Molecular cloning and characterization of the human KIN17 cDNA ...

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The N-terminal domain of kin17 protein is conserved among ively, we used an ..... are also relocalized after DNA damage: XPG protein (41),. 3.Herrlich,P.
Carcinogenesis vol.21 no.9 pp.1701–1710, 2000

Molecular cloning and characterization of the human KIN17 cDNA encoding a component of the UVC response that is conserved among metazoans

Patricia Kannouche3, Philippe Mauffrey, Ghislaine Pinon-Lataillade, Marie Genevie`ve Mattei1, Alain Sarasin2, Leela Daya-Grosjean2 and Jaime F.Angulo4 Laboratoire de Ge´ne´tique de la Radiosensibilite´, De´partement de Radiobiologie et de Radiopathologie, Direction des Sciences du Vivant, Centre d’Etudes de Fontenay-aux-Roses, CEA, 60-68 Avenue du Ge´ne´ralLeclerc, BP 6, 92265 Fontenay-aux-Roses Cedex, 1Unite´ INSERM 491 ‘Ge´ne´tique Me´dicale et De´veloppement’, Faculte´ de Me´decine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5 and 2Laboratoire de Ge´ne´tique Mole´culaire, UPR 42 CNRS-IFC1 Institut de Recherches sur le Cancer, BP 8, 94801 Villejuif, France 3Present

address: MRC Cell Mutation Unit, Sussex University, Falmer, Brighton BN1 9RR, UK 4To

whom correspondence should be addressed Email: [email protected]

We describe the cloning and characterization of the human KIN17 cDNA encoding a 45 kDa zinc finger nuclear protein. Previous reports indicated that mouse kin17 protein may play a role in illegitimate recombination and in gene regulation. Furthermore, overproduction of mouse kin17 protein inhibits the growth of mammalian cells, particularly the proliferation of human tumour-derived cells. We show here that the KIN17 gene is remarkably conserved during evolution. Indeed, the human and mouse kin17 proteins are 92.4% identical. Furthermore, DNA sequences from fruit fly and filaria code for proteins that are 60% identical to the mammalian kin17 proteins, indicating conservation of the KIN17 gene among metazoans. The human KIN17 gene, named (HSA)KIN17, is located on human chromosome 10 at p15–p14. The (HSA)KIN17 RNA is ubiquitously expressed in all the tissues and organs examined, although muscle, heart and testis display the highest levels. UVC irradiation of quiescent human primary fibroblasts increases (HSA)KIN17 RNA with kinetics similar to those observed in mouse cells, suggesting that up-regulation of the (HSA)KIN17 gene after UVC irradiation is a conserved response in mammalian cells. HSAkin17 protein is concentrated in intranuclear focal structures in proliferating cells as judged by indirect immunofluorescence. UVC irradiation disassembles HSAkin17 foci in cycling cells, indicating a link between the intranuclear distribution of HSAkin17 protein and the DNA damage response. Introduction UV radiation produces DNA lesions that perturb DNA metabolism and activate multiple and complex signal transduction pathways (1). In mammalian cells, the UV response counterAbbreviations: BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; FCS, foetal calf serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IIF, indirect immunofluorescence; MEM, minimum Eagle’s medium; NLS, nuclear localization signal; PBS, phosphate-buffered saline; 3⬘-UTR, 3⬘-untranslated region; 5⬘-UTR, 5⬘-untranslated region. © Oxford University Press

balances these deleterious effects, thereby allowing cell recovery and improving survival (2). Of the 100 or so reported UV-responsive genes, nearly 60% are involved in general processes, including cell adhesion, membrane receptors and lipid metabolism, and ~40% participate in cell growth, of which ⬍5% play a role in DNA repair [calculated from data describing the mammalian genes inducible at the mRNA or protein level by radiation or DNA-damaging agents reported by Friedberg et al. (1), Ch. 13, pp. 602–603, table 13-2]. The primary effect of UV is activation of growth factor/receptor kinases at the cell membrane level (3), although the late UVresponsive genes appear to be directly induced by a nuclear signal produced by UV-induced DNA damage (4). The molecular basis of the nuclear components of the UV response remains to be elucidated. We have identified (MMU)Kin17, a mouse gene coding for a 45 kDa nuclear DNA-binding protein which seems to be a novel component of the UV response (5,6). Initially, MMUkin17 protein was identified using antibodies raised against bacterial RecA protein. The primary structures of RecA and MMUkin17 are different. The cross-reactivity is due to a 40 residue domain displaying 49% homology with the C-terminal region of RecA, a domain involved in the regulation of DNA binding and in the SOS response (7,8). The biochemical activity of MMUkin17 protein has not been determined yet but it has been proposed to participate in illegitimate recombination or in regulation of gene expression (9,10). We showed that MMUkin17 protein forms intranuclear foci and that its overexpression reduces DNA synthesis and inhibits growth of mammalian cells (11,12). UVC irradiation of growth-arrested mouse fibroblasts increases (MMU)Kin17 RNA levels and the cellular concentration of MMUkin17 protein, indicating participation of the (MMU)Kin17 gene in the UVC response (5). Furthermore, γ-irradiation of cultured rat cells induces the accumulation of kin17 protein, suggesting a role in a general DNA damage response (13). Kannouche et al. reported that MMUkin17 protein forms nucleoplasmic foci (7,11) similar to those observed for proteins involved in DNA repair (14–17), replication (18) and RNA splicing, as judged by indirect immunofluorescence (IIF) or electron microscopy (19,20). These proteins are located in particular areas of the nucleus, like the DNA attachment sites or peri- and interchromatin regions (20). This distribution allows rapid access to their substrates, thus facilitating DNA replication and transcription (11,14,18). Here we report the first characterization of the (HSA)KIN17 cDNA and localization of the (HSA)KIN17 gene on chromosome 10, at p15–p14. The (HSA)KIN17 gene is ubiquitously expressed in human tissues and in cultured cells. The encoded protein forms intranuclear foci in proliferating cells. UVC irradiation increases (HSA)KIN17 RNA levels and changes the distribution of HSAkin17 protein. Our results indicate that participation of the (HSA)KIN17 gene in the response to UVC is conserved among mammals and suggest that there is a correlation between the subnuclear localization of HSAkin17 protein and the DNA damage response. 1701

