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Absence of P16 and Truncation of ARF Tumor Suppressors in Chickens

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Absence of P16 and Truncation of ARF Tumor Suppressors in Chickens Absence of p16INK4a and truncation of ARF tumor suppressors in chickens Soo-Hyun Kim*, Michael Mitchell†, Hideta Fujii‡, Susana Llanos*§, and Gordon Peters*¶ *Molecular Oncology, †Computational Genome Analysis, and ‡Developmental Genetics Laboratories, Cancer Research U.K., London Research Institute, Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved November 14, 2002 (received for review September 13, 2002) The INK4b-ARF-INK4a locus on human chromosome 9p21 (Human Whereas INK4a operates upstream of pRb (3, 4), the ARF Genome Organization designation CDKN2B-CDKN2A), and the cor- protein functions upstream of p53 by binding directly to MDM2 responding locus on mouse chromosome 4, encodes three distinct and protecting p53 from MDM2-mediated degradation (1, 2). products: two members of the INK4 cyclin-dependent kinase in- Current thinking is that the INK4a͞ARF locus plays a key role hibitor family and a completely unrelated protein, ARF, whose in cellular defenses against hyperproliferative signals and stress. carboxyl-terminal half is specified by the second exon of INK4a but For example, INK4a accumulates in human diploid fibroblasts in an alternative reading frame. As INK4 proteins block the phos- (HDFs) that undergo replicative senescence, either as a conse- phorylation of the retinoblastoma gene product and ARF protects quence of telomere attrition or in response to oncogenic Ras p53 from degradation, the locus plays a key role in tumor suppres- (9–11). Similarly, ARF accumulates as mouse embryo fibro- sion and the control of cell proliferation. To gain further insights blasts (MEFs) approach their replicative limits and in response into the relative importance of INK4a and ARF in different settings, to a variety of oncogenes (12–14). However, there are clear we have isolated and characterized the equivalent locus in chick- differences in the relative importance of INK4a and ARF in cells ens. Surprisingly, although we identified orthologues of INK4b and from different lineages or species (14–17) and in the way they are ARF, chickens do not encode an equivalent of INK4a. Moreover, the regulated. For example, Ras induces ARF in MEFs but not in reading frame for chicken ARF does not extend into exon 2, HDFs (15, 18, 19), whereas pRb represses INK4a in HDFs but because splicing occurs in a different register to that used in not in MEFs (10, 20). There have also been suggestions that the mammals. The resultant 60-aa product nevertheless shares func- sequences encoded by exon 2 make different contributions to the tional attributes with its mammalian counterparts. As well as intracellular localization and function of ARF in the two species indicating that the locus has been subject to dynamic evolutionary (21, 22). pressures, these unexpected findings suggest that in chickens, the To gain further insight into these questions, we sought to tumor-suppressor functions of INK4a have been compensated for isolate the equivalent locus from chicken, both because it by other genes. represents an intermediate between fish and man in evolutionary terms and because of the relative resistance of chicken cells to ͞ immortalization in tissue culture, similar to HDFs. After char- he INK4a ARF locus has an important role in the control of acterizing 18 kb of genomic DNA and two groups of cDNA Tcell proliferation and in tumor suppression (1, 2) and is clones from late-passage chicken embryo fibroblasts (CEFs), we incapacitated in a variety of familial and sporadic cancers (3, 4). conclude that the chicken INK4b-ARF-INK4a locus is able to INK4a The INK4a product, p16 , functions as an inhibitor of encode an equivalent of p15INK4b and a truncated yet functional cyclin-dependent kinases (Cdks) 4 and 6 (5), hence the official version of ARF specified only by exon 1␤. Surprisingly, a partial designation CDKN2A. These Cdks, along with D-type cyclins, duplication of exon 1␤ has replaced exon 1␣, and we find no regulate the phosphorylation of the retinoblastoma protein evidence that chicken cells contain a p16INK4a orthologue. (pRb) in the late G1 phase of the cell cycle (6). Interestingly, p16INK4a is the prototype of a family of INK4 proteins, each Materials and Methods comprising between 3 and 5 ankyrin repeats (3, 4), orthologues Cells. Primary CEFs were grown at 37°C in DMEM supple- of which have been identified in a variety of mammals as well as mented with heat-treated 10% (vol͞vol) FCS and 2% chicken in Fugu and Xiphophorus fish (7, 8). All members of the gene serum (GIBCO͞BRL). The U20S human osteosarcoma family isolated thus far show the same exon 1–exon 2 splice cell line and the NARF-2 derivative line in which human junction and, in mammals, INK4b and INK4a occur in a con- p14ARF is expressed from an isopropyl-␤-D-thiogalactoside served tandem arrangement on human chromosome 9p21 and (IPTG)-regulated promoter were cultured as described (23). syntenic