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FEBS Letters 581 (2007) 1166–1172

Gene expression of -dependent kinase subunit Cks2 is repressed by the tumor suppressor but not by the related p63 or

Karen Rothera,b, Markus Dengla, Jana Lorenza, Katrin Tscho¨pa, Ralf Kirschnera,b, Joachim Mo¨ssnera, Kurt Engelanda,* a Medizinische Klinik II, Max-Bu¨rger-Forschungszentrum, Universita¨t Leipzig, Johannisallee 30, D-04103 Leipzig, Germany b Interdisziplina¨res Zentrum fu¨r Klinische Forschung (IZKF) Leipzig, Max-Bu¨rger-Forschungszentrum, Universita¨t Leipzig, Johannisallee 30, D-04103 Leipzig, Germany

Received 17 November 2006; revised 12 February 2007; accepted 13 February 2007

Available online 26 February 2007

Edited by Varda Rotter

occur normally in Cks2/ spermatocytes and oocytes, this Abstract Cks2 proteins are essential components of cyclin/cy- clin-dependent kinase complexes and contribute to con- arrest is most likely caused by an inability to enter trol. We identify Cks2 as a transcriptional target downregulated I of rather than a checkpoint-mediated I ar- by the tumor suppressor p53. Cks2 expression was found to be rest. However, overexpression of Cks1 as well as of Cks2 could repressed by p53 both at the mRNA and the levels. p53 abrogate metaphase I arrest. Thus, incomplete development of downregulates transcription from the Cks2 in a dose- germ cells in Cks2-deficient cells results from the absence of dependent manner and in all cell types tested. This repression ap- Cks1 expression rather than from a germ cell specific function pears to be independent of p53 binding to the Cks2 promoter. In of Cks2 [5]. contrast to p53, neither p63 nor p73 proteins can repress Cks2 A principal factor in regulation of growth arrest as well as transcription. Thus p53, rather than its homologues p63 and is the tumor suppressor protein p53. The of p73, may contribute to control of the first metaphase/anaphase p53 is mutated in the majority of human tumors and acts by transition of mammalian meiosis by downregulation of Cks2 expression. engaging in complexes with other proteins or functions as a Ó 2007 Federation of European Biochemical Societies. Pub- . p53 functions as a transactivator and lished by Elsevier B.V. All rights reserved. repressor. One prominent example of a transactivated target gene is the cdk inhibitor p21WAF1/CIP1 [6]. Other target Keywords: Cks2; Tumor suppressor p53; p63; p73; Cyclin- such as several regulators of the G2/M checkpoint like cyclin dependent kinase subunit 2; Transcriptional repression B, cdc25C and cdk1 are repressed by p53 [7–10]. Many inves- tigations concentrated on solving the mechanism of the p53- dependent repression without identifying one mechanism which would yield a general explanation for the regulatory de- tails. Most repressed genes do not have p53-consensus site in 1. Introduction their promoters. For the cdk1 and cdc25C promoters we and others have shown that p53-dependent repression can be med- The cyclin-dependent kinase subunit (Cks) family is com- iated through CCAAT-boxes [7,9,10].Lo¨hr et al. implicated WAF1/CIP1 posed of small proteins (9–18 kDa). They are essential compo- induction of in the transcriptional downregula- nents of cyclin/cyclin-dependent kinase complexes and thereby tion by p53 [11]. In contrast to common somatic tumors, p53 contribute to cell cycle control [1]. The first member of this gene mutations with less than 3% occur seldom in germ cell tu- family isolated was the product of suc1 (suppressor of cdc2 mors. The high level of wild-type p53 found in these tumors mutation), which is necessary for the function of cell cycle reg- may contribute in part to the chemosensitivity of germ cell tu- ulatory protein kinase p34cdc2 in fission yeast [2]. Homologues mors [12]. Recently, it has been shown that p53 may be in- of Suc1, named Cks, were identified since then in many species volved in homologous recombination and/or checkpoint and share extensive sequence conservation. Co-immunoprecip- control by directly binding to DMC1 protein to repress geno- itation studies have shown tight binding of Cks proteins to mi- mic instability in meiotic germ cells [13].With p63 and p73 two totic cyclin-dependent kinases [3]. Mammalian cells express genes coding for proteins homologous to p53 have been iden- two paralogs like the human Cks1 and Cks2 proteins sharing tified. The p53, p63 and p73 proteins share a similar basic do- 81% identity in their amino acid sequence [1]. main structure and display substantial amino acid identity in Cks2 may be able to compensate for some of the functions their DNA-binding domains. p63 and p73 proteins do not rep- lost when Cks1 is deleted in Cks1/ mice [4]. Cks2/ ani- resent classical tumor suppressors as their genes are rarely mals are found to be viable but sterile in both sexes. This ste- found mutated or deleted in tumors. Unlike p53-deficient mice, rility was found to be due to an arrest of the germ cells at p63 and p73 knockouts do not show enhanced cancer suscep- metaphase I of meiosis. As all events of meiotic I tibility but exhibit severe developmental abnormalities. How- ever, some of the functions of p63 and p73 overlap with the role of p53. For example, all three proteins can activate tran- WAF1/CIP1 *Corresponding author. Fax: +49 341 9712209. scription of several of the same genes such as p21 E-mail address: [email protected] (K. Engeland). and Bax [14,15].