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Materials and methods Cells and culture conditions MRC-5 fibroblasts (21) were obtained from Dr M.Mezzina (IRSC, Villejuif, France), LoVo cells (22) from Dr M.-F.Poupon (Curie, Paris, France), K562 and HL60 cells (ATCC CCL-243 and CCL-240) from Dr W.H.Fridman (Curie, Paris, France), human mammary HBL100 cells (23) from Dr G.Goubin (Curie, Paris, France), HeLa cells (ATCC CCL2) from Dr E.May (CEA, Fontenay aux Roses, France) and Boleth cells (24) from Dr G.Frelat (CEA, Fontenay aux Roses, France). Human primary fibroblasts, MRC-5 fibroblasts and HBL100 cells were cultured in minimum Eagle’s medium (MEM), 10% foetal calf serum (FCS) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), 10% FCS, and antibiotics. LoVo, Boleth, HL-60 and K562 cells were cultured in RPMI 1640, 15% FCS and antibiotics. HeLa cells were made quiescent by 72 h incubation of confluent cells in DMEM, 0.25% FCS, whereas human primary fibroblasts were arrested by 24 h incubation in MEM, 0.5% FCS. Cloning of (HSA)KIN17 cDNA One microgram of RNA from Boleth cells was reverse transcribed using oligo(dT)16 (GeneAmp; Roche Molecular Systems, USA). The mixture of cDNAs was amplified by PCR using oligonucleotides A (5⬘-TCAAAGACAACTGTTGCTGGC-3) and B (5⬘-ATACCTTCAACTCTGCGTCCTT-3⬘) from (MMU)Kin17 cDNA. The 1000 bp DNA fragment, called P1000, was subcloned in plasmid pMOSBlue (Amersham Life Science) to give pMOSBlueP1000. 32P-labelled P1000 DNA was used to screen a λgt11 cDNA library from human testis. Plaques (2.5⫻105) were plated on Escherichia coli Y1090 bacteria and transferred to Hybond-N membranes (Amersham Life Science). The seven positive clones obtained were further characterized as described (6). Rapid amplification of cDNA ends (RACE) from human prostate enabled the isolation of 230 bp of the 5⬘-region (Marathon-Ready cDNAs; Clontech) using primer C (5⬘-CCTGGTGCTGGAATTACTGTCT-3⬘) for initial amplification and primer D (5⬘-CTCTGATGAGATTCGGACATACAAT-3⬘) for the nested reaction. Thereafter, the (HSA)KIN17 cDNA was amplified by PCR using oligonucleotides E (5⬘-AGAAAGTGATCGCTGCCG-

TGGT-3⬘) and F (5⬘-GCGAACACCAATTTGATGCTTTAAGA-3⬘) and introduced into pMOSBlue, generating plasmid pMOSBlue-HSAKin17, here named PK1. DNA fragments were sequenced using the Thermo Sequenase dye terminator cycle sequencing kit (Amersham) in an ABI 373 automatic sequencer with vector- and gene-specific primers. The chromatograms were analysed with EditView and SeqEd (Applied Biosystems, France). Gene mapping by in situ hybridization Chromosome spread preparations were made from phytohaemagglutininstimulated human lymphocytes cultured for 72 h. 5-Bromodeoxyuridine was added (final concentration 60 µg/ml) for the last 7 h of culture to ensure post-hybridization chromosomal banding of good quality. The DNA of pMOSBlueP1000 was tritium labelled by nick translation to a specific activity of 1⫻108 d.p.m./µg The radiolabelled probe was hybridized to metaphase spreads at a final concentration of 100 ng/ml hybridization solution as previously described (25). After coating with nuclear track emulsion (NTB2; Kodak), the slides were exposed for 20 days at 4°C, then developed. To avoid any slipping of silver grains during the banding procedure, chromosome spreads were first stained with buffered giemsa solution and metaphases were photographed. R-banding was then performed by the fluorochrome–photolysis– giemsa method and metaphases were rephotographed before analysis. Overproduction of kin17 protein In E.coli. The ORF of (HSA)KIN17 cDNA was placed in vector pET19b under control of the T7 promoter, in-frame with a 5⬘-end sequence coding for six histidine residues (Invitrogen, USA). The resulting plasmid, pET19b(His)6(HSA)KIN17, coding for a chimeric protein of 49 kDa called PK17, was introduced into BL21 bacteria. The production of (His)6–HSAkin17 protein was induced with IPTG and monitored by western blot as previously described (26). In HeLa cells. The ORF of (HSA)KIN17 cDNA was inserted in pCMVDT21, placed downstream of the CMV promoter (7), producing plasmid pCMV(HSA)KIN17. HeLa cells grown to 50% confluence were transfected with pCMV(HSA)KIN17 using Fugene 6 (Boehringer Mannheim). After 20– 30 h incubation in complete medium, cells were processed for western blot or IIF as described below.