regions on mouse chromosome 4 and rat chromosome Cells were transiently transfected by calcium phosphate precip- 5, suggesting that they evolved by a gene-duplication event (3). itation and harvested after 48 h. Retroviral infection of the TIG3 Because there is no evidence for such a duplication in Fugu (8), strain of HDFs expressing the ecotropic virus receptor was as it appears that INK4b was the primordial gene at this locus. described (24). However, the INK4a locus has the highly unusual capacity to encode two structurally and functionally different proteins. Two Bacterial Artificial Chromosome (BAC) and cDNA Libraries. The transcripts, designated ␣ and ␤, are produced; they initiate at chicken BAC library was obtained from the UK Human Genome separate promoters and incorporate different first exons (1␣ and 1␤) spliced to a common second exon (1, 2). Whereas the MEDICAL SCIENCES ␣-transcript specifies p16INK4a, the ␤-transcript encodes p14ARF This paper was submitted directly (Track II) to the PNAS office. (p19ARF in mouse), so-called because the second exon is trans- Abbreviations: HDF, human diploid fibroblast; CEF, chicken embryo fibroblast; BAC, bac- Ϫ terial artificial chromosome; MTAP, methylthioadenosine phosphorylase; Cdk, cyclin- lated in the 1 (alternative) reading frame to that used to dependent kinase; HA, hemagglutinin. INK4a ␤ generate p16 . As exon 1 is poorly conserved and has no Data deposition: The sequences reported in this paper have been deposited in the GenBank obvious relatives in the current databases, its evolutionary database (accession nos. AY138245–AY138247). origins remain unknown. For example, did exon 1␤ originally §Present address: Ludwig Institute for Cancer Research, St. Mary’s Hospital, London W2 belong to a different gene and move to the INK4a͞b locus at 1PG, United Kingdom. some point after the INK4a͞b duplication? ¶To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0135557100 PNAS ͉ January 7, 2003 ͉ vol. 100 ͉ no. 1 ͉ 211–216 Downloaded by guest on October 1, 2021 Mapping Project Resource Center. The cDNA library was constructed from CEFs at Ϸ50 population doublings by using the ␭ZAP-II cDNA synthesis kit according to the manufacturer’s protocols (Stratagene). After screening of 1.35 ϫ 106 recombi- nant phages (unamplified), 170 plaques showed positive signal, of which 50 were purified and analyzed further. DNA and RNA Analyses. CEF genomic DNA was isolated by using the Easy-DNA kit (Invitrogen) and analyzed by Southern hy- bridization performed at either 60°C (normal stringency) or 55°C (low stringency) following standard procedures (25). Total and polyadenylated RNA was prepared from late passage CEFs by using the RNeasy kit (Qiagen, Chatsworth, CA) and the Poly(A) Pure kit (Ambion, Austin, TX), respectively. RNA blots were hybridized at 42°C in ULTRAhyb buffer (Ambion) and washed according to the manufacturer’s protocol. The PCR primer sequences used to generate the probes described in Fig. 2A and the conditions for the touch-down RT-PCR of methyl- Fig. 1. Organization of the INK4b͞ARF͞INK4a locus in different species. (A) thioadenosine phosphorylase gene (MTAP) are detailed in Schematic representations (not to scale) of the INK4b, ARF, INK4a, and MTAP Supporting Text, which is published as supporting information on genes in Fugu, humans, and chickens. INK4b exons are shown as black, ARF as the PNAS web site, www.pnas.org. cross-hatched, INK4a as stippled, and MTAP as empty boxes. The arrows indicate the direction of transcription. Dotted lines denote the splicing for the Plasmid Construction. The longest chicken ARF cDNA was used ARF transcript. (B) Restriction map of the chicken INK4͞ARF locus. Exons are as a template for PCR by using primers that introduced a BglII depicted as in A. All XbaI (X), HindIII (H), BamHI (B), SfiI (Sf), BglII (Bg), and PstI site at the presumed 5Ј end of chicken ARF (excluding the ATG) (P) sites are indicated. The locations of critical NotI and XhoI fragments and an XbaI site downstream of potential termination codons in described in the text are shown below the map. The two sets of differently all three reading frames of exon 2 (nucleotides 150–816 in the shaded boxes above the map denote regions of the genomic sequence that are directly duplicated (see Fig. 6A). sequence deposited under accession no. AY138245). The result- ant 683-bp product was cloned into the pEGFP-C3 vector (CLONTECH) to create an in-frame fusion protein with GFP Ch precipitation, we used SMP14 for MDM2, Y-11 for HA-tagged at its N terminus, designated GFP-ARF . A second plasmid proteins (sc-805, Santa Cruz Biotechnology), and H-22 for Cdk4 was generated comprising nucleotides 147–326 of chicken Ј Ј (sc-601, Santa Cruz Biotechnology). Methods used to visualize ARF cDNA flanked by a 5 BglII site and a 3 KpnI site. The human ARF and the various GFP fusion proteins by direct and 196-bp PCR product was cloned into the pEGFP-N1 vector indirect immunofluorescence, and to assay p53 stabilization by (CLONTECH) in which the initiation codon for the GFP has ARF, were described (26).
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