A 5′ Extended IFN-Stimulating Response Element Is Crucial for IFN- −γ Induced Tripartite Motif 22 Expression via Interaction with IFN Regulatory Factor-1 This information is current as of October 1, 2021. Bo Gao, Yaxin Wang, Wei Xu, Zhijian Duan and Sidong Xiong J Immunol 2010; 185:2314-2323; Prepublished online 14 July 2010; doi: 10.4049/jimmunol.1001053 Downloaded from http://www.jimmunol.org/content/185/4/2314 References This article cites 49 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/185/4/2314.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology A59 Extended IFN-Stimulating Response Element Is Crucial for IFN-g–Induced Tripartite Motif 22 Expression via Interaction with IFN Regulatory Factor-1 Bo Gao,* Yaxin Wang,* Wei Xu,* Zhijian Duan,* and Sidong Xiong*,† Interferon-g is crucial for the noncytopathic clearance of hepatitis B virus. In our previous study, we demonstrated that an IFN-g– inducible molecule, tripartite motif (TRIM) 22, played an important role in antiviral immunity against hepatitis B virus. However, the molecular mechanism of TRIM22 induction by IFN-g is still unclear. In this study, we identified a novel cis-element termed 59 extended IFN-stimulating response element (59 eISRE) that was crucial for IFN-g inducibility of TRIM22 through transfection assays with luciferase reporter constructs and EMSAs. The 59 eISRE consists of an ISRE-like motif (ACTTTCGTTTCTC) and a 6-bp sequence (AATTTA) upstream of it, and all three thymine triplets of this cis-element (AATTTAACTTTCGTTTCTC) were revealed to contribute to the IFN-g inducibility of TRIM22 by site-directed mutagenesis. Further studies showed that upon IFN-g Downloaded from stimulation, the 59 eISRE could be bound by IFN regulatory factor-1 (IRF-1), but not by STAT1, as demonstrated by supershift analysis and an ELISA-based transcription factor assay. Moreover, overexpression of IRF-1 significantly induced TRIM22 expres- sion, whereas silencing of IRF-1 with specific short interference RNA abolished IFN-g–induced TRIM22 expression in HepG2 cells, indicating an IRF-1–dependent expression of TRIM22. Taken together, it was demonstrated in this study that a novel cis-element, 59 eISRE, was crucial for the IFN-g–induced transcriptional activity of the TRIM22 gene via interaction with IRF-1. The Journal of Immunology, 2010, 185: 2314–2323. http://www.jimmunol.org/ ripartite motif (TRIM) family proteins are involved in In our previous study, we demonstrated that one of the TRIM diverse cell processes, including apoptosis, differentiation, family members, TRIM22 (also named Staf50), could inhibit T and transcriptional regulation (1, 2). They are characterized hepatitis B virus (HBV) gene expression and replication efficiently by a combination of RING, B-box, and coiled-coil domains (1). The by significantly inhibiting the activity of HBV core promoter in RING domain of many TRIM proteins has been shown to possess a RING domain-dependent manner (13). We also demonstrated that E3 ubiquitin ligase activity (2–5), whereas the B-box and coiled- TRIM22 was a RING finger E3 ubiquitin ligase (4), and its E3 coil domains may be involved in protein interactions and homo/ ligase activity was responsible for its antiviral activity against ence- heterodimerization (1, 2). Recent studies have demonstrated that phalomycocarditis virus as reported by another research group (14). by guest on October 1, 2021 many members of the TRIM family play important roles in innate Additionally, several studies indicated that TRIM22 possessed anti- antiviral immunity. For example, TRIM5a has been shown to block retroviral activity depending on certain cell types (15–17). the infectivity of a range of different retroviruses in a species- IFNs have an important role in immune system to defend against specific manner (6, 7); TRIM25 is essential for RNA helicase viral infections. They consist of two main classes of related cyto- RIG-I–mediated antiviral activity (5, 8); TRIM28 can inhibit the kines: type I IFNs and type II IFNs. Although there are many type I replication of murine leukemia viruses or related retroelements in IFNs, including IFN-a, IFN-b, IFN-l, etc., IFN-g is the only type II embryonic cells by transcriptional silencing (9, 10). It is speculated IFN family member (18–20). The biological effects of both IFN- that the TRIM family may represent a new, widespread class of a/b and IFN-g are mediated by IFN-stimulated genes (ISGs), but proteins involved in antiviral innate