Fig. 1. Sequences of mouse and human KIN17 cDNAs and the deduced proteins. The nucleotides of the human and mouse cDNAs are shown in the top and bottom rows of each line. Both sequences include 65 nt of the 5⬘-UTR, the whole ORF and the known nucleotides of the 3⬘-UTR. The nucleotides are numbered beginning with the first nucleotide of the cDNA. The human and mouse kin17 protein sequences (393 and 391 residues, respectively) are indicated between the nucleotide sequences. Identical residues are indicated by a dot in the mouse sequence. The zinc finger motif (amino acids 28–50) and the region homologous to RecA (amino acids 163–201) are underlined. The bipartite nuclear localization signal (NLS, amino acids 240–257) and the ARE motifs in the 3⬘-UTR are marked in bold. The EMBL accession numbers of the human and mouse sequences are AJ005273 and X58472, respectively.

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UVC induces relocalization of UVC irradiation conditions Human primary fibroblasts were irradiated with a germicidal lamp at 254 nm at 0.2 J/m2/s. Dosimetry was performed with a UV radiometer CX-254 (Vilber Lourmat, Marne la Valle´ e, France). Prior to irradiation, confluent cells were incubated for 24 h in MEM, 0.5% FCS. The medium was removed and cells were washed with phosphate-buffered saline (PBS) before irradiation. Control cells were treated in the same way but not irradiated. At the indicated times, cells were harvested and RNA was extracted. HeLa cells were irradiated

Fig. 2. The N-terminal domain of kin17 protein is conserved among metazoans. Comparisons with the EST databases indicated a high degree of identity among human, mouse, fruit fly and filarial kin17 proteins. The ETS sequences were aligned using MACAWZ v.2.0.2. Letter code prefixes are: HSA, Homo sapiens; MMU, Mus musculus; DME, Drosophila melanogaster; BMA, Brugia malayi. Identities are shown at the bottom. Of 62 residues, 42 are identical and six similar. The EMBL accession numbers are: HSA, AJ005273; MMU, X58472; DME, DME6528; BMA, AA110577.

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during the exponential growing phase. Alternatively, they were irradiated 20 h after transfection, rinsed in PBS at the indicated time and processed for IIF. IIF microscopy Cells fixed in cold methanol/acetone (30:70% v/v) for 10 min at –20°C were rehydrated in PBS for 15 min at room temperature. After 60 min incubation with pAb2064 or pAbanti-RecA antibodies diluted in PBS containing 3% bovine serum albumin (BSA), cells were extensively washed with PBS. Thereafter, they were incubated for 45 min with Cy2-conjugated affiniPure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) diluted 1:500 in PBS, 3% BSA. After washing three times for 5 min with PBS, the nuclear DNA was stained with 0.2 µg/ml DAPI and cells were mounted with Glycergel (Dako) as described (7). No significant signal attributable to secondary antibody alone was detected. Cells were photographed with a Zeiss Axiophot2 Photomicroscope equipped with phase contrast and epifluorescence optics using Plan-Neofluar lenses. Images were recorded on Kodak films. Alternatively, we used an on-chip thermoelectronically cooled charge coupled device camera (Coolview; Photonic Science, UK). After scanning the preparations by eye, representative images were saved as grey scales and then coloured using KS300 software (Zeiss, Germany). Antibodies The antibodies used were characterized in Kannouche et al. (7). The endogenous HSAkin17 protein was detected by IIF with rabbit polyclonal antibody pAb2061 or pAb2064 raised against mouse kin17 protein (1:200), whereas overproduction of HSAkin17 protein was monitored using two rabbit polyclonal antibodies independently raised against RecA protein, pAbanti-RecA (1) and (2) (1:300). In western blots, HSAkin17 protein was detected using pAb2064 or pAbanti-RecA diluted 1:2000. DNA and RNA analysis DNA purification and the standard Southern procedure were previously described by Tissier et al. (6). RNAs were isolated using RNA-B™ (Bioprobe Systems, France) as described in Kannouche et al. (5). Alternatively, (HSA)Kin17 RNA levels in tissues and tumor cell lines were determined using commercial northern blots containing 2 µg poly(A)⫹ RNA (MTN; Clontech). The filters were hybridized as previously described (5). The autoradiographic films were scanned in an Agfa Arcus TM plus scanner (Agfa-Gevaert). The signals were normalized with those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin RNAs using NIH IMAGE v.1.6. The DNA probes were: (i) a PstI fragment of the GAPDH cDNA, described in Fort et al. (27); (ii) a 2.0 kb BamHI–EcoRI fragment of human β-actin cDNA (Clontech); (iii) the 1000 bp fragment of (HSA)KIN17 cDNA (P1000). DNA probes were 32P-labelled using a Random Priming DNA Labelling Kit (Boehringer Mannheim). The specific activities ranged from 3 ⫻ 108 to 8⫻108 c.p.m./µg DNA.

