ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3

This information is current as Qian Feng, Ying Miao, Jun Ge, Yukang Yuan, Yibo Zuo, of September 27, 2021. Liping Qian, Jin Liu, Qiao Cheng, Tingting Guo, Liting Zhang, Zhengyuan Yu and Hui Zheng J Immunol 2018; 201:675-687; Prepublished online 25 May 2018;

doi: 10.4049/jimmunol.1800285 Downloaded from http://www.jimmunol.org/content/201/2/675

References This article cites 37 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/201/2/675.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

ATXN3 Positively Regulates Type I IFN Antiviral Response by Deubiquitinating and Stabilizing HDAC3

Qian Feng,*,†,1 Ying Miao,*,†,1 Jun Ge,*,1 Yukang Yuan,*,†,1 Yibo Zuo,*,† Liping Qian,*,† Jin Liu,*,† Qiao Cheng,*,† Tingting Guo,*,† Liting Zhang,*,† Zhengyuan Yu,‡ and Hui Zheng*,†

Ataxin-3 (ATXN3) belongs to the Josephin family of deubiquitinases. So far, ATXN3 is majorly linked to the neurodegenerative disease, Machado–Joseph disease. The role of ATXN3 in the antiviral function has not been explored, and the in vivo deubiquiti- nating activity of ATXN3 remains largely unknown. In this study, we report that ATXN3 is an important positive regulator of type I IFN (IFN-I)–mediated antiviral activity in murine primary lung cells and human epithelial and fibroblast cell lines. We clarify that ATXN3 does not promote IFN-I production, but enhances the IFN-I–mediated signaling pathway. Furthermore, ATXN3 physically interacts with histone deacetylase 3 (HDAC3) and upregulates the level of HDAC3 . Moreover, ATXN3 deubi- Downloaded from quitinates HDAC3, thereby enhancing HDAC3 protein stability. Interestingly, the interaction between ATXN3 and HDAC3 increases during viral infection, which promotes IFN-I–induced signaling in murine primary lung cells. Finally, we reveal the ATXN3/HDAC3 axis–mediated regulation of IFN-I antiviral response. These findings reveal a novel biological function of ATXN3 and an important antiviral mechanism by which the deubiquitinase ATXN3 positively regulates IFN-I antiviral response, and they may provide a novel strategy for enhancing IFN-based antiviral therapy. The Journal of Immunology, 2018, 201: 675–687. http://www.jimmunol.org/

biquitination, as a posttranslational modification, par- studied (6, 7). In general, K48-linked polyubiquitination modifi- ticipates in various cellular biological processes, such as cation results in the degradation of substrate (4, 6), U protein degradation, signal transduction, cell cycle, and whereas K63-linked polyubiquitination plays important roles in apoptosis (1–3). has 76 aa and can be covalently at- regulating endocytosis, DNA repair, protein kinases activation, tached to protein substrates (4). There are seven lysine residues, and so on (8, 9). including K6, K11, K27, K29, K33, K48, and K63, on the ubiq- Ubiquitination of a protein is a rigorous and reversible process. uitin (5). Among them, only K48 and K63 have been extensively The levels of protein ubiquitination are also precisely regulated by deubiquitinases. There are ∼100 deubiquitinases in the human by guest on September 27, 2021 genome (10–12), which have been divided into at least five major *Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123, families: the ubiquitin-specific protease (USP) family, the ovarian China; †Jiangsu Key Laboratory of Infection and Immunity, Soochow University, tumor family, the ubiquitin C-terminal hydrolase family, the Suzhou 215123, China; and ‡Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou 215006, China Machado–Joseph disease (MJD; the Josephin domain) family, and 1 the JAB1/MPN/Mov34 metalloenzyme family. Q.F., Y.M., J.G., and Y.Y. contributed equally to this work. Ataxin-3 (ATXN3) belongs to the MJD family, containing the ORCIDs: 0000-0002-6148-1907 (Z.Y.); 0000-0002-4325-4946 (H.Z.). N-terminal Josephin domain, two ubiquitin-interacting motifs, and Received for publication February 26, 2018. Accepted for publication May 8, 2018. polyglutamine (polyQ) (13). The Josephin domain is a cysteine This work was supported by National Natural Science Foundation of China Grants protease. Cys14, His119, and Asn134 in the Josephin domain are 31570865, 31770177, and 81702048, the Program of 1000 Young Talents, Chang- jiang Scholars and Innovative Research Team in University of Ministry of Education close to each other (see Fig. 7A) and are critical to ATXN3 cat- of China Grant PCSIRT-IRT1075, and the Priority Academic Program Development alytic activity (14–16). So far, the studies on ATXN3 are focused of Jiangsu Higher Education Institutions. on the function of its polyQ domain. It has been demonstrated that H.Z. and Z.Y. conceived and designed the projects; Q.F., Y.M., J.G., and Y.Y. per- the expansion of polyQ could result in lesion and death of neu- formed most of the experiments and data analysis; Y.Y., Q.C., Y.Z., L.Q., and J.L. contributed to cellular experiments and plasmid purification; T.G. and L.Z. provided rons, called MJD (17). Additionally, several studies reported that technical support; and H.Z. and Q.F. wrote the manuscript. ATXN3 can interact with some proteins, including CHIP, Parkin, Address correspondence and reprint requests to Prof. Hui Zheng or Dr. Zhengyuan and VCP, to take part in different physiological processes (18–22). Yu, Institutes of Biology and Medical Sciences, Soochow University, Ren-ai Road However, whether or how ATXN3 plays roles in cellular antiviral 199, Suzhou Industrial Park, Suzhou 215123, China (H.Z.) or Department of Oncol- ogy, The First Affiliated Hospital of Soochow University, Shizi Street 188, Suzhou defense still remains unexplored. 215006, China (Z.Y.). E-mail addresses: [email protected] (H.Z.) or The IFN family plays a critical role in the defense against [email protected] (Z.Y.) pathogenic microbes (23–27). There are three IFN families: type I Abbreviations used in this article: ATXN3, ataxin-3; CHX, cycloheximide; FH- (IFN-I or IFN-a/b), type II (IFN-g), and type III (IFN-l) IFNs. In ATXN3, Flag-HA–tagged ATXN3; HA, hemagglutinin; HA-ub, HA-ubiquitin; HDAC3, histone deacetylase 3; H1N1, influenza A virus; IFN-I, type I IFN; ISG, the classic IFN-I signaling pathway, IFN-I members bind to their IFN-stimulated ; ISRE, IFN-stimulated response element; mIFN-b, mouse IFN- receptor (the IFNAR1 and IFNAR2) and induce tyrosine phos- b; MJD, Machado–Joseph disease; polyQ, polyglutamine; qPCR, quantitative real- time PCR; SeV, Sendai virus; shATXN3, shRNA against human ATXN3; shHDAC3, phorylation and activation of the JAK family (JAK1/TYK2), shRNA against human HDAC3; shRNA, short hairpin RNA; TCID50, 50% tissue which subsequently activates STAT proteins. Phosphorylated culture–infective dose; TM, triple ; USP, ubiquitin-specific protease; VSV, STAT1 and STAT2 combine with IFN regulatory factor 9 to form a vesicular stomatitis virus; WT, wild-type. transcription complex. This complex translocates into the nucleus Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 to bind to the IFN-stimulated response elements (ISRE) located www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800285 676 ATXN3 DEUBIQUITINATES HDAC3 TO PROMOTE IFN SIGNALING within IFN-stimulated gene (ISG) promoters, which finally in- N-ethylmaleimide (10 mM) was added into the above lysis buffer when the duces expression of a series of ISGs to perform IFN-activated ubiquitination of protein was detected. Equivalent protein quantities were biological functions (28, 29). subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were then blocked with 5% nonfat In this study, we show that ATXN3 potentiated cellular antiviral milk or 5% BSA for 0.5 h at room temperature and then incubated with the responses. We found that ATXN3 did not promote IFN-I production, but primary Abs: anti-Flag (F7425; Sigma-Aldrich), anti-HA (ab9110; Abcam), enhanced IFN-I–mediated antiviral defense. Interestingly, the length of anti-ubiquitin (sc-8017; Santa Cruz Biotechnology), anti-GAPDH (AB-M- the polyQ tract of ATXN3 had no influence on its antiviral activity. M001; Hangzhou Goodhere Biotechnology), anti-Myc (M20002H; Abmart), anti–a-tubulin (66031-1-Ig; Proteintech), anti–pY701-STAT1 (9167; Cell ATXN3 positively regulated IFN-I–mediated antiviral signaling during Signaling Technology), anti–VSV-G (sc-66180; Santa Cruz Biotechnology), viral infections. We further demonstrated that ATXN3 physically anti–b-actin (66009-1-Ig; Proteintech), anti-HDAC3 (sc-11417; Santa Cruz interacted with histone deacetylase 3 (HDAC3). Moreover, ATXN3 Biotechnology), anti-ATXN3 (13505-1-AP; Proteintech), and anti-HA could remove both K48-linked and K63-linked polyubiquitination of (H1N1) (11684-T56; Sino Biological). Then the polyvinylidene difluoride 3 HDAC3 and therefore increased the level and the stability of HDAC3 membrane was washed three times with PBST (1 PBS and 1% Tween 20) and then subjected to secondary Abs (HRP-conjugated goat anti-rabbit or protein. Furthermore, ATXN3 promotes cellular antiviral responses that goat anti-mouse [Bioworld]) for 1 h at room temperature and visualized with are dependent on HDAC3 in IFN-I signaling. Our findings identify ECL Prime (Thermo Scientific). The densities of Western blots were ana- HDAC3 as an in vivo substrate of the deubiquitinase ATXN3, and they lyzed with the ImageJ program (http://rsbweb.nih.gov/ij/download.html). reveal a novel biological function of ATXN3, which promotes the RNA isolation and real-time PCR understanding of cellular antiviral innate immunity. Total RNAs were extracted from cells using TRIzol reagent (Invitrogen). The cDNA was produced by reverse transcription using oligo(dT) and Materials and Methods analyzed by quantitative real-time PCR (qPCR) with IFIT1, ISG15, ISG54, Downloaded from Cell culture and reagents SeV, H1N1, VSV, b-actin, IFN-a,andIFN-b primers using SYBR Green Supermix (Bio-Rad Laboratories). The primer sequences were as follows: HeLa, A549, HEK293T, HT1080, PC9, and HepG2 cells were cultured in SeV, 59-GATGACGATGCCGCAGCAGTAG-39 and 59-CCTCCGATGT- DMEM (HyClone Laboratories) supplemented with 10% FBS (Life CAGTTGGTTCACTC-39; H1N1, 59-TTCTAACCGAGGTCGAAACG-39 Technologies) and antibiotics (100 U/ml penicillin and 100 mg/ml strep- and 59-ACAAAGCGTCTACGCTGCAG-39;IFIT1,59-CACAAGC- tomycin) at 37˚C and 5% CO2. Cells were transfected using LongTrans CATTTTCTTTGCT-39 and 59-ACTTGGCTGCATATCGAAAG-39;ISG15,

