Oncogene (2000) 19, 2619 ± 2627 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Complex roles of Stat1 in regulating gene expression Chilakamarti V Ramana1, Moitreyee Chatterjee-Kishore1, Hannah Nguyen1 and George R Stark*,1 1Department of Molecular Biology, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio, OH 44195 USA Stat1 is a fascinating and complex protein with multiple, by which Stat1 is activated in response to IFNs and yet contrasting transcriptional functions. Upon activa- the nature of its interaction with other proteins and tion, it drives the expression of many genes but also speci®c gene targets are determined by the type of suppresses the transcription of others. These opposing IFN. The pathway leading to activation by Type II characteristics also apply to its role in facilitating IFN begins when IFNg binds to its receptor IFNGR, crosstalk between signal transduction pathways, as it catalyzing activation of Jak1 and Jak2 through auto- participates in both synergistic activation and inhibition and trans-phosphorylation. Already bound to the of gene expression. Stat1 is a functional transcription cytoplasmic domains of the IFNg receptor subunits factor even in the absence of inducer-mediated activation, (Bach et al., 1996; Kotenko et al., 1995; Sakatsume et participating in the constitutive expression of some al., 1995; Kaplan et al., 1996), the activated Jaks genes. This review summarizes the well studied involve- phosphorylate the receptor on speci®c tyrosine ment of Stat1 in IFN-dependent and growth factor- residues, including Y440 of IFNGR1, creating a dependent signaling and then describes the roles of Stat1 docking site for the recruitment of Stat1 via its Src- in positive, negative and constitutive regulation of gene homology 2 (SH2) domain (Greenlund et al., 1994, expression as well as its participation in crosstalk 1995; Igarashi et al., 1994). Other Stat family between signal transduction pathways. Oncogene members are activated by various cytokines through (2000) 19, 2619 ± 2627. similar recruitment mechanisms, making the IFNg signal transduction pathway a generic model for Stat- Keywords: constitutive transcription; negative regula- mediated cytokine signaling. tion; interferons; growth factors; crosstalk Receptor-bound Stat1 is activated through phos- phorylation of Y701 by the Jaks, leading to the formation of homodimers by reciprocal phosphotyr- osine-SH2 domain interactions (Schindler et al., 1992; Signal transducers and activators of transcription Shuai et al., 1992, 1993). The Stat1 homodimers then (Stats) comprise a family of transcription factors translocate to the nucleus by a mechanism that discovered through their key involvement in interferon depends on the GTPase activity of Ran/TC4 (Sekimoto (IFN) signaling. Their activation and mechanisms of et al., 1997), where they bind to the promoters of action represent the ®rst examples of direct signaling to IFNg-responsive genes via gamma activated sequence the nucleus by extracellular polypeptide ligands with- (GAS) elements (Darnell et al., 1994; Schindler and out the involvement of second messengers, tyrosine Darnell, 1995). Recent crystallographic studies reveal phosphorylation of transcription factors, and transcrip- that Stat1 homodimers interact with DNA over a tion factors that make direct contact with both 15 bp region, with relatively few contacts between the membrane-bound receptors and DNA (reviewed by Stat1 side chains and the DNA bases (Chen et al., Darnell et al., 1994; Schindler and Darnell, 1995; 1998). The DNA binding speci®city of Stat1 homo- Leaman et al., 1996a; Stark et al., 1998). Since the dimers may be aected additionally by their interac- initial discovery of Stats 1 and 2, the family has been tions with other transcription factors (Bluyssen et al., expanded to seven members. These play vital roles in 1995; Look et al., 1995). For most IFNg-inducible signaling pathways induced by many dierent cyto- genes, Stat1 binding results in the activation of kines and growth factors that regulate cell growth and expression. The transcriptional activity of Stat1 is dierentiation, the immune response, antiviral activity, enhanced at some point in the activation pathway by and homeostasis. This review focuses on the diverse the phosphorylation in the cytoplasm of serine 727 functions of Stat1. through a process that depends on Jak2 and the Pyk2 tyrosine kinase (Wen et al., 1995; Zhu et al., 1997; Takaoka et al., 1999; Kovarik et al., 1998). Stat1 and IFN-dependent signaling As shown in Figure 1, the activation of Stat1 by Type I IFN (IFNa/b) is initiated by ligand binding to IFN-dependent signaling involves members of four the IFNAR receptor, resulting in activation through protein families: (1) the Type I and II IFN receptors; auto- and trans-phosphorylation of Jak1 and Tyk2 (2) the receptor-associated Janus protein tyrosine (Colamonici et al., 1994; Gauzzi et al., 1996). Activated kinases (Jaks); (3) the Stats; and (4) members of the Jak1 and Tyk2 catalyze the sequential phosphorylation interferon