Fig. 3. Localization of the (HSA)KIN17 gene at chromosome 10p14–15 by in situ hybridization. Ideogram of human chromosome 10 indicating the distribution of the silver grains detected after hybridization with (HSA)KIN17 cDNA.

Protein preparation and western blot analysis Scraped cells were resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 0.5 M NaCl, 1 mM EDTA, 2 mM DTT and 0.5% Triton X-100), sonicated twice

Fig. 4. Expression of (HSA)KIN17 RNA in human tissues. A northern blot containing 2 µg human poly(A)⫹ RNA from the indicated tissues (MNT, Clontech) was hybridized at 42°C overnight with the 32P-labelled probe P1000 (upper) and then reprobed with actin cDNA (lower). After hybridization, the filters were rinsed twice for 20 min in 2⫻ SSC, 0.1% SDS at room temperature then in 2⫻ SSC, 0.1% SDS at 60°C for 20 min, followed by twice in 0.1⫻ SSC, 0.1% SDS at 60°C for 15 min, and were then subjected to autoradiography. Arrows to the left indicate the transcript size.

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Fig. 5. Expression levels of (HSA)KIN17 RNA in tumoral cells. (A) Detection of (HSA)KIN17 RNA. A northern blot containing 2 µg poly(A)⫹ RNA from the indicated human tumoral cells (MNT, Clontech) was hybridized as described in Figure 4. (B) The (HSA)KIN17 gene is not amplified in K562 cells. Ten micrograms of genomic DNA extracted from HL60, K562 and HBL100 cells (lanes a–c, respectively) were digested with EcoRI and analysed by Southern blot at 65°C with radiolabelled P100 DNA. After hybridization overnight, the filters were rinsed twice for 20 min in 2⫻ SSC, 0.1% SDS at 65°C followed by twice in 0.1⫻ SSC, 0.1% SDS at 65°C for 30 min and then subjected to autoradiography. Molecular weight markers (kb) are indicated on the left.

for 6 s then centrifuged at 20 000 g for 30 min at 4°C. The proteins were separated by 11% SDS–PAGE and analysed by western blot as described (5).

possesses two AUUUA motifs known to regulate mRNA stability of proliferation-related genes (29).

Results

Structural conservation of kin17 protein among metazoans The amino acid sequence of HSAkin17 protein revealed a zinc finger motif from residues 28 to 50 which is 100% identical to the domain that mediates the binding of MMUkin17 to double-strand DNA (30). The central region, residues 163– 201, was 92% identical to the RecA homologous region known to be recognized by anti-RecA antibodies (Figure 1). A bipartite nuclear localization signal (NLS), between residues 240 and 257, able to direct the protein into the nucleus (see below), was 73% identical to the mouse NLS (31). We noted the presence of putative protein kinase C phosphorylation sites as well as a putative O-glycosylation site (SSKSSTL) near the NLS region. Scanning the EMBL EST database showed that 5⬘ cDNA fragments from the fruit fly Drosophila melanogaster and from the nematode Brugia malayi code for polypeptides 68% identical to the HSAkin17 protein (Figure 2). A protein from the yeast Schizosaccharomyces pombe presented 40% identity. No significant similarities were detected with bacteria. We conclude that kin17 protein is conserved among mammals and that its functional domains are also conserved among eukaryotes.

Molecular cloning of the (HSA)KIN17 cDNA Cross-hybridization of the (MMU)Kin17 cDNA with several mammalian genomic DNAs suggested a high conservation of the gene (6). We transcribed RNAs from human cells and used oligonucleotides corresponding to the (MMU)Kin17 cDNA to amplify by PCR a 1000 nt DNA fragment, named P1000, which was 87.5% identical to (MMU)Kin17 cDNA. The radiolabelled P1000 DNA probe hybridized to seven of 2.5⫻105 plaques of a cDNA library from human testis. The seven cDNA inserts lacked the 5⬘-region of the ORF. Therefore, we amplified a 5⬘ DNA fragment 230 bp in length by RACE. The reconstructed sequence of (HSA)KIN17 cDNA was 1528 bp long and contained an ORF coding for a protein of 393 amino acids with a calculated Mr of 45 345 Da and an isoelectric point of 9.1 (Figure 1). The human and mouse KIN17 cDNAs were 81% identical and encoded polypeptides of 393 and 391 amino acids, respectively, which were 92.4% identical. The nucleotides surrounding the initiation ATG codons at the beginning of each ORF of the human and mouse KIN17 cDNAs are similar to the translation initiation sequence CC(A/G)CCATGG described by Kozak (28). Transfection of human KIN17 cDNA into mammalian cells inhibited cell proliferation as previously described for the mouse KIN17 cDNA (11,12), indicating that they produce similar biological effects. The 5⬘-untranslated region (5⬘-UTR), ORF and 3⬘untranslated region (3⬘-UTR) of the human and mouse KIN17 cDNAs presented respective identities of 38.5, 86 and 66%, indicating preferential conservation of the coding region. The 3⬘-UTR of (HSA)KIN17 RNA contains 72% (A⫹U) and 1704

