Polyomavirus Small T Antigen Transactivates Genes by Its Ability to Provoke the Synthesis and the Stabilization Of

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Polyomavirus Small T Antigen Transactivates Genes by Its Ability to Provoke the Synthesis and the Stabilization Of Oncogene (2007) 26, 6356–6360 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc SHORT COMMUNICATION Polyomavirus small T antigen transactivates genes by its ability to provoke the synthesis andthe stabilization of MYC B Klucky1 and E Wintersberger Division of Molecular Biology, Department of Medical Biochemistry, MFPL, Medical University of Vienna, Vienna, Austria DNA tumor viruses are capable of driving quiescent cells polyomaviruses, but it is so far unproven that these into the cell cycle. In case of polyomaviridae, two viral viruses play a causative role in the formation of cancers. proteins, the large andthe small (ST) T antigens are However, polyomaviruses have been extremely helpful responsible for this outcome. ST interacts with the protein in studies of cell cycle regulation and cell transforma- phosphatase PP2A andwith chaperons of the dnaKtype tion. This includes the discovery of important cellular andleadsto the transactivation of several genes, which regulators such as p53 or the retinoblastoma protein play a role in S-phase induction. One of these is the pRB (Moran, 1993). transcription factor myelocytomatosis (MYC), which by All polyomaviruses code for two major early proteins, itself is an important regulator of growth. Microarray the small (ST) and the large (LT) tumor antigen. analysis has revealedseveral ST-inducedgenes, which are Polyomaviruses can infect a variety of cell types also targets of MYC; hence, ST may induce these genes including differentiated and hence growth arrested cells. via MYC. Experiments shown here are in line with this As they depend on the cellular machinery for replication assumption. MYC-regulatedgenes are inducedby ST at of their DNA, the T antigens meet this prerequisite by later times than MYC anda MYC responsive promoter is interfering with the growth control of the host cell, stimulatedby ST. Regulation of MYC occurs through driving cells into the S-phase of the cell cycle. These signal transduction pathways, which are co-ordinated by reactions may form the basis for the tumorigenic PP2A suggesting that they may be targets of ST. Here, capacity of these viruses. we show that this is the case as important kinases involved The contribution of LT oncogene to S-phase induc- in these pathways appear in the active phosphorylated tion consists of the transactivation of E2F-regulated form in the presence of ST. Inhibition of these kinases genes, which requires interactions of the viral protein interferes with MYC induction and inhibition of MYC with the pocket proteins (pRB and its relatives p130 and activity blocks ST-mediated transactivation. p107; Harbour and Dean (2000)) with chaperons such Oncogene (2007) 26, 6356–6360; doi:10.1038/sj.onc.1210458; as HSC70 (Sullivan and Pipas, 2002) and with the published online 16 April 2007 coactivator proteins CBP/p300 (Nemethova et al., 2004). Keywords: polyomavirus; T antigens; MYC; S-phase The main cellular interactors of ST antigen are the induction protein phosphatase PP2A (Yang et al., 1991) and chaperons. PP2A is trimeric, consisting of a catalytic subunit C and two regulatory subunits A and B. B The multiple-stage process of cancer development is subunits constitute a family of proteins whereof driven by genetic and epigenetic events. Cancer can also individual members are assumed to confer substrate be associated with virus infection, for instance, with specificity to the phosphatase (Millward et al., 1999; members of the small DNA tumor virus family. The role Virshup, 2000). ST proteins can replace distinct B of human papillomaviruses in the development of subunits (Chen et al., 2004), which results in an carcinomas of the cervix is well documented and a inactivation of PP2A with respect to specific substrates. contribution of polyomaviridae, Simian virus 40 (SV40) Among these substrates is, for instance, p27Kip1 an or the human polyomaviruses, BKV and JCV, in the inhibitor of the cyclin-dependent kinase cdk2 (Porras etiology of human cancers has frequently been con- et al., 1999; Schu¨ chner and Wintersberger, 1999). ST has sidered (Reiss and Khalili, 2003). Up to 80% of been shown to stimulate signal transduction pathways the human population carries information of human (Sontag et al., 1993; Frost et al., 1994; Howe et al., 1998) and to transactivate genes, for example telomerase, Correspondence:Professor E Wintersberger, Division of Molecular cyclin D1 (Watanabe et al., 1996) and cyclin A (Porras Biology, Department of Medical Biochemistry, Medical University of et al., 1996; Schu¨ chner et al., 2001). Thus, ST Vienna, Dr. Bohrgasse 9, Vienna A-1030, Austria. contributes importantly to S-phase induction. Indeed it E-mail:[email protected] was found that neither LT nor ST by itself can induce an 