npg
Genomic imprinting of p57KIP2, a cyclin-dependent kinase inhibitor, in mouse
Izuho Hatada & Tsunehiro Mukai
National Cardiovascular Center Research Institute, 5-7-1, Fujishiro-dai, Suita, Osaka 565, Japan
Correspondence should be addressed to I.H.
p57KIP2 is a potent tight-binding inhibitor of several G, cyclin/Cdk complexes, and is a negative regulator of cell proliferation1.2. The gene encoding human p57KIP is located on chromosome 11p15.5 (ref. 2), a region implicated in both sporadic cancers and Beckwith-Wiedemann syndrome, a familial cancer syndrome, marking it a tumour suppressor candidate. Several types of childhood tumours including Wilm’s tumour, adrenocortical carcinoma and rhabdomyosarcoma display a specific loss of maternal 11p15 alleles, suggesting that genomic imprinting3-8 plays an important part9-12. Genetic analysis of the Beckwith-Wiedemann syndrome has indicated maternal carriers as well as suggested a role in genomic imprinting13. Here, as a first step towards elucidating the genesis of human cancers in this region, we showed that a mouse homologue of p57KIP2 is genomically imprinted. The paternally inherited allele is transcriptionally repressed and methylated. This murine gene maps to the distal region of chromosome 7, within a cluster of imprinted genes, including insulin-2, insulin-like growth factor- 2, H19 and Mash2 (refs 14-18).
To clarify whether the p57KIP2 gene is expressed from one or both parental alleles, we analyzed p57KIP2 for allelic differences between the two mouse parental strains, C57BL/6 and PWK, by searching for sequence polymorphisms in products generated by RT-PCR19 and found a 24-bp deletion (cDNA nucleotides 877 to 900) in the PWK p57KIP2 (Fig. la). We analyzed by RT-PCR the mRNA phenotypes of newborn mice (whole bodies), adult heart, lung, skeletal muscle, kidney and liver derived from F, progeny of the reciprocal cross between C57BL/ 6 and PWK; that is, (PWK× C57BL/6)F, and (C57BL/6x PWK)F,. Only a 198-bp fragment corresponding to the PWK allele could be detected in (PWK x C57BL/6)F , and only a 222-bp fragment corresponding to the C57BL/6 allele could be detected in neonatal (C57BL/6 x PWK)F, mice (Fig.1b). We eliminated the possibility of genomic contamination by running concurrent PCR assays of RNA samples that had not been reverse transcribed; these controls invariably gave negative results. The identical results were obtained from the mRNA from the various tissues mentioned above (Fig. 1b). These findings indicate that p57KIP2 is expressed only from the maternal allele and is repressed in the paternal allele.
We next examined whether this gene is subject to allele- specific methylation, by performing PCR followed by digestion of genomic DNA with the methylation-sensitive restriction enzyme Hhal (refs 20,21). Primers were designed to amplify a 222-bp PCR product that included a translated region (nucleotides 877 to 900 of the cDNA) and three Hhal sites. These are the same primers used for RT-PCR. These primers amplified a region which has a feature unique to imprinted genes, that is, iterated repeats in the CpG island as observed in other genes including Igf2, Igf2r, SNRPN and U2af1-rs1. Such regions were
a
1@
20
30
40
50
60
70
sa
90
gccpogtat gagctoggaa ctgagccggt ctctgagcag ggcgaggage agggcgcaga gccpgtcgog gagaaggacg aggagccgga
100
110
120
130
14@
150
168
170
180
ggoggagcag ągcącggage eggtegagga gcagggtgeg gagccodice aggagcagea togggagceg gtegaggage aggacgagoal
190
200
210
220
230
24@
250
260
270
tcaagagcag egcggccagg agctgaagga ccagcctete t
b
Neonate (whole body)
12345678
222bp
198bp
Reverse transcriptase
+
Heart
Lung
Skeletal muscle
12345678 1234567812345678
222bp
198bp
Reverse transcriptase +
Kidney
Liver
12345678 12345678
222bp
198bp
Reverse transcriptase
+
subjected to mono-parental methylation22. Indeed, before directly demonstrating the imprinting of the p57KIP2 gene, we had first found repeats in the CpG island of the gene, suggesting that this unique characteristic could be used as a marker to search for imprinted genes. The two parental alleles were distinguished by the length polymorphism on RT-PCR (see above). Only a 222-bp
(PWK × C57BL/6)F (C57BL/6 x PWK)F
C57BL/6 PWK
222bp
198bp
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a
D7Mit12
Kip2
Igf2
☒
☒
☒
D7Mit47
☒
☒
49 49
2
1
0
0
0
1
7
b
D7Mit12
2.9
1.0
Igf2,
Kip2
D7Mit47
fragment corresponding to the C57BL/6 allele could be detected in (PWK x C57BL/6)F1, and only a 198-bp fragment corresponding to the PWK allele could be detected in (C57BL/6 x PWK)F, (Fig. 2). This indicates that the Hhal sites are methylated exclusively on the paternal allele, which is consistent with a functional role of DNA methylation in the repression of gene expression.