The (HSA)KIN17 gene is located at human chromosome 10p15–p14 The pMOSBlueP1000 DNA was radiolabelled and hybridized to human chromosomes as described (25). Among the 100 metaphase cells examined after in situ hybridization, 257 silver grains were associated with chromosomes and 51 of these (19.8%) were located on chromosome 10. Thirty-eight grains out of 51 (74.5%) mapped to the p15–p14 region of the chromosome 10 short arm. This result locates the (HSA)KIN17

UVC induces relocalization of

Fig. 6. Detection of HSAkin17 protein in cell extracts. Protein extracts separated by 11% SDS–PAGE were analysed by western blot using pAb2064 (A) or pAbanti-RecA (B) anti-kin17 antibodies. (A) Detection of HSAkin17 protein in total extracts of human cells. Protein extracts from HeLa cells transfected with pCMV(HSA)KIN17 (lane 1), human fibroblasts MRC-5 (lane 2) and LoVo, human colon cancer (lane 3) and HeLa cells (lane 4) were analysed by western blot using pAb2064 raised against the MMUkin17 protein. (B) Anti-RecA antibodies recognize the exogenous HSAkin17 protein. Escherichia coli BL21 (DE3) cells transformed either with PK17 coding for (His)6–HSAkin17 protein (lane 1) or with the pET19b plasmid (lane 2) were lysed and the extracts analysed by western blot using pAbanti-RecA antibodies. The chimeric protein (His)6–HSAkin17 has a molecular mass of 49 kDa (lane 1), which is 4 kDa higher than endogenous HSAkin17 protein. The 39 kDa band revealed in E.coli extracts corresponds to RecA protein (lanes 1 and 2). HeLa cells transfected either with plasmid pCMV(HSA)KIN17 (lane 3) or with plasmid pCMVDT21 without the insert cDNA (lane 4) were analysed under the same conditions.

gene in the 10p15–10p14 region of the human genome near the Vim and Ilra2 genes (32,33; Figure 3). The Vim, (MMU)Kin17 and Ilra2 genes have been mapped to mouse chromosome 2 (26,34), making it likely that the (HSA)KIN17 gene is within a syntenic region of human chromosome 10. Ubiquitous expression of (HSA)KIN17 RNA in human tissues We detected a major band of 1.8 kb corresponding to (HSA)KIN17 RNA under stringent hybridization conditions in pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, heart, spleen, thymus, prostate, testis, ovary, small intestine, colon (mucosal lining) and peripheral blood leukocytes (Figure 4, upper). Therefore, (HSA)KIN17 RNA appears to be ubiquitously transcribed, although each tissue has a particular expression level. The signal of β-actin RNA showed that the variations observed were not due to degradation or to differences in the loaded RNA (Figure 4, lower). Furthermore, the 1.8 kb band of (HSA)KIN17 RNA appeared to be more than 4-fold higher in heart, skeletal muscle and testis as compared with kidney or liver. The 1.8 kb transcript was barely detectable in kidney, brain, lung, liver and colon. We noted a faint minor band of 4.4 kb or higher molecular weight in pancreas, skeletal muscle, heart, spleen, thymus, prostate, testis, ovary and small intestine (Figure 4). The intensity of the 4.4 kb band was 10-fold lower than that of the major 1.8 kb (HSA)KIN17 RNA. Taking into account that anti-kin17 antibodies detect a major band with an apparent molecular weight of 45 kDa and several minor