regulatory factor (IRF) family of transcrip- of Y466 of IFNAR1 (Krishnan et al., 1996), Y660 of tion factors. As depicted in Figure 1, the mechanisms Stat2, and Y701 of Stat1. Heterodimerization of activated Stat1 and Stat2 is coordinated with their dissociation from the receptor, followed by binding of *Correspondence: GR Stark the heterodimer to p48, a member of the IRF family of Functions of Stat1 CV Ramana et al 2620 cytokines such as growth hormone, epidermal growth factor and interleukin-10, all of which activate Stat1 and Stat3 in vitro, are not substantially aected in Stat1-null mice (Meraz et al., 1996; Durbin et al., 1996). However, recent work of Lee et al. (2000) reveals that the lack of Stat1 does aect lymphocyte survival and proliferation in mice independently of the role of Stat1 in the response to IFNg. In summary, these studies con®rm the paramount role of Stat1 in IFN-dependent signaling and the related physiology. Roles of Stat1 in signaling in response to growth factors and cytokines Stat1 has been implicated as a mediator of biological responses to a variety of growth factors and cytokines, based on ligand-dependent tyrosine phosphorylation and activation, although these observations alone do not prove that Stat1 is essential for signal transduction. Jak1, Stat1 and Stat3 are phosphorylated in response to epidermal growth factor (EGF). However, Jak1 is not required either for Stat activation or for the Figure 1 Activation of Stat1 in IFNg and IFNa signaling. IFNg signaling involves ligand-induced oligomerization of the IFNg induction of the c-fos gene (Leaman et al., 1996b). receptor subunits (IFNGR1 and IFNGR2) leading to the Furthermore, Stat activation in response to EGF in phosphorylation and activation of Jak1, Jak2, IFNGR1 and Jak2- or Tyk2-null cells is normal. In cells transfected Stat1. Stat1 homodimers translocate to the nucleus and bind to with a kinase-dead mutant EGF receptor, there is no GAS elements. The IFNa/b-mediated association of IFNAR1 and Stat activation in response to EGF and c-fos is not 2 stimulates the phosphorylation and activation of Tyk2, Jak1, IFNAR1, Stat1 and Stat2. Stat1-2 heterodimers, in conjunction induced, indicating that the kinase activity of the with p48, form the trimeric complex ISGF3, which translocates to receptor is required, directly or indirectly, for these two the nucleus and binds to ISRE elements. Alternatively, Stat1 responses. Furthermore, transient transfections in Hela homodimers and Stat1-2 heterodimers translocate to the nucleus cells using a c-fos promoter lacking the Stat regulatory and bind to GAS elements element (SIE), indicate that this element may play a minor role in the induction of c-fos by EGF in these cells (Leaman et al., 1996b). Similarly, in Stat1-null transcription factors, to form the ISGF3 complex. mice, the induction of c-fos by EGF is not altered in ISGF3 translocates into the nucleus by an unknown the liver (Meraz et al., 1996). mechanism and binds to interferon-stimulated response PDGF-induced phosphorylation of Jaks and Stats is elements (ISREs) in the promoters of IFNa/b-inducible independent of any single Jak but does require receptor genes. Stat1 homodimers and Stat1-2 heterodimers not tyrosine kinase activity (Vignais et al., 1996). Mutagen- associated with p48, also formed in response to IFNa/ esis studies revealed that Stat activation by the PDGF b, bind to GAS elements to activate gene expression. receptor requires the juxtamembrane phosphorylation For example, in the case of the IRF1 gene, IFNa- sites but not Src tyrosine kinase activation. The induced Stat1-2 heterodimers stimulate expression by activation of MAP kinase and induction of c-fos is binding to a GAS element without p48 (Li et al., 1996). correlated with Stat but not Src activation by the However, based on studies in p48-null human cells, PDGF receptor (Sachsenmaier et al., 1999). Cell-free ISGF3 is by far the more important factor, activating studies have further shown that the activation of Stat1 most IFNa/b-inducible genes (John et al., 1991; requires it to interact with the PDGF receptor directly, Bluyssen et al., 1995). The levels of p48 and, therefore, whereas Stat3 activation additionally requires Jak the relative abundance of the various Stat1 complexes proteins (Vignais and Gilman, 1999). vary widely among dierent cell types. The binding of growth hormone leads to dimerization Analysis of the ISRE-ISGF3 interactions in the 6 ± of its receptor, accompanied by the phosphorylation and 16 and 9 ± 27 promoters demonstrate that ISGF3 activation of Jak1, Jak2, Stat1, Stat3 and Stat5 (Han et interacts with DNA over a 35 bp region (Dale et al., al., 1996). Jak2 but not Jak1 is required for the growth 1989; Imam et al., 1990). DNA contacts are made by hormone-dependent phosphorylation events that are Stat1 and p48, while Stat2 serves as the main provider required to couple the receptor to Stat-dependent of transcriptional activation (Qureshi et al., 1995; signaling pathways and also to pathways involving the Bluyssen and Levy, 1997).