(Ucallm) or polyethyleneimine (Polysciences) according to the manufac- 59-GGGACCTGACGGTGAAGATG-39 and 59-CGCCGATCTTCTGGGT- http://www.jimmunol.org/ turers’ instructions. Human IFN-a was purchased from PBL Interferon GAT-39;ISG54,59-CACCTCTGGACTGGCAATAGC-39 and 59-GTCAG- Source. Mouse IFN-b was purchased from R&D Systems. Cycloheximide GATTCAGCCGAATGG-39;VSV,59-ACGGCGTACTTCCAGATGG-39 (CHX), MG132, puromycin, Polybrene, and other chemicals were pur- and 59-CTCGGTTCAAGATCCAGGT-39; b-actin, 59-ACCAACTGGGAC- chased from Sigma-Aldrich. GACATGGAGAAA-39 and 59-ATAGCACAGCCTGGATAGCAACG-39; IFN-a,59-TGGGAACAGAGCCTCCTAGA-39 and 59-CAGGCACAAG- Constructs GGCTGTATTT-39;andIFN-b,59-CATTACCTGAAGGCCAAGGA-39 and 59- Flag-hemagglutinin (HA)–tagged ATXN3 (FH-ATXN3) was obtained from CAGCATCTGCTGGTTGAAGA-39. The relative expression of the target Addgene. Flag-HDAC1, Flag-HDAC2, and Flag-HDAC3 were gifts from Dr. was normalized to b-actin mRNA. The results were analyzed from three 6 T. Kang (Sun Yat-sen University Cancer Center, Guangzhou, China). The independent experiments and are shown as the average mean SD. lentiviral packaging plasmids psPAX2 and pMD2G were gifts from Dr. S. He Reporter gene assay by guest on September 27, 2021 (Soochow University). HA-ubiquitin (HA-ub), HA-R48k, HA-R63K, IFN-b– luciferase, ISRE-luciferase, and Renilla plasmids were described previously For analyses of IFN-b production, cells were cotransfected with the IFN- (30). All short hairpin RNAs (shRNAs) against human ATXN3 (shATXN3) b–luciferase together with Renilla plasmids and FH-ATXN3 or control plasmids were purchased from GeneChem (Shanghai, China). The plasmids. After 48 h, cells were infected with SeV for 2 h and then the shRNAs against human HDAC3 (shHDAC3) (no. 1) plasmid was a gift from infection medium was moved. Cells were washed twice and fed with fresh Dr. Z. Yuan (Institute of Biophysics, Chinese Academy of Sciences), and the medium to incubate for 20 h. To detect IFN-a–induced transcriptional shHDAC3 (no. 2) plasmid was a gift from Dr. H. You (Xiamen University). activity, cells were transfected with the ISRE-luciferase together with HA-ATXN3-20Q and HA-ATXN3-79Q were gifts from Dr. H. Wang and Renilla plasmids and FH-ATXN3 or shATXN3 or indicated plasmids. Z. Ying (College of Pharmaceutical Sciences, Soochow University). Myc- After 48 or 72 h, cells were treated with or without IFN-a overnight. The ATXN3wasgeneratedusingPCRamplificationfromtheFH-ATXN3plas- luciferase activity was measured using the Dual-Luciferase reporter assay mid and cloned into the Myc-N1 vector. Myc-ATXN3-C14A/H119D/N134S system (E1910; Promega). triple mutation (TM) was generated by a QuickChange site-directed mutagen- esis kit (Stratagene). All plasmids were confirmed by DNA sequencing. ELISA Virus and viral infection The concentrations of human IFN-a in culture supernatants were measured by ELISA kits (E-EL-H2532c; Elabscience) according to the manufac- Vesicular stomatitis virus (VSV), VSV-GFP, Sendai virus (SeV), and in- turer’s instructions. fluenza A virus (H1N1, PR/8/34) were described previously (31). To determine the antiviral effect of IFN-a, cells were transfected with FH- Isolation and transfection of mouse primary lung cells ATXN3 or shATXN3. Forty-eight or seventy-two hours after transfection, The primary mouse lung cells were isolated from C57BL/6 mouse lung a cells were treated with IFN- (60 IU/ml) for 22–24 h. After washing twice, tissues, which were cut into pieces and digested by erythrocyte lysis buffers. cells were infected with VSV-GFP for 2 h. Then the medium was moved After centrifugation, cells were collected and cultured. 293T cells were and cells were cultured with fresh medium for a continuous 20 h. Cells transfected with shRNAs (2.5 mg), psPAX2 (1.6 mg), and pMD2G (0.9 mg) were analyzed by fluorescence microscopy or FACS directly, or analyzed and then cultured with fresh medium for 72 h. The culture supernatant was by Western blotting using an anti–VSV-G Ab. collected and filtered (Millex-GP, 0.22 mm). The primary mouse lung cells Fluorescence microscopy and FACS analysis were then infected with the packaged lentiviruses in the presence of Pol- ybrene (8 mg/ml) for 12 h and then cultured for 24 h. After infection, the Cells infected with VSV-GFP were imaged with an upright fluorescence cells were cultured for 72 h in 10% FBS complete DMEM containing microscope. Magnification was 3200. For flow cytometry analysis puromycin (1.5 mg/ml) for further experiments. (FACS), cells were collected with cold 13 PBS and were acquired in a FACSCalibur (BD Biosciences) equipped with a 488-nm argon laser and a Fifty percent tissue culture–infective dose assay 635-nm red diode laser. Data were analyzed with FlowJo software VSV viral titers were determined by a standard 50% tissue culture–infective (FlowJo, Ashland, OR). dose (TCID50) assay. Briefly, 293T cells were transfected with either FH- Western blots ATXN3 or shATXN3 and then were infected with VSV for 20 h. Cultural supernatants containing the VSV viruses were diluted with DMEM serially Cells were harvested in Nonidet P-40 lysis buffer (pH 7.4; 50 mM Tris-HCl, and then were put on the monolayer of Vero cells in 96-well plates. The 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 50 mg/ml PMSF). TCID50 was calculated by the Spearman–Ka¨rber algorithm. The Journal of Immunology 677

