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PATZ1 Is a DNA Damage-Responsive Transcription Factor That Inhibits P53 Function

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PATZ1 Is a DNA Damage-Responsive Transcription Factor That Inhibits P53 Function PATZ1 Is a DNA Damage-Responsive Transcription Factor That Inhibits p53 Function Nazli Keskin,a,b Emre Deniz,a,b Jitka Eryilmaz,a* Manolya Un,a Tugce Batur,a Tulin Ersahin,c,d Rengul Cetin Atalay,c,d Shinya Sakaguchi,e Wilfried Ellmeier,e Batu Ermana,b Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkeya; Sabanci University Nanotechnology b c Research and Application Center-SUNUM, Istanbul, Turkey ; Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey ; Informatics Institute, Downloaded from Middle East Technical University, Ankara, Turkeyd; Division of Immunobiology, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austriae Insults to cellular health cause p53 protein accumulation, and loss of p53 function leads to tumorigenesis. Thus, p53 has to be tightly controlled. Here we report that the BTB/POZ domain transcription factor PATZ1 (MAZR), previously known for its tran- scriptional suppressor functions in T lymphocytes, is a crucial regulator of p53. The novel role of PATZ1 as an inhibitor of the p53 protein marks its gene as a proto-oncogene. PATZ1-deficient cells have reduced proliferative capacity, which we assessed by transcriptome sequencing (RNA-Seq) and real-time cell growth rate analysis. PATZ1 modifies the expression of p53 target genes http://mcb.asm.org/ associated with cell proliferation gene ontology terms. Moreover, PATZ1 regulates several genes involved in cellular adhesion and morphogenesis. Significantly, treatment with the DNA damage-inducing drug doxorubicin results in the loss of the PATZ1 transcription factor as p53 accumulates. We find that PATZ1 binds to p53 and inhibits p53-dependent transcription activation. We examine the mechanism of this functional inhibitory interaction and demonstrate that PATZ1 excludes p53 from DNA bind- ing. This study documents PATZ1 as a novel player in the p53 pathway. ild-type (WT) p53 is a stress-responsive, sequence-specific mouse embryonic stem cells (7). Yet another ChIP-Seq study re- on September 28, 2017 by BILKENT UNIVERSITY Wtranscription factor that inhibits the cell cycle, promotes vealed that of the 3,193 genes with nearby p53 REs, 432 were senescence, and, if the insult to cellular health is not resolved, regulated by DOX in mouse embryonic fibroblasts (MEFs) (8). induces apoptotic death (1). p53 mutations that allow cells to In the present study, we have identified the role of a BTB-ZF escape this death are the most common genetic event in human (broad-complex, tramtrack, and bric-à-brac zinc finger) tran- cancer (2). Alternative mechanisms of escape rely on the overex- scription factor, PATZ1 (POZ-AT hook-zinc finger) that inhibits pression of MDM2 and MDMX, major negative regulators of p53 the ability of p53 to bind to its REs. PATZ1 has an N-terminal that keep p53 levels low under unstressed conditions through BTB domain for protein interaction, a central DNA binding AT ubiquitination-mediated degradation (3). Moreover, many DNA hook domain, and a second, C-terminal, zinc finger (ZF) DNA tumor viruses encode proteins that can inactivate p53 (2). binding domain (9–11). A recent ChIP-Seq study identified that However, the list of p53 regulators is far from complete. Vari- the PATZ1 binding consensus can be either AT rich or GC rich ous stress conditions result in the upregulation of p53, con- (12). The specificity for two different consensus sequences is likely trolled by feedback loops, posttranslational modifications, and due to two different (AT hook and ZF) DNA binding domains. epigenetic changes. In the present study, we aimed to find new Members of the BTB-ZF transcription factor family control players that modify p53 function. diverse yet potentially related processes in cancer, development, p53 is composed of an N-terminal transactivation domain and stem cell biology. PATZ1 can interact with other BTB-ZF (through which it interacts with MDM2), a proline-rich domain, central DNA binding and tetramerization domains, and a C-ter- transcription factors such as BCL-6 and Bach to control important minal regulatory domain. p53 binds response elements (REs) in cell fate decisions (13, 14). While PATZ1 was originally identified nuclear DNA, which results in stress-induced changes (either up- as a lymphocyte specific c-Myc-regulating transcription factor, its or downregulation) in gene expression. Tetrameric p53 binds REs that are composed of two half-sites, each with a consensus of RR RCWWGYYY, separated by 0 to 21 bases (where R is a purine, Y is Received 9 December 2014 Returned for modification 28 December 2014 Accepted 25 February 2015 a pyrimidine, and W is either an A or T) (4). p53 REs can be Accepted manuscript posted online 9 March 2015 subcategorized as those that respond to low levels of wild-type Citation Keskin N, Deniz E, Eryilmaz J, Un M, Batur T, Ersahin