Oncogene (2012) 31, 1442–1458 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE The proto-oncoprotein KR-POK represses transcriptional activation of CDKN1A by MIZ-1 through competitive binding KM Lee1, WI Choi1, DI Koh1, YJ Kim1, BN Jeon1, JH Yoon1, CE Lee2, SH Kim2, JOh2 and MW Hur1 1Department of Biochemistry and Molecular Biology, Brain Korea 21 Project for Medical Science, Severance Biomedical Research Institute, Yonsei University School of Medicine, SeoDaeMoon-Ku, Seoul, Korea and 2Department of Biological Science, Sungkyunkwan University, Suwon, Korea The BTB/POZ family of proteins has been implicated in Introduction multiple biological processes, including tumourigenesis, DNA damage responses and cell cycle progression and The BTB/POZ domain proteins were originally found in development. MIZ-1 (Myc-interacting zinc-finger protein Drosophila melanogaster, bric-a-brac, tramtrack and 1) is known to activate transcription of CDKN1A. broad complex transcription regulators (Koonin et al., We recently found that a kidney cancer-related POK 1992; Aravind and Koonin 1999) and in pox virus zinc- transcription factor, KR-POK, is highly expressed in finger proteins. The BTB/POZ domain proteins have an kidney, brain and bone marrow cancer tissues and is a evolutionarily conserved protein–protein interaction potential proto-oncoprotein. Mouse Kr-pok represses domain at their N-termini, called the BTB/POZ domain transcription of the CDKN1A by acting on the proximal (Bardwell and Treisman, 1994; Albagli et al., 1995). promoter. The BiFC/FRET assay, co-immunoprecipita- These proteins have been identified in organisms ranging tion and glutathione S-transferase-fusion protein pull- from human to yeast. About 40 of the 194 human BTB/ down assay indicate that MIZ-1 and Kr-pok interact POZ domain, regulatory proteins are classified as POK via their POZ domains. Oligoucleotide pull-down assays family proteins. POK proteins are made up of an and chromatin immunoprecipitation assays revealed that N-terminal POZ domain, which has an important role in MIZ-1 binds to the proximal GC-box#3 (bp, À55 to À63) forming homo- or hetero-dimers and interacting with and the MIZ-1-binding elements, MRE-A (bp, À90 to other proteins, and a C-terminal Kru¨ppel-type (C2H2) À64) and MRE-B (bp, À27 to À17). Interestingly, MIZ-1 zinc-finger domain, which recognizes and binds to also binds to the distal p53-binding elements. Kr-pok binds specific DNA sequences. These proteins have been to the proximal GC-box#1 (bp, À95 to À100) and #3 (bp, implicated in many biological processes, and in parti- À55 to À63) relatively strongly. It also shows weak cular, some of the POK proteins were shown to act as binding to the MREs and the distal p53-binding elements. major regulators of apoptosis (Yamochi et al., 1999), Kr-pok competes with MIZ-1 in binding to these elements differentiation, development (Farkas et al., 1994; Barna and represses transcription by inhibiting MIZ-1/p300 et al., 2000), transcription (Chang et al., 1996; Dong recruitment, which decreases the acetylation of histones et al., 1996; Dhordain et al., 1997; Lin et al., 1998; H3 and H4. Our data indicate that Kr-pok stimulates cell Deltour et al., 1999; Huynh et al., 2000; Maeda et al., proliferation by interfering with the function of MIZ-1 in 2005; Jeon et al., 2008; Choi et al., 2008) and CDKN1A gene transcription using a mechanism that is oncogenesis (Chen et al., 1993; Kerckaert et al., 1993; radically different from other MIZ-1-interacting proteins, Maeda et al., 2005). such as B-cell lymphoma 6, c-Myc and Gfi-1. Promyelocytic leukaemia zinc-finger null mice display Oncogene (2012) 31, 1442–1458; doi:10.1038/onc.2011.331; severe defects in limb development and germ stem cell published online 1 August 2011 maintenance (Barna et al., 2000; Costoya et al., 2004). T-helper-inducing POZ/Kru¨ppel-like factor, also known Keywords: MIZ-1; Kr-pok; CDKN1A; p21; competition; as cKrox, has recently been reported as a master cell proliferation regulator of T-cell lineage commitment (He et al., 2010). B-cell lymphoma 6 (Bcl-6), promyelocytic leu- kaemia zinc finger and hypermethylated in cancer-1 have been implicated in non-Hodgkin’s lymphoma, acute promyelocytic leukaemia and spontaneous malig- Correspondence: Dr MW Hur, Department of Biochemistry and nant tumours, respectively (Chen et al., 1993; Kerckaert Molecular Biology, Brain Korea 21 Project for Medical Science, et al, 1993; Issa et al., 1997). Along with others, we Severance Biomedical Research Institute, Yonsei University recently found that FBI-1 stimulates cell proliferation School of Medicine, 134 ShinChon-Dong, SeoDaeMoon-Ku, Seoul and is a potent proto-oncoprotein (Maeda et al., 2005; 120-752, Korea. E-mail: [email protected] Jeon et al., 2008; Choi et al., 