0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.02.028 K. Rother et al. / FEBS Letters 581 (2007) 1166–1172 1167

2. Materials and methods TCA CTC AGG TC-30; DNp63-hum-for, 50-CTG GAA AAC AAT GCC CAG AC-30; DNp63-hum-rev, 50-GTG GAA TAC GTC CAG 0 0 2.1. Plasmids and cloning of the Cks2 promoter GTG GC-3 ; GAPDH-hum-for, 5 -GAC CCC TTC ATT GAC CTC 0 0 0 The human wild-type Cks2 promoter fragment, containing 654 AAC-3 ; GAPDH-hum-rev, 5 -CAC GAC GTA CTC AGC GCC-3 . nucleotides upstream of the start codon, was obtained by PCR ampli- These primer pairs result in PCR products of 314 bp (Cks2), 159 bp fication with the primers Cks2_human_for, 50-GGG GTA CCC CCC (p53), 303 bp (p73), 206 bp (TAp63), 296 bp (DNp63) and 186 bp AGC CGA CCA CGA AGC CG-30, and Cks2_human_rev, 50-CAT (GAPDH). Quantitative analysis was performed employing GAPDH GCC ATG GTG CTG GCG CGC TGC AGA AAA TG-30 and as a standard and LightCycler analysis software as described [8]. The cloned (KpnI/NcoI) in pGL3-Basic (Promega). The resulting clone mRNA levels of untreated cells were set at 100%. was sequenced yielding identical nucleotide sequences which was sub- mitted to the data base (GenBank Accession No. EF026653). 2.5. Chromatin immunoprecipitation assays Human p53-expression plasmids, pCMV-p53wt and pCMV- ChIPs of Cks2 and p21WAF1/CIP1 promoters were carried out as de- p53mut, were generously provided by Vogelstein [16]. The wild-type scribed [25]. Samples were analyzed after 35 PCR cycles with the fol- and mutant expression constructs of p73a, p73b, TAp63a, TAp63c, lowing primers: ChIP-Cks2hum-for, 50-CGA CCG ACT ACG TCA TA*p63a,TA*p63c, DNp63a and DNp63c have been described previ- TCA CC-30 and ChIP-Cks2hum-rev, 50-CCT CGC AAA GTC CGC ously [17]. The plasmid YA13m29 expresses a dominant-negative mu- TTC C-30 (GenBank Accession No. EF026653). Primers for amplifica- tant of transcription factor subunit NF-YA and has been described tion of the human p21WAF1/CIP1 promoter fragment have been de- previously [18]. The control plasmid pRL-null (Promega) contains a scribed [25]. cDNA coding for Renilla luciferase.