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bands of lower molecular weights in all the human cells we have analysed until now, we conclude that the 4.4 kb RNA may correspond to a premature unspliced form of (HSA)KIN17 RNA or may indicate an alternative splicing particular to these tissues. Differential expression of (HSA)KIN17 RNA in several tumor cell lines KIN17 RNA was detected by northern hybridization in all the human cell lines examined (Figure 5). Non-tumorigenic cells presented low (HSA)KIN17 RNA levels which are comparable to those detected in kidney, brain, lung, liver and colon (Figure 4, upper) and to those observed in primary human fibroblasts (Figure 8, upper, NI). Similar low levels were detected in several human cancer cells, like HL60 (promyelocytic leukaemia) and Raji (Burkitt’s lymphoma) (Figure 5A, upper) cells, whereas it was 15-fold higher than the basal level in K562 cells (chronic myelogenous leukaemia) and at least 10fold higher in SW480 cells (colorectal adenocarcinoma) (Figure 5A, upper). The integrity and the equivalent amounts of poly(A)⫹ RNA in each slot were confirmed with the β-actin cDNA probe (Figure 5A, lower). We tested whether the observed differences in K562 cells could be due to rearrangement of the (HSA)KIN17 locus. We have grown K562 cells in our laboratory and confirmed that they presented high expression levels of (HSA)KIN17 RNA as compared with HL60 and HBL100, both of which displayed low levels of (HSA)KIN17 RNA and were, respectively, tumorigenic and non-tumorigenic (data not shown). These results indicate that the high expression level of (HSA)KIN17 RNA in K562 cells is independent of the growth conditions of the cells used for the northern blot (commercial versus our preparation). The genomic DNAs of K562, HL60 and HBL100 cells were purified and analysed by Southern blot. We observed a very similar hybridization pattern, ruling out the possibility of an amplification of the (HSA)KIN17 locus (Figure 5B). We conclude that (HSA)KIN17 RNA expression may be up-regulated in K562 cells by a transcriptional or post-transcriptional mechanism. HSAkin17 protein is localized in intranuclear foci in proliferating cells The great similarity of human and mouse kin17 proteins prompted us to test whether antibodies directed against the mouse kin17 protein recognize the human form. We observed a 45 kDa band by western blot of protein extracts from different human cells as well as in cells transfected with pCMV(HSA)KIN17, confirming the predicted ORF (Figure 6A, lanes 1–4). The preimmune serum did not reveal any signal (data not shown). Therefore, the pAb2064 antibody recognizes the endogenous HSAkin17 protein in total cell extracts. We also investigated whether pAbanti-RecA still recognizes the RecA homologous region of HSAkin17 in spite of the amino acid substitutions. Extracts of HeLa cells transfected with pCMV(HSA)KIN17 presented a major band of 45 kDa corresponding to HSAkin17 protein (Figure 6B, lane 3). Cells transfected with the vector pCMV alone did not show any immunoreacting material (Figure 6B, lane 4). Bacterial extracts expressing the chimeric protein (His)6–HSAkin17 showed a 49 kDa band corresponding to the calculated Mr (Figure 6B, lane 1). We conclude that pAbanti-RecA barely recognizes HSAkin17 protein in human extracts. The signal can be observed only above a given HSAkin17:total protein ratio, rendering its detection difficult. We then analysed the subcellular localization of HSAkin17 protein in exponentially growing 1705

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Fig. 7. HSAkin17 protein is localized in intranuclear foci in proliferating cells. Exponentially growing HeLa cells (a–c) or confluent cells serum-deprived for 3 days in DMEM containing 0.25% FCS (d) were fixed, permeabilized and then processed for IIF using the pAb2064 antibody (1:200). All images were captured under the same conditions. Proliferating HeLa cells presented a speckled pattern (a), whereas the signal was uniformly distributed in mitotic cells, indicated by an arrow (b). DAPI staining of the DNA corresponding to the same cell is marked by an arrow (c). Quiescent cells showed very weak staining (d). Bar 10 µm.

HeLa cells and in serum-starved cells by IIF with the pAb2064 antibody. Proliferating cells in interphase presented uniform and low overall nucleoplasmic staining together with more intense intranuclear punctated fluorescence (Figure 7). A thin perinuclear region was heavily stained, indicating that HSAkin17 protein preferentially accumulated near or in the nuclear membrane and in bright foci dispersed through the nucleoplasm but excluded from the nucleoli (Figure 7a). Mitotic cells showed a few lightly stained dots surrounded by uniform labelling throughout the cell, excluding the chromosomes (Figure 7b and c, indicated by an arrow). Quiescent cells were poorly labelled. Staining was uniformly distributed in the 1706

whole cell (Figure 7d). pAb2061, raised independently against MMUkin17 protein, gave similar results (not shown). We conclude that arrested cells weakly express HSAkin17 protein. In contrast, proliferating cells in interphase display the highest levels of HSAkin17 protein distributed in the perinuclear region and in intranuclear foci. Foci are disassembled during mitosis, indicating redistribution of HSAkin17 protein during the cell cycle. (HSA)KIN17 RNA levels increase in quiescent primary fibroblasts after UV irradiation We investigated whether the (HSA)KIN17 gene is up-regulated in response to UVC in confluent primary fibroblasts. We

UVC induces relocalization of

Fig. 8. UVC irradiation increases (HSA)KIN17 RNA levels in primary human fibroblasts. Confluent primary fibroblasts serum-starved for 24 h were irradiated at 15 J/m2 and compared with non-irradiated cells (marked NI). Cells were further incubated using the same medium and harvested at the indicated times to be analysed by northern blot with (HSA)KIN17 and GAPDH radiolabelled cDNA at 42°C as described in Figure 4. The corresponding autoradiographs are shown. The (HSA)KIN17 signals were quantified, normalized, expressed in arbitrary units and plotted.

wanted to check whether up-regulation of (HSA)KIN17 RNA is a characteristic of normal human cells and not a response activated during cell immortalization or transformation. Serum deprivation for 24 h avoided any possible interference with the increase in (HSA)KIN17 RNA inherent to cell proliferation (5). Growth-arrested cells were irradiated with UVC and RNA was monitored by northern hybridization. The (HSA)KIN17 RNA level remained constant for 6 h and increased 3-fold within 16 h after irradiation (Figure 8, upper). Twenty-four hours later it decreased to the basal level as compared with non-irradiated cells (Figure 8, lower and histogram). These results are representative of two independent experiments performed with foreskin fibroblasts at early passages. We conclude that UVC irradiation increases (HSA)KIN17 RNA levels in human primary fibroblasts. UV irradiation provokes the redistribution of HSAkin17 protein in proliferating cells UVC irradiation does not change the total amount of MMUkin17 protein as shown by western blot (5). We wondered whether UVC affects the nucleoplasmic distribution of HSAkin17 protein in proliferating HeLa cells as determined by IIF. Before