2009). We also showed that Received 23 January 2011; revised 31 May 2011; accepted 22 June 2011; FBI-1 increases FASN gene expression and thereby published online 1 August 2011 provides phospholipid cell membrane components to Kr-pok, transcriptional repressor of CDKN1A KM Lee et al 1443 meet the lipid needs of rapidly proliferating cancer cells In colon cancer cells, inhibition of p21 expression by c- (Choi et al., 2008). Along with others, we have shown Myc-MIZ-1 changes the p53-dependent response from that POK proteins, including FBI-1 (Choi et al., 2009), cell cycle arrest to apoptosis (Seoane et al., 2002). MIZ-1 ZBTB2 (Jeon et al., 2009), ZBTB4 (Weber et al., 2008), also interacts with BCL-6, another POK protein, via ZBTB5 (Koh et al., 2009) and BCL6 (Peukert et al., their POZ-domains, and this interaction is important in 1997) have major influences on the regulation of the p53 the transcriptional repression of CDKN1A and in the pathway. development of Bcl (Phan et al., 2005). Additionally, Among the genes of p53 pathway, the cyclin- Gfi-1 and MIZ-1 form a ternary complex with c-Myc dependent kinase inhibitor p21 is a downstream and together repress CDKN1A. c-Myc has been shown regulator of the ARF-HDM2-p53-p21 pathway and is to induce lymphomagenesis. It has been suggested that a major regulator of cell cycle arrest in mammalian cells the ternary complex of Gfi-1, MIZ-1 and c-Myc is (el-Deiry et al., 1993; Gartel and Radhakrishnan, 2005; important in the regulation of cell cycle and has an Toledo and Wahl, 2006). The p53 pathway has a effect on lymphomagenesis through the transcriptional crucial role in mediating growth arrest when cells are regulation of CDKN1A (Liu et al., 2010). exposed to DNA-damaging agents (el-Deiry et al., 1994; Using BiFC/FRET analysis, we recently found that Ogryzko et al., 1997). The CDKN1A gene, mainly MIZ-1 interacts with the POK protein Kr-pok (also regulated at the transcriptional level, is a transcriptional called ZBTB36, ZNF857C in humans) via its POZ target of the tumour-suppressor p53. Whereas induction domain. KR-POK (kidney cancer-related POZ-domain of p21 by exposure to DNA-damaging agents results in Kru¨ppel zinc-finger protein) is a proto-oncoprotein that G1-, G2- or S-phase arrest, repression of p21 expression is highly expressed in majority of malignant kidney has a variety of outcomes depending on the cellular cancer tissues, and there is a strong correlation between context (Gartel, 2009). p21 suppresses oncogenesis by kidney cancer development and KR-POK expression negatively regulating cell growth, as has been demon- levels. Murine KR-POK, indicated as Kr-pok, stimu- strated in chronically damaged liver and renal epithelial lates cell proliferation and represses the transcription of cells (Willenbring et al., 2008). CDKN1A and by recruiting co-repressor—HDAC com- POK family proteins, and the molecular interac- plexes, causing deacetylation of histones H3 and H4 tions among POK proteins, have potential roles in (unpublished data). Accordingly, molecular interactions cancer development or cell differentiation by regulating between Kr-pok and MIZ-1 can have significant effects CDKN1A gene transcription, as have been elegantly on cell cycle regulation by MIZ-1. shown for MIZ1 and BCL6 in Bcl (Phan et al., 2005; MIZ-1 has an antagonistic function with Kr-pok in Weber et al., 2008; Choi et al., 2009; Jeon et al., 2009; the transcriptional regulation of CDKN1A and other Koh et al., 2009). Although the POK proteins Myc- genes of the p53 pathway. In this study, we investigated interacting zinc-finger protein 1 (MIZ-1), ZBTB4 and whether the two proteins, MIZ-1 and Kr-pok, have BCL6 are known to interact with each other (Phan et al., important roles in regulating the transcription of 2005; Weber et al., 2008), the partnerships and the CDKN1A. Our data suggest that Kr-pok competes with functions of most of the POZ domain proteins are MIZ-1 to bind to the CDKN1A proximal regions, largely unknown. Accordingly, to analyse and under- including MIZ-1-binding sites, and in this way interferes stand the biological functions carried out by POZ- with transcriptional activation by MIZ-1. The binding domain proteins and by molecular interactions among competition between Kr-pok and MIZ-1 on the POZ domains, we are in the process of determining proximal promoter of the CDKN1A is functionally virtually all of the protein interactions of every human significant and has an important role in cell proliferation POZ-domain protein using BiFC/FRET analysis and cancer development. (Kim et al., 2007). MIZ-1 (also called ZBTB17) interacts with c-Myc, ZBTB4, BCL-6 and Gfi-1 and recruits interacting proteins to the CDKN1A promoter (Peukert et al., Results 1997; Phan et al., 2005; Weber et al., 2008; Basu et al., 2009).