2.2. Cell culture, transfections and luciferase assay Parental HCT116 cells and HCT116 cells with targeted deletion in 3. Results and discussion both p53 alleles [19] as well as inducible cell line D.P53 A2 (DLD-1- tet-off-p53) was generously provided by Vogelstein [20]. The latter cell 3.1. Expression of Cks2 mRNA is downregulated after increased line is a derivative of the colorectal carcinoma cell line DLD-1, which has endogenous mutant p53 alleles. Expression of wild-type p53 is reg- p53 expression ulated by a modified tetracycline (tet)-regulated system The human colorectal adenocarcinoma cell line DLD-1 is (tet-off-system) [8,20,21]. negative for functional p53 protein. We employed a stably HCT116, DLD-1, SaOS-2, HeLa, HS766T and C33A cells were cul- transfected DLD-1 cell line which allows for tet-off-regulated tured and transiently transfected as described [8,22–24]. Unless other- wise noted, transfections were carried out using 300 ng of Cks2 expression of wild-type p53 [8,20]. In control experiments we promoter construct and 25 ng of p53, p73 or p63 expression plasmids had confirmed that after tet-off induction for 9 h expression and pRL-null control plasmid per assay. DNA amounts were held con- of p53 mRNA and protein increases [8]. To search for hitherto stant using pUC19 plasmid. Luciferase activity was measured 24 h unidentified p53 target genes, we had performed a DNA after transfection. Firefly luciferase was normalized to Renilla lucifer- microarray hybridization experiment using RNA from these ase activity in order to compensate for variability in transfection effi- ciencies. DLD-1-tet-off-p53 cells without and 9 h after induction of p53 expression [25]. We found Cks2 mRNA to be downregu- 2.3. Western blotting and oligonucleotide microarray analysis lated 9.7-fold when wild-type p53 was overexpressed relative Frozen cell pellets were lysed in TRIZOLÒ Reagent (Invitrogen) and to the uninduced state. As controls and consistent with previ- proteins were extracted as suggested by the manufacturer. Protein con- ous observations mRNA levels for known p53 target genes like centrations were determined with the Bio-Rad protein assay and p21WAF1/CIP1 and were found to be upregulated and pre- 100 lg of total cell lysate per sample was separated in a 15% SDS– polyacrylamide gel. Proteins were transferred onto a polyvinylidene viously described targets like , , cdk1 and difluoride transfer membrane (Hybond-P; Amersham Pharmacia Bio- cdc25C were downregulated [25]. Furthermore, under the tech) and blocked with 5% BSA/TBS-Tween 20. Antibodies employed experimental conditions employed changes in expression of were the anti-Cks2 mouse monoclonal antibody (1F7G5, 3 lg/ll, p53 target genes precede the changes due to impending apop- Zymed), the anti-p53 mouse monoclonal antibody (DO-1, 1:2000, ) and the anti-b-actin monoclonal antibody (AC-1, 1:2000, tosis [17]. Following the microarray analysis as an indicator, Sigma). Goat-anti-mouse IgG conjugated with horseradish peroxidase we confirmed the p53-dependent decrease of the Cks2 mRNA (Pierce) was used as a secondary antibody and chemiluminescent sig- levels by real-time RT-PCR quantification in two independent nals were detected by SupersignalÒ West Dura Extended Duration experiments yielding an average of 10.1-fold downregulation Chemiluminescent Substrate Kit (Pierce). Oligonucleotide microarray of Cks2 mRNA. analysis was performed as described previously [25].