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irradiation, 60% of the cells presented a stained perinuclear region and a less intense fluorescence uniformly distributed throughout the nucleoplasm with an average of 15–20 foci/ nucleus in the 200 cells analysed (Figure 9A, NI). Between 0.5 and 2 h after irradiation, 100% of cells presented 15–20 foci/nucleus that were easily distinguishable (Figure 9A, 0.5, 1 and 2 h). At 6 h, the mean number of foci decreased to an average of 7–10 foci/nucleus in 60% of cells (Figure 9A, 6 h). Between 13 and 24 h the nucleoplasm was uniformly labelled and only 10% of cells displayed ⬍10 foci/nucleus. Forty-eight hours after UVC irradiation, the fluorescence was comparable with that of non-irradiated cells (Figure 9A, NI compared with 48 h). We did not precisely measure any differences in the diameters of the observed foci. We conclude that HSAkin17 protein is preferentially directed into focal structures within 2 h after irradiation, followed by a slow and massive dispersal 13 h later. Since pAb2064 antibody recognizes the C-terminal end of the protein (7), we sought to test whether a UV-induced conformational change of HSAkin17 protein renders the recognized epitope less accessible, thus generating the observed diffuse staining. We used pAbantiRecA, recognizing the core of kin17 protein, in IIF experiments. Cells were transfected to overproduce HSAkin17 protein, irradiated and processed for IIF. Before irradiation, HSAkin17 protein formed 15–30 foci/nucleus and was also detectable throughout the nucleoplasm with a distribution similar to that observed for the endogenous protein (Figure 9B, NI, compared with A, NI). One hour after irradiation, the overall nucleoplasmic fluorescence slightly decreased and foci were more distinguishable than in non-irradiated cells (Figure 9B, NI compared with 1 h). At 15 h there were 5–10 foci/nucleus, a decrease comparable with that observed in the case of the endogenous protein 13 h after irradiation (Figure 9A, 13 h). We obtained similar results using pAb2061 and pAb2064, eliminating the possibility that the observed redistribution is produced by a differential accessibility to the epitope due to a conformational change of HSAkin17 protein (data not shown). We conclude that after UV irradiation the redistributions of endogenous and exogenous HSAkin17 proteins are probably governed by the same mechanism and that the dispersal of HSAkin17 protein represents a response of cycling cells to the UVC-induced damage. Discussion We report characterization of the human KIN17 cDNA encoding a 45 kDa nuclear protein that forms intranuclear foci during proliferation and is redistributed in the nucleoplasm during the cell cycle. UVC provokes the relocalization of HSAkin17 protein, suggesting its participation in the cellular response to DNA damage. The KIN17 gene is remarkably conserved among metazoans, indicating its participation in a conserved function. The (HSA)KIN17 locus is situated on human chromosome 10 p14–10p15 near the Il2r and Vim genes in a region syntenic with mouse chromosome 2. In the human genome, rearrangements in chromosome 10 are the most frequent genetic abnormality in glioblastomas (35). In particular, 10p modifications are frequently correlated with a pattern of anomalies like growth and mental retardation, dolichocephalic skull, high forehead, frontal bossing and others (36). Further studies will determine whether (HSA)KIN17 plays a role in these diseases. The (HSA)KIN17 gene codes for a 1.8 kb RNA which 1707

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Fig. 9. UVC irradiation causes the relocalization of HSAkin17 protein in exponentially growing HeLa cells. (A) The effect on endogenous HSAkin17 protein. Cells irradiated during the exponentially growing phase at 15 J/m2 and further incubated in the same medium for 0.5, 1, 2, 6, 13, 24 and 48 h were fixed and processed for IIF using pAb2064 antibody. A representative field at each time point is shown. Bar 10 µm. (B) UVC changes the distribution of the overproduced HSAkin17 protein. HeLa cells transfected with pCMV(HSA)KIN17 were irradiated 20 h later at a UVC dose of 15 J/m2 and fixed as in (A). The overproduced HSAkin17 protein was detected by IIF using pAbanti-RecA antibody. Representative staining patterns indicated by an arrow show the decrease in HSAkin17 foci number 15 h after UVC. Bar 10 µm. NI, non-irradiated.

correlates well with the length of the cloned (HSA)KIN17 cDNA and with the apparent molecular weight of the detected protein. The (HSA)KIN17 RNA is ubiquitously expressed in human cells at levels characteristic for each tissue. Among the cultured cells tested, K562, tumorigenic cells derived from a chronic myelogenous leukaemia, and SW480 cells, from a colorectal adenocarcinoma, present the highest levels of (HSA)KIN17 RNA. The locus (HSA)KIN17 is not amplified in K562 cells and we failed to detect changes in the restriction pattern, suggesting that the increased expression levels may be due to a point mutation or to an increased transcription rate. Recently, Blattner et al. (37) have reported that UV irradiation increases the half-life of mouse KIN17 RNA, raising the possibility that transcript stability may account for the high 1708

levels of (HSA)KIN17 transcript in K562 cells. It should be noted that mouse and human KIN17 RNAs share two AUUUA motifs present in the 3⬘-UTR that are known to regulate transcript stability (29). This is a particular aspect of the remarkable evolutionary conservation of the KIN17 gene. Indeed, the degree of phylogenetic identity between the Homo sapiens and D.melanogaster kin17 proteins is ⬎60% in a 200 amino acid overlap corresponding to 50% of the whole ORF. (HSA)Kin17 cDNA belongs to a group of eight reported ORF coding for remarkably similar eukaryotic kin17 proteins. A weak but suggestive homology was observed with several mammalian DNA- or RNA-binding proteins. In lower eukaryotes, the identity decreases to 40%, making it difficult to determine at what stage KIN17 appeared during evolution.