immunity (11, 12). the induction of ISGs by IFN-g is often more complex than that of IFN-a/b, largely due to the fact that many of the IFN-g–stimulated *Institute for Immunobiology, Department of Immunology, Shanghai Medical Col- genes are induced with variable kinetics unlike those stimulated by lege of Fudan University, Shanghai; and †Institute of Biology and Medical Sciences, IFN-a/b, and the expression of some IFN-g–regulated genes Soochow University, Suzhou, People’s Republic of China requires de novo protein synthesis (20–23). Received for publication March 31, 2010. Accepted for publication June 6, 2010. To date, nearly 70 TRIM family members have been identified, This work was supported by grants from the National Natural Science Foundation of and two classes of TRIMs can already be distinguished: IFN- China (30890141, 30872355, 30671952), the Science and Technology Commission of Shanghai Municipality (09JC1401800), the Doctoral Fund of Youth Scholars of inducible TRIMs and constitutive TRIMs (12). The IFN-inducible Ministry of Education of China (2009071120060), the Major State Basic Research TRIMs are involved in a broad range of biological processes that Development Program of China (2007CB512401), and the Program for Outstanding Medical Academic Leader of Shanghai (LJ06011, 07JC14004). are associated with innate immunity, and the constitutive TRIMs, such as TRIM1 and TRIM28, can also block virus replication at Address correspondence and reprint requests to Dr. Sidong Xiong, Institute of Bi- ology and Medical Sciences of Soochow University, 199 Ren ai Road, Suzhou, early stages. The two classes of TRIMs may contribute to the over- 215006, P.R. China. E-mail address: [email protected] all antiviral defense (11, 12). TRIM22 was demonstrated to be one Abbreviations used in this paper: Act.D, actinomycin D; ChIP, chromatin immuno- of the most strongly induced TRIMs in HepG2 cells after treatment precipitation; CHX, cycloheximide; eISRE, extended IFN-stimulating response ele- ment; GAS, IFN-g activation site; GBP, guanylate-binding protein; HBV, hepatitis B with IFNs in our previous study (13). Similar to our results, Barr virus; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; ISRE, IFN-stimulating et al. (15) found that TRIM22 was the most upregulated TRIM response element; NP-40, Nonidet P-40; siRNA, short interference RNA; TRIM, queried on the microarray prepared from IFN-treated human tripartite motif. osteosarcoma cells. Additionally, TRIM22 was also reported to Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 be strongly induced by IFNs in several other cell lines, such as www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001053 The Journal of Immunology 2315 Hela, MCF-7, and Daudi, etc. (17, 24). However, at present, little is plasmid was mixed with 3 ml Lipofectamine 2000 in serum-free medium known about how IFNs regulate TRIM22 gene expression. and added into HepG2 cells. In all transfection assays, pCMV–b-gal was cotransfected to normalize the transfection efficiency. Cells were incubated In this paper, we mainly focused our study on the molecular for 6 h at 37˚C in the presence of the transfection complex, washed twice mechanisms of transcriptional regulation of TRIM22 gene by with serum-free medium, and grown in fresh supplemented medium. For IFN-g. We identified a novel cis-element termed 59 extended IFN- IFN-g–treated samples, IFN-g was added 24 h posttransfection, and the stimulating response element (eISRE), which was crucial for the cultures were incubated for another 24 h. The cells were harvested, and the IFN-g inducibility of TRIM22. In addition, the 59 eISRE was also luciferase activity in the cell lysates was determined with the Luciferase Reporter Assay System (Promega). Reporter activities are presented as implicated in the constitutive transcriptional activity of TRIM22. means 6 SD of at least three independent experiments. Furthermore, we demonstrated that IFN regulatory factor-1 (IRF-1) could bind to the 59 eISRE both in vitro and in vivo and played a key Nuclear extract preparation role in the transcriptional regulation of TRIM22. A total of 5 3 106 IFN-g–treated or untreated HepG2 cells were washed in cold PBS and resuspended in 500 ml buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF). Materials and Methods Postincubation for 15 min on ice, cells were lysed in 0.6% Nonidet P-40 Cell culture and stimulation (NP-40) by vigorous vortex for 10 s and centrifuged at 5000 rpm for 5 min.