2.4. RNA extraction and real-time RT-PCR analysis 3.2. p53 downregulates transcription from the Cks2 promoter in Extraction of total RNA and real-time RT-PCR mRNA quantifica- a dose-dependent manner and in different cell types tion with the LightCycler system (Roche) including calculations have In order to elucidate whether this decrease in Cks2 mRNA is been described [8,9]. Primers were used at 1 lM with 3 mM MgCl2 dependent on p53 as a regulator of transcription, we examined and 50 ng of RNA template in the QuantiTect SYBRÒ Green RT- PCR system (Qiagen) according to the manufacturer’s instructions. the regulation of the Cks2 promoter. We amplified human Each cycle of PCR included 15 s of denaturation at 94 °C, 20 s of pri- DNA upstream of the Cks2 coding region and cloned a mer annealing at 62 °C (Cks2) or 55 °C (p53, p73, TAp63 and DNp63) 645 bp promoter fragment into a firefly luciferase-expressing and 15 s of extension/synthesis at 72 °C. At the end of the extension reporter plasmid (Cks2-Luci). Cks2-Luci together with expres- steps at 72 °C (Cks2) or 79 °C (p53, p73, TAp63 and DNp63) fluores- sion constructs for wild-type or mutant p53 was cotransfected cence of each sample was measured to allow quantification of the mRNA. Gene-specific primers used for RT-PCR: Cks2-hum-for, in p53-negative SaOS-2 cells. Increasing amounts of wild-type 50-CAT TAC ATG ATT CAT GAG CCA G-30; Cks2-hum-rev, 50- p53 led to a downregulation of the Cks2 reporter. In contrast, GTT TCA GTA AAC TCA AAC AGG CAC-30; p53-hum-for, the expression of a DNA-binding deficient mutant of p53 at 0 0 0 5 -ATG GAG GAG CCG CAG TCA GAT C-3 ; p53-hum-rev, 5 - the highest concentration had only a minor effect on the CCA TTG TTC AAT ATC GTC CGG G-30; p73-hum-for, 50-AAG ACA TGC CCC ATC CAG ATC-30; p73-hum-rev, 50-CAT GAA Cks2 reporter (Fig. 1A). In addition, similar experiments were GTT GTA CAG GAT GGT G-30; TAp63-hum-for, 50-ATG TCC performed in different cell lines including human cervical car- CAG AGC ACA CAG AC-30; TAp63-hum-rev, 50-CAC ATG GGG cinoma cells HeLa and C33A, human colon carcinoma cells 1168 K. Rother et al. / FEBS Letters 581 (2007) 1166–1172

DLD-1 A 120 Cks2 promoter (total cell lysate) 100 _ + p53wt induction 80 Cks2 60

40 p53

20

luciferase relative light units % β-actin 0 p53wt - 1 ng 5 ng 25 ng - p53mut - - - - 25 ng Fig. 2. Western blot analysis of Cks2 expression after induction of p53wt. DLD-1-tet-off-p53 cells without () and with (+) induction of B 120 Cks2 promoter wild-type p53 expression for 9 h. Total cell lysate was prepared and 100 lg of protein was used for each lane. Samples were analyzed with