The human p57KIP2 gene maps to 11p15.5 (ref. 2), and Igf2, Ins2 and H19have all been placed in this region. The short arm of chromosome 11 shares a region of homology with the very distal region of mouse chromosome 7. Igf2, Ins2 and H19 are located in this mouse chromosome region and are tightly linked, suggesting that the mouse p57KIP2 gene will also reside in this region. Our mapping results place p57KIP2 in the distal region of mouse chromosome 7 linked to D7Mit12, Igf2 and D7Mit47 (Fig. 3a,b). The ratios of the total numbers of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and most likely gene order were:
centromere-D7Mit12-(3/102)-p57KIP2_(0/102)-Igf2- (1/102)-D7Mit47
The recombination frequencies (expressed as genetic distance in centiMorgans (cM) +/- the standard error) were:
D7Mit12-2.9+/-1.7-p57KIP2, Igf2-1.0+/-1.0-D7Mit47
Although not shared by all imprinted genes, one feature of these products is that they are involved in regulation of growth, as is true for Igf2, Ins1, Ins2, Igf2r, H19 and Mas. The product of p57KIP2 also shares this criteria because overexpression of p57KIP2 arrests the cells in G, (ref. 2), suggesting that this feature also could become a marker by which to search for imprinted genes. Embryos with maternal duplication of distal chromosome 7 die in late
gestation or at birth with marked reduction in both fetal and placental size23,24. This reduction in growth may be caused at least partly by the dosage effect of the p57KIP2 gene product in maternal disomy mice, although the possibility of effects by other imprinted genes clustered in this region cannot be excluded.
The chromosomal location of human p57KIP2, 11p15.5, marks it as a candidate tumour suppressor2. This region has been investigated intensively because of the frequent loss of heterozygosity at this locus in a number of human cancers including breast, bladder, lung, ovarian, kidney and testicular carcinomas25. Several types of childhood tumours, including Wilms’ tumour, adrenocortical carcinoma and rhabdomyosarcoma, display a specific loss of the maternal 11p15 alleles, suggesting that genomic imprinting plays a role in these cancers9-12. In addition, rearrangements in the 11p15 region have been linked to Beckwith-Wiedemann Syndrome, which is characterized by numerous growth abnormalities including macroglossia, gigantism, visceromegaly, exomphalos and an increased risk of childhood tumours. Genetic analysis indicated maternal carriers, also suggesting that genomic imprinting plays a role in this syndrome. Thus, it is possible that the p57KIP2 gene, whose paternal allele is repressed by genomic imprinting, could be a tumour suppresor like the other cyclin-dependent kinase inhibitor, p16, on chromosome 9p21 (ref. 26). If the maternal allele is inactivated by chromosome loss or rearrangement, the complete loss of p57KIP2 in the cell could possibly result in tumorigenesis due to acceleration of the cell cycle.
Methods
RT-PCR. Total RNA prepared using the acid-phenol method27 was applied to RT-PCR as described previously19. The primer sequences used for amplifying the 222-bp product were: 5’-GCCGGGTGATG- AGCTGGGAA-3’ and 5’-AGAGAGGCTGGTCCTTCAGC-3’ (forward and reverse, respectively).
Hhal-sensitive PCR assay. Genomic DNA was digested with Xbal in addition to Hhal to reduce the DNA size and thus facilitate PCR. The sequences of the primers used were 5’-GCCGGGTGATGAGC- TGGGAA-3’and5’-AGAGAGGCTGGTCCTTCAGC-3’ (forward and reverse, respectively).
Genetic mapping. We mapped the p57KIP2 locus by interspecific backcrossing using progeny derived by mating ((C57BL/6 x M. spretus)F1 xC57BL/6) mice. DNAs ofinterspecific backcross progeny were obtained from the UK Human Genome Mapping Project Resource Centre. A total of 102 mice were used to map the p57KIP2 locus. The p57KIP2 locus was amplified by PCR using primers of the following sequences: 5’-ATGGAGGTGGACAGCGAGTC-3’ and 5’-AGAGAGGCTGGTCCTTCAGC-3’ (forward and reverse, respectively). We found an Aval site in the amplified C57BL/6 DNA, but no such site in amplified M. spretus DNA. Therefore, we utilized this polymorphism for the genotyping of backcross mice. A description of the polymorphisms for loci linked to p57KIP2 has been reported previously28,29.
Acknowledgements
We thank S. Ohishi and K. Tohyama for technical assistance. This work was supported by a Grant-in Aid for Scientific Research from the Ministry of Education and Science, Special Coordination Funds for promoting Science and Technology (Encouragement System of COE) from the Science and Technology Agency, a Research Grant for Aging from the Ministry of Health and Welfare of Japan.
Received 9 June; accepted 25 July 1995.
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