1Current address:DKFZ, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg D-69221, Germany. S-phase in growth-arrested mouse or human fibroblasts, Received 13 June 2006; revised 26 February 2007; accepted 2 March but that the two proteins together do so quite effectively 2007; published online 16 April 2007 (Ogris et al., 1992; Porras et al., 1999). This result was Polyoma ST transactivates genes through induction of MYC B Klucky and E Wintersberger 6357 substantiated more recently when it was found that of ST-induced MYC, their mRNA levels should increase stable transfection of a combination of oncogenic Ras, later than that of MYC. As shown in Figure 1a, this is telomerase and SV40 LT and ST antigen can transform indeed the case. In Swiss 3T3 mouse fibroblasts, which human cells; LT alone was not sufficient for this conditionally express ST under the direction of the reaction (Yu et al., 2001; Hahn et al., 2002). MMTV promoter (Ogris et al., 1992), ST mRNA is Microarray analysis was carried out to more fully induced already 4 h after addition of dexamethasone to comprehend the capacity of ST and LT proteins to serum-starved cells. MYC mRNA also increases already deregulate the transcriptional program of the host cell 4 h after addition of dexamethasone and reaches a (Klucky et al., 2004). This showed that both proteins maximum level after 20 h. mRNAs for telomerase affect transcription of a surprisingly large number of (TERT) and cyclin D1 increase later, which is in line genes. Interestingly, among the proteins found tran- with the assumption that they are targets of MYC. If scriptionally upregulated by ST was the transcription this were so, one would expect that a reporter gene factor myelocytomatosis (MYC). Intact binding sites for PP2A and chaperons (J domain) were found essential for this reaction. This finding led us to propose that the a S3T3 STwt induction of a variety of genes seen in the microarray 0h 28h 0h 4h 8h 16h 18h 20h 24h 28h Dex study may be a consequence of the upregulation of 30 MYC (Klucky et al., 2004). 25 MYC dimerizes with MAX to bind specifically to a 20 DNA sequence, called E-box, present in promoters of 15 many genes. Thereby MYC controls the expression 10 MYC mRNA 5 of genes involved in proliferation, differentiation and Fold Induction apoptosis (Levens, 2003; Pelengaris and Khan, 2003; 0 Patel et al., 2004). We found concurrencies by compar- 7 ing data of the microarray analysis of cells expressing ST 6 with the results of genome-wide investigations of MYC- 5 regulated genes. For instance, like MYC, PyST upregu- 4 3 lates several ribosomal proteins, heat shock proteins, 2 nucleolin, cyclins and telomerase (Klucky et al., 2004). 1 Fold Induction Induction of all of these presumptive MYC targets was Cyclin D1 mRNA 0 found to depend on an interaction of ST with PP2A and chaperons. If these genes were indeed secondary targets 20 16 12 Figure 1 PyST induction of MYC target genes depends on an intact PP2A and chaperon binding site. (a) Time course of 8 induction of mRNA for cyclin D1, TERT and ST. Swiss 3T3 TERT mRNA 4 fibroblast and derived cell lines, conditionally expressing PyST wild Fold Induction 0 type or mutants under the control of the Dexamethasone-inducible MMTV promoter, was grown as described (Ogris et al., 1992). 30 Growth arrest and further treatment of cells, isolation of RNA and 25 quantitative PCR were carried out as described previously (Klucky et al., 2004), using Light Cycler Fast Start DNA Master SYBR 20 Green I Kit as recommended by the supplier (Roche, Applied 15 Science Corp.). Primer sequences:MYC forward:5 0-GCCCGCG 10 0 0 ST mRNA CCCAGTGAGGATA-3 , reverse:5 -GCGGCGGCGGTGAGG 5 TC-30; mTERT forward:5 0-TACCAACCCCATCAGGCAAAT Fold Induction -30, reverse:5 0-TGGGCTGGTGTTCAAGGCATC-30, cyclin D1 0 forward:5 0-CTGTGCGCCCTCCGTATCTTA-30, reverse:5 0- 0 0 GGCGGCCAGGTTCCACTTGAG-3 ; ST forward:5 -CA GG b ST max CATATAAGCAGCAGTC-30; reverse:5 0-CATCTCGGGTTGG TGTTC-30; b-actin forward:5 0-TGGCACCACACCTTCTACA MYC ATGAG-30; reverse:5 0 CAAGAAGGAGGCTGGAAAAGAG-30. E-box Luc b-Actin was used for normalization of the data. (b) ST activates myc-responsive elements. The pMYC-TA-LUC vector (Clontech, Palo Alto, CA, USA), was used to measure the stimulation of transcription from a MYC-responsive promoter by ST. The MYC- 8 luciferase vector was co-transfected with a pCIneo mammalian 6 expression vector, containing ST wild type or mutants defective in binding PP2A (PP2AÀ) or chaperons (STJÀ), into REF 52 cells. 4 After serum starvation of the cells for 48 h, expression of luciferase 2 was measured. CMV b-galactosidase was co-transfected to control for transfection efficiency. ST wild type, but not the mutants, 0 responsive element transactivates the transcription factor MYC and in turn mediates FoldI nduction of myc- mock STwt PP2A- STJ- strong expression of the reporter luciferase gene. Equal expression of ST was verified by Western blot analysis using an antibody α PyST against the N-terminus of ST.
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