e 100 antibodies directed against human Cks2, p53 and b-actin. 80 60 p73a and p73b. Neither p73a or p73b nor any of the p63 splice 40 light units % variants tested were able to change transcription from the luciferase relativ 20 Cks2 promoter like p53 when compared to their DNA-binding 0 deficient and transcriptionally inactive mutant forms (Fig. 3A). SaOS-2 Hela HCT116 HS766T C33A Since cotransfections with p63 and p73 variants did not yield a change in expression from the Cks2 promoter, we made sure in p53mut p53wt control experiments that transient transfection of SaOS-2 cells Fig. 1. p53 downregulates transcription from Cks2 promoter in a indeed lead to expression of each p63 and p73 splice variant dose-dependent manner. Either a plasmid coding for wild-type p53 or mRNA from the transfected plasmids (Fig. 3B). After real- a DNA-binding mutant of p53 was cotransfected along with the Cks2 time RT-PCR samples were analyzed on agarose gels yielding promoter reporter in transient cotransfection assays. All experiments were standardized to Renilla luciferase activity expressed from a strong induction of p53-family members in transfected cells. cotransfected pRL-null vector. Experiments were carried out in No specific PCR product was detected in non-transfected con- triplicate with standard deviations indicated. (A) The Cks2-Luci trol cells which therefore appeared negative for p53, p63 and construct carrying the human Cks2 promoter was cotransfected in p73 mRNA. As another control, GAPDH mRNA levels re- SaOS-2 cells with p53mut or increasing amounts of p53wt. The total mained constant giving a quality control for the RNA prepa- amount of DNA transfected was held constant by adding pUC19. Cotransfection with pUC19 vector exclusively, served as a control and rations (Fig. 3B). Moreover, we had found p73a, p73b, set to 100%. (B) Different cell lines were transfected with the Cks2 TAp63c and TA*p63c able to activate transcription from a promoter reporter together with wild-type or mutant p53-expressing p21WAF1/CIP1 reporter [9,17]. This confirms them to be trans- plasmids. Maximum expression for each cell line was adjusted to 100%. criptionally active but unable to alter expression from the Cks2 promoter. Inability of the other p63-splice variants to re- press the Cks2 promoter may be due to absence of the transac- HCT116 and human pancreatic carcinoma cells HS766T. tivation domain (DNp63a,DNp63c) or to deficiency in Wild-type p53 downregulates transcription from the Cks2 pro- transcriptional activity by an inhibitory function of the SAM moter in all cell systems (Fig. 1B). domain (TAp63a,TA*p63a) as described for p73 [26].

3.3. Cks2 protein levels are also reduced after p53 induction 3.5. Repression of Cks2 transcription appears to be independent Lysates from DLD-1-tet-off-p53 cells before and after wild- of p53 binding to the Cks2 promoter type p53 expression were prepared and Cks2 protein levels Transcriptional activation by p53 is generally associated were compared. Western blot analysis shows a moderate with p53 binding to the regulated promoter. The classical reduction of Cks2 protein expression already 9 h after p53 p53-binding site exists of two palindrome pentamers, PuPu- induction (Fig. 2). This slight effect is dependent on the lag PuC(A/T)(T/A)GPyPyPy, separated by 0–13 bp [27]. In case phase after mRNA decrease and stability of the Cks2 protein of transcriptional repression, direct binding of p53 to a consen- and was consistently detected in several independent experi- sus site in the downregulated promoter usually is not detected. ments. Cks2 protein levels may further decrease with longer Since human as well as mouse Cks2-promoter reporter-con- periods of p53 induction. However, we had observed earlier structs were repressed in transient transfection assays by that after induction of p53 for more than 9 h a significant num- wild-type p53 (data not shown) we performed an in silico anal- ber of cells have been become apoptotic [17]. ysis of human and mouse Cks2 promoter sequences (Fig. 4). This comparison revealed that neither a classical p53-consen- 3.4. Expression of p73 or p63 proteins does not alter sus element nor alternative p53-binding sites were conserved transcription from the Cks2 promoter between the two species [28–31]. Furthermore, we searched To test Cks2 promoter responsiveness to several p63 and p73 for other known consensus sites to be conserved in both pro- splice variants we employed plasmids coding for wild-type moters. However, in these short promoter sequences we only TAp63a, TAp63c,TA*p63a,TA*p63c, DNp63a, DNp63c, found three conserved CCAAT-boxes potentially binding the K. Rother et al. / FEBS Letters 581 (2007) 1166–1172 1169

A Repression of the Cks2 promoter 10,0 9,09.0 8,0 7,07.0 6,0 5,05.0 4,0 3,03.0 2,0 1,01.0 fold change (repression) 0,0 α β α γ α γ α γ 3 3 p533 6 6g 63 63 p5 p73 p73 p p3g p73a p73b *p63 Np63a p6N TAp63 TAp63 Δ Δ TAp63a TAp63g TA*p63aTA*p TATA*p DN DN

