An IFN regulatory factor-2 DNA-binding domain dominant negative mutant exhibits altered cell growth and gene expression YR Rubinstein 1 , PH Driggers 2 , VV Ogryzko 3 , AM Thornton 4 , K Ozato 3 and CH Pontzer* ,1 1 Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, MD 20742, USA; 2 Department of Obstetrics and Gynecology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland, MD 20814, USA; 3 Laboratory of Molecular Growth Regulation, National Institute of Child Health and Human Development, NIH, Bethesda, Maryland, MD 20892, USA; 4 Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892, USA In order to study interferon regulatory factor (IRF) family mediation of cell growth regulation, we estab- lished U937 cell lines stably transfected with a truncated form of IRF-2 lacking the transcriptional repressor domain. The truncated IRF-2 contained the DNA binding domain (DBD) and bound the ISRE. Phenoty- pically, the IRF-2 DBD transfectants exhibited reduced cell growth, altered morphology and increased cell death. Consistent with alterations in growth characteristics, the IRF-2 DBD transfectants constitutively expressed higher levels of the cyclin dependent kinase inhibitor p21 WAF1/Cip1 than did control clones. The level of p21 WAF1/Cip1 expression was positively correlated with the level of DBD expressed, as well as with the level of growth inhibition in these clones. DBD expression also correlated with expression of other members of the growth regulatory complex, cyclin dependent kinase 2 and cyclin A, but not proliferating cell nuclear antigen. These results imply active repression by IRF-2 to keep p21 WAF1/Cip1 transcriptionally silent. Oncogene (2000) 19, 1411 ± 1418. Keywords: interferon; interferon regulatory factor; cell growth Introduction Interferons (IFNs) are known to modulate expression of a number of proteins that mediate their antiproli- ferative and antitumor activities (Pestka et al., 1987). The signal transduction cascades leading to expression of proteins involved in growth regulation are initiated by binding of IFNs to their cell surface receptors. Type I IFNs (IFNa, IFNb, IFNo, and IFNt) all act via binding to the same receptor (Stewart et al., 1987). Upon receptor engagement, the JAK1 and TYK2 tyrosine kinases phosphorylate STAT-1a, -1b and STAT-2 proteins. These activated STAT proteins, together with p48, form the IFN-stimulated gene factor 3 (ISGF3) complex. ISGF3 can then bind to conserved sequences present in the promoter of type I IFN- responsive genes, IFN-stimulated response elements (ISRE) (Darnell et al., 1994). In addition to STAT proteins, members of the IFN regulatory factor (IRF) family of transcription factors are induced and activated in response to interaction of type I IFNs with their receptors. The IRF family includes, among others, IRF-1, IRF-2, IRF-3, IFR-4, IFN consensus sequence binding protein (ICSBP) and p48 (Harada et al., 1998; Pitha et al., 1998). IRF-1 and p48, act as transcriptional activators while others, such as IRF-2 and ICSBP, function as repressors. All of these factors exhibit high homology in their DNA-binding domains (DBD) and interact with a conserved element, termed, IRF-E. Numerous studies have indicated that the regulation of cell growth depends on the ratio between IRF-1 and IRF-2, and any change that interferes with the balance of these two factors can result in a signi®cant eect on cell growth (Taniguchi et al., 1995; Kirchho et al., 1993; Tanaka et al., 1994). Over expression of IRF-2 in NIH3T3 cells results in oncogenic transformation of the cells, which revert to non-transformed phenotype when IRF-1 is over expressed (Harada et al., 1993). The presence of IRF-1 is critical for expression of some IFN- inducible genes. For example, induction of both guanylate binding protein and inducible nitric oxide synthase is IRF-1-dependent (Matsuyama et al., 1993). In contrast, IFN-induced expression of other genes, such as RNA-dependent protein kinase (PKR) and 2'5' oligoadenylate synthetase (2'5' OAS), both of which in¯uence cell growth (Meurs et al., 1993; Koromilas et al., 1992; Rysiecki et al., 1989; Hassel et al., 1993), appears to be independent of the IRF-1-mediated pathway of transcriptional activation. It has been suggested that despite the lack of signi®cant eect of IRF-1 on induction of these genes, their expression may be in¯uenced by alterations in the IRF-1:IRF-2 balance during the cell cycle (Lamphier and Taniguchi, 1994). The passage of the cell from one phase of the cell cycle to another is dependent on the activation and subsequent inactivation of the cyclins and their inhibitors. p21 WAF1/Cip1 is a universal cyclin dependent kinase (Cdk) inhibitor (El-Deiry et al., 1993; Harper et al., 1993; Xiong et al., 1993). Paradoxically, it has also been shown to be important in the activation of Cdks. Low levels of p21 WAF1/Cip1 expression prompt the association of cyclins and their kinases into active complexes, while high levels of p21 WAF1/Cip1 result in formation of a complex containing multiple p21 WAF1/Cip1 subunits with inactivation of Cdks (Zhang et al., 1994). The promoter of p21 WAF1/Cip1 has been found to have a number of IRF-1 binding sites (Taniguchi et al., 1995). The p21 WAF1/Cip1 gene is also regulated by p53, and an IRF-1 binding site has also been found in the promoter region of the p53 gene, which has an important role in Oncogene (2000) 19, 1411 ± 1418 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc *Correspondence: CH Pontzer Received 17 August 1999; revised 16 December 1999; accepted 13 January 2000