UVC induces relocalization of

However, there are several mammalian nuclear proteins involved in essential DNA processes, like tumour suppressor p53 (38) and DNA-damage sensor protein poly(ADP-ribose) polymerase (39), which lack a yeast counterpart, indicating that some essential DNA transactions are particular to mammals and are missing in lower eukaryotes. The import of kin17 protein into the nucleus of proliferating cells has been observed in human reconstructed skin (40) and after serum stimulation of mouse quiescent fibroblasts (5). During mitosis, HSAkin17 foci disappear and the protein is uniformly distributed throughout the whole cell, excluding chromosomes. The foci are restored at the end of mitosis, emphasizing that the redistribution of kin17 protein is cell cycle-dependent. We entertain the idea that kin17 protein binds tightly to the nuclear matrix through a C-terminal domain, since it has previously been shown that: (i) extraction of MMUkin17 protein from cell lysates needs high salt concentrations (7); (ii) overexpression of MMUkin17 protein alters the pattern of nucleoplasmic foci morphology (11); (ii); (iii) deletion of the C-terminal-end of kin17 protein leads to a uniform nucleoplasmic distribution of MMUkin17 protein (11). However, after UVC irradiation the HSAkin17 protein seems to be released into the nucleoplasm. Several human nuclear proteins distributed in intranuclear foci are also relocalized after DNA damage: XPG protein (41), BRCA1 protein (42), PCNA (43), hMre11–hRad50 complex (15) and HsRad51 protein (16). We have previously reported that kin17 foci do not overlap with the ‘replicative factories’ formed by PCNA or with the foci of HsRad51 and BRCA1 (11). Although the biological role of these foci is not known, it has been shown that hRad50 foci are formed at DNA repair sites (17). In contrast, XPG and BRCA1 foci are dispersed after UV irradiation (41,42). It seems to us that the different equilibria between the focal and the dispersed forms reflect distinct roles in the DNA damage response. We hypothesize that HSAkin17 foci are assembly centres of proteins, with a yet undefined physiological function. Subsequent dispersal of HSAkin17 after UVC-induced damage may correspond to recruitment of the protein for a putative protective function. Such a relocalization has been described for transcription factors and RNA-processing enzymes. When transcription is minimal they are localized in foci. Increased transcription led to changes in their phosphorylation states and to a physiological relocalization (14,19). The following experimental evidence supports the hypothesis that HSAkin17 protein is part of a multicomponent RNA– protein complex: (i) the MMUkin17 and large T-Ag proteins co-locate in several intranuclear foci (11) and T-Ag is associated with the hnRNP network (20); (ii) two-hybrid analysis suggests that kin17 interacts with DDX1 protein, a putative RNA helicase which is co-amplified with MYCN in a subset of retinoblastoma and neuroblastoma tumours and derived cell lines (Mauffrey, unpublished results; 44); (iii) the tissue expression pattern of the DBP2 gene encoding a human RNA helicase (45) is identical to that of (HSA)KIN17. The dispersion of HSAkin17 protein 13 h after UVC irradiation and the slow accumulation kinetics of (HSA)KIN17 RNA are indicative of participation in the late phase of the response to UV as defined by Herrlich et al. (3). It has been shown that the late genes, like p53, are activated by the presence of UVinduced lesions (4). Recently, Blattner et al. have shown that in mouse cells accumulation of (HSA)KIN17 RNA is independent of p53. Nevertheless, this accumulation may be directly related to the amount of UV-induced DNA damage

HSAkin17

protein

(37). The accumulation kinetics of human and mouse KIN17 RNAs are very similar, making it likely that the KIN17 gene is part of an evolutionarily conserved response to UVC. Analysis of NER-deficient human cells should help us gain further insight into these phenomena. Acknowledgements We thank C.de la Roche Saint Andre´ for communicating the sequence of DMEkin17 protein before publication. We are indebted to J.H.J.Hoeijmakers for the human testis cDNA library, J.Feunten for anti-BRCA1 antibody, P. Radicella for the prostate cDNAs and C.Radding for anti-Rad51 antibody. We thank Drs M.-F.Poupon, E.May and M.Mezzina for providing cell lines. We are grateful to Dr M.Kress and B.Dutrillaux for helpful discussions. The suggestions of Dr E.Bruford from the HUGO Nomenclature Committee were greatly appreciated. P.Kannouche benefited from fellowships from the INSTN, CEA and Electricite´ de France. Infobiogen ([email protected] ) provided us with the computer programs and the Human Genome Consortium with several human cDNA fragments. This work was made possible by funds provided by contracts ARC no. 6060 and EDF no. 8702 to A.J.F.

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