T, Cetin Atalay R, p53, those that respond to stabilized p53 after stress, and those Sakaguchi S, Ellmeier W, Erman B. 2015. PATZ1 is a DNA damage-responsive that respond to p53 with mutations in its DNA binding domain. A transcription factor that inhibits p53 function. Mol Cell Biol 35:1741–1753. recent study found 160 p53 REs controlling 129 genes in the hu- doi:10.1128/MCB.01475-14. man genome (5). Another study using chromatin immunopre- Address correspondence to Batu Erman, [email protected]. cipitation (ChIP) with paired-end ditag (PET) sequencing identi- * Present address: Jitka Eryilmaz, SANKO Textile, Inegol, Bursa, Turkey. fied 542 functional p53 binding sites in the human genome (6). N.K. and E.D. contributed equally to the manuscript. Similarly, a study using ChIP followed by RNA sequencing (ChIP- Copyright © 2015, American Society for Microbiology. All Rights Reserved. Seq) found that 3,697 target genes were bound and differentially doi:10.1128/MCB.01475-14 regulated by p53 in doxorubicin (DOX) (Adriamycin)-treated May 2015 Volume 35 Number 10 Molecular and Cellular Biology mcb.asm.org 1741 Keskin et al. expression and function are not restricted only to T lymphocytes Healthcare-Open Biosystems) was modified and transferred into the (15–19). Early embryonic expression is likely functional, as pLMP retroviral expression plasmid (EAV4679; GE Healthcare-Open Patz1Ϫ/Ϫ mice are born at a non-Mendelian frequency and are Biosystems), using EcoRI and XhoI restriction sites. Retrovirus produc- small in size (16, 17). PATZ1 is expressed in human colon and tion, infection, and selection were performed as stated above. Silencing of PATZ1 expression in NIH 3T3 cells was confirmed by Western blotting. testicular cancer cell lines and is deleted or translocated to the Ϫ/Ϫ Ewing sarcoma gene (EWS) in small-cell sarcoma (9, 17, 20). The Luciferase assays. HCT116 p53 , HEK293T, and HeLa cells were cotransfected with plasmids expressing p53, PATZ1, or PATZ1Alt with PATZ1 protein was recently shown to be an inhibitor of p53- firefly luciferase reporter constructs that contained either the pG13 pro- dependent endothelial cell senescence and was also shown to in- moter (Addgene plasmid 16442), the mG15 promoter (Addgene plasmid teract with p53 (17, 21). 16443), the p21 gene promoter (Addgene plasmid 16451) (26), or the We undertook the present study to identify factors that control Puma gene promoter (Addgene plasmid 16591) (27), together with the Downloaded from p53 function. We used state-of-the-art techniques such as tran- renilla luciferase-encoding pRL-SV40 plasmid (Promega). Cells were har- scriptome sequencing (RNA-Seq) and real-time cell growth ex- vested 48 h after transfection and analyzed with the Dual Luciferase re- periments to show that PATZ1 promotes cellular proliferation. porter kit (catalog number E1960; Promega). Promoter activity was ana- We discovered that PATZ1 is regulated by DNA damage and can lyzed by normalizing firefly luciferase activity to renilla luciferase activity bind to and inhibit the transcriptional activity of p53. We charac- and adjusted to the fold increase over background. terized the interaction of p53 with PATZ1 and its alternatively Expression plasmids and oligonucleotides. The cytomegalovirus spliced variants. We found that at least one of the four known (CMV) promoter-driven FLAG-p53 expression plasmid was a kind gift Patz1 gene products binds to p53 and interferes with p53 function from P. Ballar, Ege University, Izmir, Turkey. We sequenced this plasmid to ensure that the p53 cDNA that it carries is a WT allele. Patz1, Patz1Alt, http://mcb.asm.org/ by preventing p53 DNA binding. Even though previous studies on and different Patz1 truncations were cloned into pCMV-HA or pCMV- PATZ1 focused on its role in T lymphocytes, we discovered the Myc (Clontech), using the EcoRI restriction enzyme site. Patz1 and global and unbiased effects of its absence on cell proliferation, Patz1Alt cDNAs were also cloned into the pBABE-puro plasmid by using adhesion, and morphology. Collectively, our findings indicate the EcoRI restriction enzyme site. p53␣ and p53␤ expression plasmids that PATZ1 is a novel regulator of p53-dependent transcription. were a kind gift from J. C. Bourdon, University of Dundee. The sequences of oligonucleotides used in this study for amplification are available upon MATERIALS AND METHODS request. Ϫ Ϫ Real-time cell growth and doubling time analysis. Cell proliferation Generation of MEFs. WT and Patz1 / MEFs were isolated from 13.5- was measured continuously with an xCELLigence real-time cell analyzer on September 28, 2017 by BILKENT UNIVERSITY day-old embryos and analyzed at early (Ͻ5) passages. All mice were bred dual plate (RTCA DP) system (ACEA Biosciences, San Diego, CA), by and maintained in the animal facility of the Medical University of Vienna, seeding 5,000 cells into each well of an E-Plate View 16 tissue culture plate and animal experiments were done according to protocols approved by in 200 ␮lin5%CO at 37°C.
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