Immunoprecipitation indicating that the length of polyQ of ATXN3 might not be as- Cells were harvested to collect the supernatant, which was added in specific sociated with its antiviral activity. Taken together, we demonstrate Abs overnight on a rotor at 4˚C. Protein G–agarose beads (16-266; Mil- that ATXN3 is a positive regulator of IFN-I antiviral activity lipore) were washed twice and then were added in the supernatant. The during viral infections. mixture was incubated for 3 h on a rotor at 4˚C. For immunoprecipitation of Flag-tagged proteins, M2 affinity gel (A2220; Sigma-Aldrich) was ATXN3 does not affect IFN-I production during viral infection added to cell lysates and incubated for 3 h on a rotor at 4˚C. After washing We next sought to determine the mechanism by which ATXN3 five times with lysis buffer, the immunoprecipitates were analyzed by SDS-PAGE. promotes the antiviral effect of IFN-I. First, we wanted to know whether ATXN3 promotes IFN-I production during viral infection. CHX pulse-chase assay 293T cells transfected with or without FH-ATXN3 were infected To analyze the half-life of HDAC3, HEK293T cells were transfected with with SeV, which is usually used as a sensitive virus model to Flag-HDAC3, together with FH-ATXN3 or shATXN3 plasmids. Forty-eight stimulate IFN-I production. The data showed that ATXN3 did not or seventy-two hours after transfection, cells were treated with DMSO or significantly influence IFN-b promoter activity (Fig. 2A). Further- CHX (50 mg/ml) for the indicated time. Cells were then harvested and analyzed by Western blotting. more, overexpression of ATXN3 had no effect on the mRNA levels of both IFN-a and IFN-b during viral infection (Fig. 2B). To an- In vivo deubiquitination assay alyze the effect of endogenous ATXN3 on IFN-I production, Cells were transfected with indicated plasmids. Forty-eight or seventy-two HT1080 cells were transfected with shATXN3 plasmids and then hours after transfection, cells were harvested in Nonidet P-40 lysis buffer. To infected with SeV for 12 h. The results showed that knockdown of analyze the effect of ATXN3 on the ubiquitination of HDAC3, HDAC3 endogenous ATXN3 did not significantly affect virus-induced pro- Downloaded from proteins were immunoprecipitated by a specific anti-HDAC3 Ab and then duction of both IFN-a and IFN-b mRNA (Fig. 2C). Consistent with subjected to Western blotting analyses using the anti-HA or anti-ubiquitin Ab. this finding, the concentration of IFN-a protein secreted from cells infected with viruses was not obviously affected by either ATXN3 Statistical analysis overexpression (Fig. 2D) or ATXN3 knockdown (Fig. 2E). Fur- Group comparisons were analyzed by a two-tailed Student t test. Values of thermore, we used HT1080 cells, which are very sensitive to IFN p , 0.05 were considered statistically significant.

stimulation and usually used as IFN-activity reporter cells, to test http://www.jimmunol.org/ the total activity of secreted IFN-I in culture supernatants. HT1080 Results cells were stimulated for 4 h by the culture supernatants from cells ATXN3 positively regulates IFN-I antiviral efficacy with or without viral infection. Then the mRNA levels of a repre- To explore the role of ATXN3 in the innate antiviral response, we sentative ISG, IFIT1, were measured. The results showed that the used VSV,SeV,and H1N1, which have been widely used as sensitive activities of secreted IFN-I from cells with ATXN3 overexpression viral models, to assess cellular antiviral activity. Cells were trans- were similar to those from cells with empty vector (Fig. 2F), sug- fected with empty vectors or FH-ATXN3 and then infected with gesting that ATXN3 overexpression does not upregulate IFN-I either VSV, SeV, or H1N1. We found that the levels of specific viral production and secretion. Taken together, our results demonstrated RNAs were lower in cells with ATXN3 overexpression, indicating that ATXN3 did not significantly influence the production of IFN-I. by guest on September 27, 2021 that ATXN3 promotes a cellular antiviral response (Fig. 1A). Consistently, the viral titers in culture supernatants decreased when ATXN3 enhances IFN-I–mediated signaling ATXN3 was overexpressed (Fig. 1B). Additionally, knockdown of Given that ATXN3 promotes the antiviral activity of IFN-I and does endogenous ATXN3 upregulated VSV titers in culture supernatants not increase IFN-I production, we sought to determine the (Fig. 1C). We next asked whether ATXN3 promotes IFN-mediated mechanism of ATXN3 action. First, we tried to analyze whether antiviral activity. To test this possibility, 293T cells transfected with ATXN3 affects the downstream signaling pathway mediated by FH-ATXN3 were treated with IFN-a and then infected with VSV- IFN-I. As described above, once the IFN-I pathway is activated, the GFP, which is a VSV virus with the GFP gene. Using fluorescence transcription factor complex containing STAT1, STAT2, and IFN microscopy, we found that IFN-I treatment markedly decreased the regulatory factor 9 translocates into the nucleus and binds to the GFP signal, indicating that IFN-I signaling inhibits VSV-GFP in- ISRE promoters of hundreds of ISGs, and finally induces the fection (Fig. 1D). Interestingly, overexpression of ATXN3 promoted expression of a series of ISGs. To test this hypothesis, we first IFN-a–mediated antiviral activity, as shown by significantly de- analyzed the effect of ATXN3 on the ISRE promoter activity in- creased GFP signal (Fig. 1D). Additionally, we used FACS analyses duced by IFN-I. Our results showed that overexpression of ATXN3 to determine the percentage of VSV-GFP+ cells. The data also enhanced IFN-I–activated ISRE activity (Fig. 3A). Next, cells clearly demonstrated that ATXN3 overexpression enhances IFN-I– were transfected with shATXN3 plasmids. We found that knock- mediated antiviral activity (Fig. 1E). To analyze the effect of en- down of endogenous ATXN3 significantly blocked IFN-I–medi- dogenous ATXN3 on IFN-I antiviral activity, two shRNAs against ated ISRE activity (Fig. 3B). Furthermore, we analyzed the effect human ATXN3 were used and the results showed that knockdown of ATXN3 on the expression of IFN-I–activated downstream of endogenous ATXN3 obviously upregulated VSV-G protein levels ISGs. To this end, the mRNA levels of three representative ISGs, under conditions of IFN-a treatment (Fig. 1F), suggesting that IFIT1, ISG15, and ISG54, were determined. The result showed cellular ATXN3 is responsible for efficient antiviral responses me- that knockdown of endogenous ATXN3 remarkably decreased diated by IFN-I. mRNA levels of all three ISGs induced by IFN-a (Fig. 3C). Furthermore, we tried to analyze whether the MJD-associated Similarly, in mouse primary lung cells, when ATXN3 was pathogenic ATXN3 possesses the similar antiviral function as knocked down, the mRNA levels of IFIT1 induced by mouse IFN- the physiological ATXN3. To this end, HeLa cells were transfected b (mIFN-b) also decreased obviously (Fig. 3D), indicating that with either wild-type (WT) ATXN3-20Q or polyQ-expanded ATXN3 plays an important role in promoting the IFN-I–activated ATXN3-79Q, which is usually used as a representative patho- signaling pathway. genic ATXN3. Cells were then treated with IFN-a. The results Given that our above data showed that ATXN3 promotes IFN-I– showed that the effect of pathogenic ATXN3-79Q on IFN-I anti- mediated antiviral signaling, we speculated that ATXN3 could viral activity was comparable to the WT ATXN3-20Q (Fig. 1G), promote IFN-I–induced STAT1 activation. First, we detected the 678 ATXN3 DEUBIQUITINATES HDAC3 TO PROMOTE IFN SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. ATXN3 positively regulates IFN-I antiviral efficacy. (A) 293T, HeLa, and A549 cells were transfected with or without FH-ATXN3. Then 293T cells were infected with VSV. HeLa cells were infected with SeV, and A549 cells were infected with H1N1. After 20 h culture, the viral RNA levels were analyzed by qPCR. (B and C) 293T cells transfected with either FH-ATXN3 (B) or shATXN3 (C) were infected with VSV for 20 h, and VSV titers in culture supernatants were determined by a TCID50 assay. (D) 293T cells transfected with FH-ATXN3 were pretreated with or without IFN-a (60 IU/ml) for 20 h. Then cells were infected with VSV-GFP (multiplicity of infection of 0.5) for 20 h. The VSV-GFP signal was detected by fluorescence microscopy. Original magnification 320. (E) VSV-GFP levels in (D) were analyzed by FACS. (F) 293T cells transfected with control shRNAs (shCON) or two shATXN3 were pretreated with IFN-a (60 IU/ml) for 20 h, and then cells were infected with VSV for 24 h. Whole-cell extracts were analyzed by Western blots using the indicated Abs. The protein band density was quantified with ImageJ. (G) HeLa cells transfected with empty vectors, HA- ATXN3-20Q, or HA-ATXN3-79Q were treated as in (D). The viral RNA levels of VSV were analyzed by qPCR. Data are from three experiments [(A–C and G) mean and SD of three independent replicates] or representative of three independent experiments (D–F). *p , 0.05, **p , 0.01, ***p , 0.001. n.s, not significant. influence of ATXN3 on total STAT1 protein levels. We found that signaling. Therefore, we determined whether ATXN3 could affect overexpression of ATXN3 did not upregulate the levels of STAT1 IFN-I–induced p-STAT1. The results showed that ATXN3 over- protein (Fig. 3E, 3F). Additionally, ATXN3 knockdown did not expression remarkably promoted STAT1 activation (Fig. 3F). downregulate STAT1 protein levels (Fig. 3G, 3H). STAT1 phos- When endogenous ATXN3 in cells was knocked down, the levels phorylation at tyrosine 701 is essential for activation of IFN-I of p-STAT1 induced by IFN-I were robustly inhibited (Fig. 3G), The Journal of Immunology 679 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. ATXN3 does not affect the production of IFN-I. (A) 293T cells were transfected with FH-ATXN3, together with IFN-b–luciferase (IFN-b– Luc) and Renilla. The luciferase activity was measured after cells were infected with SeV for 20 h. (B) qPCR analysis of IFN-a mRNA (left) and IFN-b mRNA (right) in HT1080 cells transfected with FH-ATXN3 followed by SeV infection for 6 h. (C) HT1080 cells transfected with shATXN3 were infected with SeV for 12 h, and the mRNA levels of IFN-a and IFN-b were analyzed by qPCR. (D and E) 293T cells were transfected with either FH-ATXN3 (D) or shATXN3 (E) and then infected with VSV for 20 h. IFN-a protein in culture supernatants was analyzed by ELISA. (F) HT1080 cells transfected with or without FH-ATXN3 were infected with VSV for 10 h, and their culture supernatants were collected and used to stimulate another plate of HT1080 cells for 4 h. The mRNA levels of IFIT1 were analyzed by qPCR. Data are from three experiments (mean and SD of three independent replicates). ***p , 0.001. n.s, not significant. which is consistent with results in mouse primary lung cells antiviral response. Thus, we demonstrate that ATXN3 can enhance (Fig. 3H). Collectively, these findings strongly indicate that IFN-I–activated signaling. ATXN3 plays important roles in promoting IFN-I–induced STAT1 activation. Furthermore, to study the effect of ATXN3 on IFN-I ATXN3 upregulates HDAC3 expression at the protein level signaling during actual viral infections, HeLa cells were trans- Given that ATXN3 positively regulates the IFN-I signaling path- fected with shATXN3 plasmids and then were infected with VSV. way, we wanted to further identify the potential substrate of The results showed that knockdown of ATXN3 also inhibited ATXN3 in regulating IFN-I signaling. IFN-I signaling has been virus-induced STAT1 activation (Fig. 3I), indicating that during reported to be activated mainly through JAK kinase (JAK1/TYK2) actual viral infection ATXN3 also positively regulates the innate activation and subsequent STAT (STAT1/STAT2) activation. 680 ATXN3 DEUBIQUITINATES HDAC3 TO PROMOTE IFN SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. ATXN3 enhanced the IFN-I–mediated signaling pathway. (A) 293T cells were transfected with empty vectors or FH-ATXN3, together with ISRE-luciferase and Renilla. The luciferase activity was measured after IFN-a (500 IU/ml) treatment for 18 h. (B) 293T cells were transfected with either control shRNAs (shCON), shATXN3 no. 1, or shATXN3 no. 2, together with ISRE-luciferase and Renilla. The luciferase activity was measured after IFN-a (500 IU/ml) treatment for 18 h. (C) PC9 cells transfected with either shCON or shATXN3 were treated with IFN-a (1000 IU/ml) for 6 h, and the mRNA levels of IFIT1, ISG15, and ISG54 were analyzed by qPCR. (D) Mouse primary lung cells were infected with shATXN3-packaged lentiviruses and then were stimulated with mIFN-b (300 IU/ml) for 4 h. The mRNA levels of IFIT1 were analyzed by qPCR. (E) 293T cells were transfected with different amounts of FH-ATXN3. Whole-cell lysates were analyzed by indicated Abs. (F) 293T cells transfected with FH-ATXN3 were stimulated with IFN-a (1000 IU/ml) for 0, 30, and 60 min. Whole-cell extracts were subjected to SDS-PAGE and probed with a pY701-STAT1 (p-STAT1) Ab. (G) HepG2 cells transfected with either shCON or two shATXN3 were stimulated with IFN-a (1000 IU/ml) for 0, 30, and 60 min. Immunoblotting was performed as in (F). (H) Mouse primary lung cells were infected as in (D) and then were treated with mIFN-b (300 IU/ml) for 30 min. Immunoblotting was performed as in (F). (I) HeLa cells transfected with shCON and shATXN3 were infected with VSV for 12 h. Whole-cell extracts were analyzed by immunoblotting using the pY701-STAT1 Ab. Data are from three experiments [(A–D) mean and SD of three independent replicates] or representative of three independent exper- iments (E–I). *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 681

A recent report showed that the deubiquitinase USP13 can target upstream of STAT1 activation. By analyzing the possible inter- STAT1 and regulate STAT1 stability (31). Our previous studies acting protein with ATXN3, we noticed that ATXN3 is able to have shown that the deubiquitinase USP2a tightly controls acti- interact with HDACs family members (32). Interestingly, a recent vated pY701-STAT1 ubiquitination and stability (30). Therefore, report showed that HDACs positively regulate the IFN signaling we speculated that ATXN3 may target the signaling molecule pathway by promoting STAT1 activation (33, 34). Therefore, we Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. ATXN3 upregulates HDAC3 expression at the protein level. (A, C, and E) 293T cells were transfected with Flag-tagged HDAC1 (A), HDAC2 (C), or HDAC3 (E), together with increased amounts of FH-ATXN3. Whole-cell extracts were subjected to immunoblotting using the indicated Abs. (B, D, and F) 293T cells were transfected with Flag-tagged HDAC1 (B), HDAC2 (D), or HDAC3 (F), together with control shRNAs (shCON) or shATXN3. Protein levels of Flag-HDACs were detected by an anti-Flag Ab. (G) 293T cells were transfected with increased amounts of FH-ATXN3, and the level of en- dogenous HDAC3 was analyzed as indicated. (H) 293T cells were transfected with shCON or shATXN3. Cell lysates were analyzed by Western blots using indicated Abs. Data are representative of three independent experiments. 682 ATXN3 DEUBIQUITINATES HDAC3 TO PROMOTE IFN SIGNALING wondered whether ATXN3 is capable of upregulating HDAC 12 h. The results showed that in primary lung cells endogenous proteins. To test this hypothesis, cells were transfected with Flag- ATXN3 also interacted with endogenous HDAC3 (Fig. 5D), and HDAC1 (Fig. 4A), Flag-HDAC2 (Fig. 4C), or Flag-HDAC3 viral infection promoted interaction between ATXN3 and HDAC3 (Fig. 4E), together with increased amounts of FH-ATXN3. Our (Fig. 5D). data showed that overexpression of ATXN3 did not obviously Given that ATXN3 possesses the activity of deubiquitinases, we affect the levels of both Flag-HDAC1 and Flag-HDAC2 (Fig. 4A, questioned whether the effect of ATXN3 on HDAC3 protein levels 4C). However, ATXN3 overexpression remarkably upregulated is mediated by the ubiquitin/ system. We found that the Flag-HDAC3 protein levels in a dose-dependent manner (Fig. 4E). proteasome inhibitor MG132 was able to block the degradation of Next, endogenous ATXN3 was knocked down by shATXN3, and HDAC3 induced by ATXN3 knockdown (Fig. 6A). Furthermore, Flag-HDAC protein levels were analyzed by immunoblotting. Our we asked whether ATXN3 could regulate HDAC3 protein stabil- results showed that knockdown of ATXN3 decreased the protein ity. To answer this question, a CHX pulse-chase assay was per- level of Flag-HDAC3 (Fig. 4F), but it did not affect the protein formed. CHX is widely used for the inhibition of protein levels of both Flag-HDAC1 and Flag-HDAC2 (Fig. 4B, 4D), in- synthesis. We found that overexpression of ATXN3 remarkably dicating that ATXN3 can specifically upregulate the protein level inhibited the degradation of Flag-HDAC3 protein (Fig. 6B). When of exogenous HDAC3. endogenous ATXN3 was knocked down, the degradation rate of Furthermore, we questioned whether endogenous HDAC3 HDAC3 was markedly accelerated (Fig. 6C), indicating that protein levels can be upregulated by ATXN3. Cells were trans- HDAC3 stability is regulated by cellular ATXN3. Additionally, fected with increased amount of FH-ATXN3, and endogenous when MG132 and CHX were combined, the degradation of Flag-

HDAC3 levels were determined by immunoblotting. The result HDAC3 protein was largely blocked (Fig. 6D). Taken together, we Downloaded from showed that overexpression of ATXN3 also markedly increased the demonstrate that ATXN3 is able to interact with HDAC3 and protein level of endogenous HDAC3 (Fig. 4G). More importantly, enhances HDAC3 protein stability. when endogenous ATXN3 in cells was knocked down, the protein ATXN3 regulates both K48-linked and K63-linked level of cellular HDAC3 was robustly downregulated (Fig. 4H), ubiquitination of HDAC3 indicating that cellular HDAC3 protein levels are tightly regulated

by ATXN3. In summary, we demonstrate that ATXN3 is a positive Given that ATXN3 increases not only the protein levels but also the http://www.jimmunol.org/ regulator of HDAC3 protein levels. protein stability of HDAC3, and ATXN3 has been reported to be one member of deubiquitinases, we speculated that ATXN3 may ATXN3 interacts with and stabilizes HDAC3 regulate HDAC3 ubiquitination as a deubiquitinase. Cells were To address how ATXN3 upregulates HDAC3 protein level, we first transfected with Myc-ATXN3 and Flag-HDAC3, together with tested whether ATXN3 can interact with HDAC3. By coimmuno- HA-ub, and ubiquitination levels of Flag-HDAC3 were analyzed. precipitation and Western blot analyses, we found a strong associ- The result showed that overexpression of ATXN3 significantly ation between Flag-HDAC3 and Myc-ATXN3 in HEK293T cells reduced Flag-HDAC3 ubiquitination levels (Fig. 7A). Conversely, (Fig. 5A, 5B). To confirm this interaction, we further determined knockdown of ATXN3 upregulated ubiquitination levels of Flag- whether endogenous ATXN3 and endogenous HDAC3 interact with HDAC3 (Fig. 7B). Furthermore, we determined the effect of by guest on September 27, 2021 each other, because we demonstrated that endogenous ATXN3 af- ATXN3 on endogenous HDAC3 ubiquitination. HEK293T cells fected cellular HDAC3 levels (Fig. 4H). To this end, whole-cell were transfected with or without shATXN3, together with HA-ub. extracts from HT1080 cells were used for immunoprecipitation Endogenous HDAC3 was immunoprecipitated, and ubiquitination by a specific anti-ATXN3 Ab, and endogenous HDAC3 was de- levels of HDAC3 were analyzed by immunoblotting. The results tected by immunoblotting. As expected, our data clearly showed the showed that knockdown of ATXN3 obviously promoted ubiq- interaction between endogenous ATXN3 and endogenous HDAC3 uitination of endogenous HDAC3 (Fig. 7C), suggesting that (Fig. 5C). Next, we questioned whether viral infection affects the ATXN3 could deubiquitinate cellular HDAC3. interaction between ATXN3 and HDAC3. To answer this question, K48- and K63-linked ubiquitination of protein substrates has mouse primary lung cells were infected with VSV for 0, 6, and been extensively studied so far. Because ATXN3 can decrease

FIGURE 5. ATXN3 interacts with HDAC3. (A and B) 293T cells were transfected with Myc-ATXN3 and/or Flag-HDAC3 as indicated. Immunoprecipitation was performed using an anti-Myc Ab (A)orM2beads(B), followed by immunoblotting as indicated. (C) Endogenous ATXN3 interacts with endogenous HDAC3. HT1080 cells were harvested and endogenous ATXN3 was immunoprecipitated, and then immunoblotting was performed using an anti- HDAC3 Ab. (D) Mouse primary lung cells were infected with VSV for 0, 6, or 12 h. Whole-cell lysates were subjected to immu- noprecipitation with an anti-ATXN3 Ab and then were analyzed by Western blots using the indicated Abs. Data are representative of three independent experiments. The Journal of Immunology 683 Downloaded from http://www.jimmunol.org/

FIGURE 6. ATXN3 promotes the stability of HDAC3. (A) HeLa cells transfected with control shRNAs (shCON) or two shATXN3 were treated with by guest on September 27, 2021 MG132 (10 mM) for 12 h. Cell lysates were analyzed by Western blots using indicated Abs. (B) 293T cells were transfected with Flag-HDAC3, together with or without FH-ATXN3. Then the cells were treated with CHX (50 mg/ml) for different times. The whole-cell extracts were analyzed by immuno- blotting using the indicated Abs. (C) 293T cells were transfected with Flag-HDAC3, together with either shCON, shATXN3 no. 1, or shATXN3 no. 2. Then the cells were treated with CHX (50 mg/ml) for different times. The whole-cell extracts were analyzed as described in (B). (D) 293T cells transfected with Flag-HDAC3 were pretreated with DMSO or MG132 (10 mM) for 12 h, followed by treatment with CHX (50 mg/ml) for different times as indicated. Whole-cell lysates were analyzed by indicated Abs. Data are representative of three independent experiments. ubiquitination of HDAC3, we tried to determine whether ATXN3 (Fig. 8A). Additionally, ATXN3-TM cannot deubiquitinate can affect K48-linked and/or K63-linked ubiquitination of HDAC3. HDAC3 (Fig. 8B). These data suggest that ATXN3 upregulates To this end, two constructs, HA-ub-R48K and HA-ub-R63K, were HDAC3 protein levels dependent on its deubiquitinase activity. used to analyze the types of HDAC3 ubiquitination. Ubiquitin protein Furthermore, we studied whether ATXN3 regulates IFN-I sig- harbors seven lysines, whereas ubiquitin-R48K and ubiquitin-R63K naling and antiviral activity dependent on its deubiquitinase activity. retain only one lysine at position 48 (R48K) and position 63 Cells transfected with either Myc-ATXN3-WT or Myc-ATXN3-TM (R63K), respectively. Our data showed that overexpression were stimulated with IFN-a. The result showed that catalytically of ATXN3 removed both K48-linked and K63-linked poly- inactive ATXN3-TM cannot promote IFN-a–induced STAT1 acti- ubiquitination chains of HDAC3 (Fig. 7D). Conversely, knock- vation (Fig. 8C). Also, ATXN3-TM lost the ability to promote ISRE down of ATXN3 significantly promoted both K48-linked and activity in IFN-I signaling (Fig. 8D). Thus, we demonstrate that K63-linked ubiquitination of HDAC3 (Fig. 7E). Collectively, we ATXN3 promotes the IFN-I signaling pathway dependent on its demonstrate that ATXN3, as a deubiquitinase, regulates ubiq- deubiquitinase activity. uitination levels of HDAC3. To further analyze the effect of ATXN3 deubiquitinase activity on IFN-I antiviral response, cells were transfected with either ATXN3 regulates IFN-I antiviral response dependent on its ATXN3-WT or ATXN3-TM and then were infected with different deubiquitinase activity viruses. The results showed that viral HA protein of H1N1 was We further studied whether the effect of ATXN3 on the IFN-I significantly downregulated by ATXN3-WT, but not by the cata- antiviral response is dependent on its deubiquitinase activity. lytically inactive ATXN3-TM (Fig. 8E). Similarly, ATXN3-WT, ATXN3 protease activity is located in its Josephin domain. It has but not ATXN3-TM, inhibited VSV infection (Fig. 8F). Addi- been well demonstrated that three sites, Cys14, His119, and Asn134 tionally, the effect of ATXN3 deubiquitinase activity on IFN-I (Fig. 8A), are essential for the complete deubiquitinase activity of antiviral function was analyzed. Cells were transfected with ei- ATXN3 (14–16). Therefore, we constructed the catalytically in- ther ATXN3-WT or ATXN3-TM and then were infected with active ATXN3-C14A/H119D/N134S TM. The result showed that VSV-GFP. As expected, IFN-a treatment decreased VSV-GFP ATXN3-TM lost the ability to increase HDAC3 protein levels infection as shown by decreased GFP signal. We found that as 684 ATXN3 DEUBIQUITINATES HDAC3 TO PROMOTE IFN SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 7. ATXN3 removes both K48-linked and K63-linked polyubiquitination of HDAC3. (A) 293T cells were transfected with HA-ub, together with Myc-ATXN3 and/or Flag-HDAC3 as indicated. Flag-HDAC3 was immunoprecipitated with M2 beads for 3 h and then immunoblotting was performed as indicated. (B) 293T cells were transfected with Flag-HDAC3 and control shRNAs or shATXN3 as indicated. Then cells were treated with MG132 (10 mM) for 12 h. Cell lysates were subjected to immunoprecipitation with M2 beads and then analyzed by Western blots with an anti-ubiquitin Ab. (C) 293T cells were transfected with HA-ub and two shATXN3 as indicated. Endogenous HDAC3 was immunoprecipitated by an anti-HDAC3 Ab, and the ubiquitination level of HDAC3 was analyzed as indicated. (D) 293T cells were transfected with Flag-HDAC3, together with HA-ub-K48 (R48K) or HA-ub-K63 (R63K) and/or Myc-ATXN3 as indicated. Flag-HDAC3 was immunoprecipitated by M2 beads, and then immunoblotting was performed using indicated Abs. (E) 293T cells were transfected with Flag-HDAC3, together with HA-ub-K48 (R48K) or HA-ub-K63 (R63K) and/or shATXN3. Cells were treated with MG132 for 12 h. Immunoprecipitation was performed using M2 beads, followed by immunoblotting using an anti-HA Ab. Data are representative of three in- dependent experiments. compared with ATXN3-WT, catalytically inactive ATXN3-TM lost STAT1 activation dependent on HDAC3. Furthermore, cells were the ability to enhance IFN-a–induced antiviral activity (Fig. 8G). transfected with shHDAC3, and the effect of ATXN3 on IFN-I– Taken together, ATXN3 regulates HDAC3 protein levels and IFN-I activated ISRE promoter activity was determined. Our results antiviral response dependent on its deubiquitinase activity. showed that HDAC3 knockdown markedly inhibited the ability of ATXN3 to promote IFN-I–mediated ISRE activity (Fig. 9B). Con- The ATXN3/HDAC3 axis promotes IFN-I–mediated signaling sistently, during viral infection, knockdown of endogenous HDAC3 and antiviral activity significantly blocked the effects of ATXN3 on IFN-I antiviral ac- The aforementioned results clearly demonstrate that the deubi- tivity (Fig. 9C). Additionally, HDAC3 knockdown also inhibited the quitinase ATXN3 constitutively interacts with HDAC3 and posi- effects of ATXN3 on host antiviral responses (Fig. 9D). In sum- tively regulates cellular HDAC3 levels. In conjunction with the mary, these findings indicate that ATXN3 positively regulates previous findings showing that HDAC3 promotes IFN-I–mediated HDAC3 protein levels, thus promoting the IFN-I signaling pathway antiviral signaling (33), we speculated that the ATXN3/HDAC3 and antiviral activity. signaling axis promotes IFN-I antiviral response. To address the importance of HDAC3 in ATXN3-mediated regulation of IFN-I Discussion signaling, two shRNAs against with HDAC3 were used. The re- ATXN3 has been recognized to be an important molecule whose sults showed that knockdown of cellular HDAC3 inhibited the up- polyQ expansion causes MJD, which is an autosomal-dominant regulation effect of ATXN3 on STAT1 activation under conditions neurologic disorder. So far, most studies on ATXN3 have fo- of IFN-I treatment (Fig. 9A), suggesting that ATXN3 promotes cused on the regulatory mechanism of ATXN3 on the development The Journal of Immunology 685 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 8. ATXN3 regulates IFN-I antiviral response dependent on its deubiquitinase activity. (A) Map of the ATXN3 construction is shown (upper panel). HepG2 cells were transfected with FH-ATXN3 (WT) or ATXN3-C14A/H119D/N134S mutant (TM), and the level of endogenous HDAC3 was analyzed as indicated. (B) 293T cells were transfected with HA-ub, together with Myc-ATXN3-WT or Myc-ATXN3-TM. Endogenous HDAC3 was immunoprecipitated with an anti-HDAC3 Ab, followed by immunoblotting using an anti-HA Ab. (C) HeLa cells transfected with either Myc-ATXN3-WT or Myc-ATXN3-TM were stimulated with IFN-a (1000 IU/ml) for 30 min. Whole-cell lysates were analyzed by indicated Abs. (D) 293T cells were transfected with either Myc-ATXN3-WTor Myc-ATXN3-TM, together with ISRE-luciferase and Renilla. The luciferase activity was measured after IFN-a (500 IU/ml) treatment for 18 h. (E and F) HeLa cells (E) or 293T cells (F) transfected with either Myc-ATXN3-WT or Myc-ATXN3-TM were infected with H1N1 (E) or VSV (F), the HA protein of H1N1 was detected by Western blots (E), and the VSV mRNA level was analyzed by qPCR. (G) 293T cells transfected with either Myc-ATXN3-WT or Myc-ATXN3-TM were treated with IFN-a (60 IU/ml) for 20 h and then infected with VSV-GFP for 24 h. The VSV-GFP signal was detected by fluorescence microscopy. Original magnification 320. Data are from three experiments [(D and F) mean and SD of three independent replicates] or representative of three independent experiments (A–C, E, and G). *p , 0.05. n.s, not significant. of MJD. However, it is less clear whether or how ATXN3 plays pathway. These findings clearly demonstrate that ATXN3 pro- roles in other biological functions. In this study, we report a novel motes activation of the IFN-I signaling pathway and IFN-I–me- biological function of ATXN3, showing that ATXN3 is an im- diated antiviral defense. portant regulator of IFN-I antiviral defense during viral infections. ATXN3 is one member of deubiquitinases. However, the in vivo We find that ATXN3 actually does not affect IFN-I production but deubiquitinating substrates of ATXN3 remain largely unexplored. promotes IFN-I–induced STAT1 activation and the IFN-I signaling It has been reported that ATXN3 deubiquitinates Parkin and 686 ATXN3 DEUBIQUITINATES HDAC3 TO PROMOTE IFN SIGNALING Downloaded from http://www.jimmunol.org/

FIGURE 9. ATXN3 promotes IFN-I–mediated signaling and antiviral activity dependent on HDAC3. (A) 293T cells were transfected with either control shRNAs (shCON) or two shHDAC3, together with empty vectors or FH-ATXN3. Then cells were stimulated with IFN-a (1000 IU/ml) for 30 min. The level by guest on September 27, 2021 of pY701-STAT1 was detected by immunoblotting with an anti–pY701-STAT1 Ab. (B) 293T cells transfected with either shCON or shHDAC3, together with FH-ATXN3, ISRE-luciferase and Renilla. The luciferase activity was measured after IFN-a (500 IU/ml) treatment for 18 h. (C) Cells were transfected as in (A) and then were pretreated with IFN-a (60 IU/ml) for 20 h, followed by infection with VSV. The viral RNA levels of VSV in cells were analyzed by qPCR. (D) HepG2 cells transfected with either shCON or shHDAC3 were infected with VSV for 20 h, and viral RNA levels were analyzed by qPCR. Data are representative of three independent experiments (A) or from three experiments [(B–D) mean and SD of three independent replicates]. *p , 0.05, **p , 0.01, ***p , 0.001. n.s, not significant.

regulates the stability of Parkin (19). Another study reports that However, further evidence is needed to completely address this ATXN3 can deubiquitinate CHIP and limits ubiquitin chain length question. on CHIP substrates (18). In this study, we report that ATXN3 Recent reports demonstrate that ATXN3 can remove both K48- interacts with HDAC3. HDAC1, HDAC2, and HDAC3 have been linked and K63-linked polyubiquitination of its substrates (35–37). reported to be required for STAT1-dependent gene activation, and Additionally, ATXN3 is able to remove monoubiquitin from silencing of HDAC1, HDAC2, and HDAC3 inhibits responsive- ubiquitin-CHIP (18). In our studies, we find that HDAC3 forms ness to IFNs (33). Therefore, we speculate that ATXN3 could polyubiquitination modification. Furthermore, we show that deubiquitinate HDACs. Our data demonstrate that ATXN3 spe- ATXN3 can remove both K48-linked and K63-linked poly- cifically deubiquitinates HDAC3 and increases the level and the ubiquitination chains of HDAC3, which again demonstrates that stability of HDAC3 protein. Therefore, we identify HDAC3 as ATXN3 possesses the ability to remove different types of poly- another in vivo deubiquitinating substrate of ATXN3. ubiquitination chains. Additionally, it could be interesting to ex- Interestingly, both Parkin and CHIP are members of the E3 plore the significance of removing K63-linked ubiquitination of ubiquitin ligases, whereas HDAC3 is one member of the histone HDAC3 by ATXN3 in the future. deacetylase family. ATXN3 contains three ubiquitin-interacting In summary, we elucidated that the deubiquitinase ATXN3 plays motifs, which can bind ubiquitin and ubiquitin-like domains. an important role in promoting IFN-I–mediated antiviral defense. Therefore, these domains of ATXN3 are thought to favor functional ATXN3 physically interacts with HDAC3 and removes poly- interactions with E3 ubiquitin ligases. In this study, we identified a ubiquitination chains of HDAC3, which increases HDAC3 levels different interacting partner, HDAC3, as a non-E3 , and protein stability in cells. Therefore, ATXN3 promotes IFN-I– indicating a possible novel mechanism by which ATXN3 regulates mediated STAT1 activation through regulating HDAC3. These its deubiquitinating substrates. In the case of ATXN3-HDAC3 findings reveal a novel biological function of ATXN3 and an regulation, it may be possible that a new E3 ubiquitin ligase is in vivo deubiquitinating substrate of ATXN3, and they may involved in the deubiquitination effect of ATXN3 on HDAC3. deepen the understanding for innate antiviral defense. The Journal of Immunology 687

Acknowledgments 17. Martins, S., F. Calafell, C. Gaspar, V. C. Wong, I. Silveira, G. A. Nicholson, E. R. Brunt, L. Tranebjaerg, G. Stevanin, M. Hsieh, et al. 2007. Asian origin for We thank Dr. Serge Y. Fuchs from the University of Pennsylvania, the worldwide-spread mutational event in Machado-Joseph disease. Arch. Neu- Dr. Tiebang Kang from Sun Yat-sen University, Dr. Zengqiang Yuan from rol. 64: 1502–1508. the Chinese Academy of Sciences, Dr. Han You from Xiamen University, 18. Scaglione, K. M., E. Zavodszky, S. V. Todi, S. Patury, P. Xu, E. Rodrı´guez- and Dr. Hongfeng Wang and Zheng Ying from Soochow University for Lebro´n, S. Fischer, J. Konen, A. Djarmati, J. Peng, et al. 2011. Ube2w and ataxin-3 coordinately regulate the ubiquitin ligase CHIP. Mol. Cell 43: 599–612. important reagents. 19. Durcan, T. M., M. Kontogiannea, T. Thorarinsdottir, L. Fallon, A. J. Williams, A. Djarmati, T. Fantaneanu, H. L. Paulson, and E. A. Fon. 2011. 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