B p73 TAp63 TA*p63 ΔNp63 MCp53C αβ C α γ α γ MC α γ

1517 1200 1000 800 500 400 300 200 100

p73 TAp63 TA*p63 ΔNp63 Cp53 αβα γ α γ α γ M

1517 1200 1000 800 500 400 300 GAPDH 200 100

Fig. 3. Neither p63 nor p73 can downregulate Cks2 expression. (A) Luciferase reporter construct Cks2-Luci and expression plasmids coding for wild- type and mutant protein of nine different p53-family members (p53,p73a,p73b,TAp63a,TAp63c,TA*p63a,TA*p63c,DNp63a,DNp63c) were transiently cotransfected in SaOS-2 cells. Experimental details were the same as in Fig. 1. (B) As controls Saos-2 cells were transiently cotransfected in order to test for mRNA expression from plasmids coding for p53-family members. Total RNA was prepared 24 h after transfection, amplified by real-time RT-PCR and visualized on agarose gels. GAPDH expression was tested as RNA preparation quality control. Expected product sizes were: p53, 159 bp; p73, 303 bp; TAp63, 206 bp; DNp63, 296 bp; GAPDH, 186 bp. Control lanes C resulted from non-transfected cells. Marker lane is denoted as M. transcription factor NF-Y, two of which are in inverse direc- [7,9,10,32]. The human Cks2 promoter contains three tion (Fig. 4). CCAAT-boxes (Fig. 4) and we tested the impact of NF-Y Although we did not find a p53-consensus site in the Cks2 on Cks2 regulation by employing YA13m29, a dominant-neg- promoter, we test for direct or indirect binding of p53 to the ative mutant of NF-Y subunit NF-YA [18]. Even the highest Cks2 promoter by chromatin immunoprecipitation assays amount of YA13m29 does not significantly reduce Cks2 pro- (ChIPs) using DLD-1-tet-off-p53 cells before (control) and moter activity (Fig. 6). In control experiments we had con- after selective induction of wild-type p53. With an antibody di- firmed that after YA13m29 expression human and rected against p53, no Cks2 promoter DNA could be co-pre- cdc25C promoter activity decreases [22,24]. In the presence cipitated and subsequently PCR-amplified (Fig. 5). Binding of YA13m29 p53 is still able to repress Cks2 transcription of p53 to the p21WAF1/CIP1 promoter served as a positive con- (Fig. 6). Thus, the experiments exclude NF-Y as responsible trol. Taken together, sequence and ChIP analyses indicate that for Cks2 promoter activation and as being a mediator in p53 does not bind to the Cks2 promoter as also observed with p53-dependent repression of Cks2. other genes repressed by p53 [25]. In summary, we identified with Cks2 a transcriptional target downregulated by p53 that contributes to control of 3.6. NF-Y is not responsible for Cks2 promoter activity and does the first metaphase/anaphase transition of mammalian not influence p53-dependent repression of Cks2 meiosis. We found that p53 downregulates Cks2 expression NF-Y is a transcriptional activator binding to CCAAT- both at the transcriptional and the protein levels. This boxes and has been implicated in p53-dependent repression repression is mediated obviously neither through direct nor 1170 K. Rother et al. / FEBS Letters 581 (2007) 1166–1172

Fig. 4. Comparison of nucleotide sequences from human and mouse Cks2 promoters. Numbering is relative to the translation start codons. Identical homologous nucleotides are indicated by vertical lines. CCAAT-boxes conserved in both sequences are boxed.

through indirect binding of p53 to the Cks2 promoter. In homologues p63 and p73, may contribute to controlling contrast to p53, the related proteins p63 and p73 cannot transition of metaphase I to anaphase I in mammalian repress Cks2 transcription. Thus p53, rather than its meiosis. K. Rother et al. / FEBS Letters 581 (2007) 1166–1172 1171

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