Repression of IRF-4 target genes in human T cell leukemia virus-1 infection Yae¨l Mamane 1 , Nathalie Grandvaux 1 , Eduardo Hernandez 1 , Sonia Sharma 1 , Steve A Innocente 2 , Jonathan M Lee 2 , Nazli Azimi 3 , Rongtuan Lin 1 and John Hiscott* ,1 1 Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, and Department of Microbiology and Immunology, McGill University, Montreal Canada H3T 1E2; 2 Hamilton Regional Cancer Center, Hamilton, Ontario, Canada; 3 Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA The human T cell leukemia/lymphotropic virus-1 (HTLV-I) is the etiologic agent of adult T cell leukemia (ATL), an aggressive and fatal leukemia of CD4+ T lymphocytes. Interferon regulatory factor-4 (IRF-4) was shown previously to be constitutively expressed in T cells infected with HTLV-1. In this study, we investigated the role of IRF-4 gene regulation in the context of HTLV-1 infection using gene array technology and IRF-4 expressing T cells. Many potential IRF-4 regulated genes were identified, the vast majority of which were repressed by IRF-4 expression. Cyclin B1, a G2-M checkpoint protein identified as an IRF-4 repressed gene in the array, was further characterized in the context of HTLV-1 infection. All HTLV-1 infected cell lines and ATL patient lymphocytes demonstrated a dramatic decrease in cyclin B1 levels; subsequent analysis of the cyclin B1 promoter identified two sites important in IRF- 4 binding and repression of cyclin B1 expression. Furthermore, IRF-4-mediated repression of cyclin B1 led to a significant decrease in CDC2 kinase activity in HTLV-1 infected T cells. IRF-4 expression in HTLV-1 infected T cells also downregulated other genes impli- cated in the mitotic checkpoint as well as genes involved in actin cytoskeletal rearrangement, DNA repair, apoptosis, metastasis and immune recognition. Several of the identified genes are dysregulated in ATL and may provide important mechanistic information concerning pathways critical to the emergence of ATL. Oncogene (2002) 21, 6751 – 6765. doi:10.1038/sj.onc. 1205843 Keywords: human T cell leukemia virus; interferon regulatory factors; cyclin B1; transcription Introduction The human T cell leukemia/lymphotrophic virus-1 is the etiologic agent of adult T cell leukemia (ATL), an aggressive and fatal leukemia of CD4+ T lymphocytes and is also associated with a neurological demyelinat- ing disease, tropical spastic paraparesis (TSP) or HTLV-1 Associated Myelopathies (HAM). ATL and HAM/TSP are geographically localized to regions of the world where HTLV infection is endemic, notably southern Japan, sub-Saharan Africa, the Caribbean basin and the southwestern United States (Edlich et al., 2000; Tsukasaki et al., 2000; Yoshida, 2001). At present, it is estimated that between 10 and 20 million people worldwide are infected with HTLV. Treatments for ATL are ineffective; conventional chemotherapeutic treatments provide at most a 5% 4-year survival rate (Cann and Chen, 1996; Ohshima et al., 1999). Once diagnosed with acute ATL, the median survival time is in the order of a few months (Edlich et al., 2000; Tsukasaki et al., 2000). Based on the phenotype of ATL cells in vivo and HTLV-1 transformed T cell lines, infection of CD4+ T cells initiates a multi-step oncogenic process that is characterized by an early proliferation of HTLV-1 infected T cells, resulting in polyclonal expansion of infected cells. Over a period of decades in vivo, a growth factor independent, monoclonal population of leukemic T cells emerges, likely as a consequence of the rapid growth of a single leukemic clone bearing multiple mutations in oncogenes and/or tumor suppres- sor genes (Yao and Wigdahl, 2000; Yoshida, 2001). The oncogenic potential of HTLV-1 resides in part in the viral Tax protein, a positive regulator of viral gene transcription which disrupts cell cycle control, gene transcription and signal transduction via protein- protein interactions (Hiscott et al., 2001; Neuveut and Jeang, 2002; Yoshida, 2001). One of the targets of Tax is the NF-kB/IkB factors which control immune and growth regulatory gene transcription (Israel, 2000; Karin, 1999a,b). The ability of Tax to stimulate NF- kB activity occurs via activation of the signaling cascade controlling IkB phosphorylation specifically through interaction with IKKg subunit of the IkB kinase complex (Chu et al., 1999; Sun et al., 2000; Xiao et al., 2000; Xiao and Sun, 2000). These molecular events culminate in transcriptional dysregulation in HTLV-1 infected cells, leading to CD4+ T cell proliferation that predisposes to ATL development (Yao and Wigdahl, 2000; Yoshida, 2001). Interestingly, one of the members of the interferon regulatory factor (IRF) family – IRF-4 – was shown to be highly expressed in cells derived from patients Received 18 April 2002; revised 26 June 2002; accepted 5 July 2002 *Correspondence: J Hiscott, Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec, Canada H3T 1E2; E-mail: john.hiscott@mcgill.ca Oncogene (2002) 21, 6751 – 6765 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc