel-Test, Inc.) was used to isolate RNA from these tissue samples.
Single-strand conformational polymorphism (SSCP) analysis (20 ) was performed, with modifications as described (32 ). Six pairs of PCR primers were designed to cover the entire transcribed region of hNAP2 (Table 1 ). RNA was isolated from fetal tissues and BWS fibroblast cultures using RNAzol B (Tel-Test, Inc.). cDNA was synthesized from ~1 [mu]g RNA using AMV reverse transcriptase (Boehringer Mannheim). For each primer pair, the 5' end of one primer was labeled with [gamma]-32P-ATP using T4 polynucleotide kinase (Boehringer Mannheim). PCR reactions contained 50 nM 5' end-labeled primer, 50 nM of the other unlabeled primer, 75 [mu]M of each deoxyribonucleotide, reverse transcribed cDNA (1/10 of the RT product), and 1 U of Taq DNA polymerase (Boehringer Mannheim). The PCR reactions were performed at 94oC for 1 min, the annealing temperature (Table 1 ) for 1 min, and 72oC for 2 min (30 cycles), followed by an extension for 10 min at 72oC. The PCR products were denatured and electrophoresed on 5% polyacrylamide gels as described (32 ), at 2 W at room temperature overnight.
This work was supported by NIH Grant CA54358 and US Army Breast Cancer Research Program DAMD17-94-J-4308. We thank Dr Paul Grundy, the University of Washington Fetal Tissue Bank, the Cooperative Human Tissue Network, the Children's Cancer Group, and the Pediatric Oncology Group for tissue specimens, and J. Patey for preparing the manuscript.
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