Transcriptional Repression of IFN Regulatory Factor 7 by MYC Is Critical for Type I IFN Production in Human Plasmacytoid Dendrit

Transcriptional Repression of IFN Regulatory Factor 7 by MYC Is Critical for Type I IFN Production in Human Plasmacytoid Dendritic Cells This information is current as of September 28, 2021. Tae Whan Kim, Seunghee Hong, Yin Lin, Elise Murat, HyeMee Joo, Taeil Kim, Virginia Pascual and Yong-Jun Liu J Immunol 2016; 197:3348-3359; Prepublished online 14 September 2016; doi: 10.4049/jimmunol.1502385 Downloaded from http://www.jimmunol.org/content/197/8/3348

Supplementary http://www.jimmunol.org/content/suppl/2016/09/14/jimmunol.150238 http://www.jimmunol.org/ Material 5.DCSupplemental References This article cites 74 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/197/8/3348.full#ref-list-1

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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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Transcriptional Repression of IFN Regulatory Factor 7 by MYC Is Critical for Type I IFN Production in Human Plasmacytoid Dendritic Cells

Tae Whan Kim,*,1 Seunghee Hong,*,1 Yin Lin,* Elise Murat,* HyeMee Joo,* Taeil Kim,†,2 Virginia Pascual,* and Yong-Jun Liu*,†

Type I IFNs are crucial mediators of human innate and adaptive immunity and are massively produced from plasmacytoid dendritic cells (pDCs). IFN regulatory factor (IRF)7 is a critical regulator of type I IFN production when pathogens are detected by TLR 7/9 in pDC. However, hyperactivation of pDC can cause life-threatening autoimmune diseases. To avoid the deleterious effects of aberrant pDC activation, tight regulation of IRF7 is required. Nonetheless, the detailed mechanisms of how IRF7 transcription is regulated in

pDC are still elusive. MYC is a well-known highly pleiotropic transcription factor; however, the role of MYC in pDC function is not Downloaded from well deﬁned yet. To identify the role of transcription factor MYC in human pDC, we employed a knockdown technique using human pDC cell line, GEN2.2. When we knocked down MYC in the pDC cell line, production of IFN-stimulated genes was dramatically increased and was further enhanced by the TLR9 agonist CpGB. Interestingly, MYC is shown to be recruited to the IRF7 promoter region through interaction with nuclear receptor corepressor 2/histone deacetylase 3 for its repression. In addition, activation of TLR9-mediated NF-kB and MAPK and nuclear translocation of IRF7 were greatly enhanced by MYC depletion. Pharmaceutical inhibition of MYC recovered IRF7 expression, further conﬁrming the negative role of MYC in the antiviral response by pDC. http://www.jimmunol.org/ Therefore, our results identify the novel immunomodulatory role of MYC in human pDC and may add to our understanding of aberrant pDC function in cancer and autoimmune disease. The Journal of Immunology, 2016, 197: 3348–3359.

lasmacytoid dendritic cells (pDCs) are specialized type I cells to trigger the protective defenses of the host and help eradicate IFN–producing immune cells that sense viral RNA and pathogens. However, uncontrolled type I IFN production has been im- P DNA through endosomal TLR7 and TLR9, respectively (1–3). plicated in several human autoimmune diseases, such as systemic lupus Type I IFNs, mainly IFN-a and IFN-b, are pleiotropic cytokines that erythematosus (SLE) (7, 8), rheumatoid arthritis (9, 10), inflam- have the most potent antiviral and antitumoral activity in humans (4–6). matory bowel disease (11), or psoriasis (12). To avoid the deleterious by guest on September 28, 2021 IFNs are released by host cells for communication between immune effects of aberrant pDC activation, tight regulation of type I IFN production in pDC is essential. Importantly, production of these type I IFNs in pDCs is mainly controlled by IFN regulatory factor (IRF)7 *Baylor Institute for Immunology Research, Dallas, TX 75204; and †Sanofi, Cambridge, MA 02139 (13). Regulation of IRF7 is complex and possesses many positive 1 and negative feedback mechanisms. Transcriptional, posttranscrip- T.W.K. and S.H. are cofirst authors. tional, translational, posttranslational, and epigenetic (acetylation and 2Current address: MedImmune, Gaithersburg, MD. methylation) regulation of IRF7 have been extensively studied (14– ORCID: 0000-0003-2156-334X (E.M.). 21). Nonetheless, detailed mechanisms of how IRF7 transcription Received for publication November 10, 2015. Accepted for publication August 22, 2016. and activation are directly regulated in pDC are still unclear. This work was supported by National Institutes of Health Grant R01AI097348-03 MYC is a well-known highly pleiotropic transcription factor. (to Y.-J.L.). MYC is a basic helix-loop-helix leucine zipper transcription factor T.W.K. designed research, performed experiments, analyzed data, and wrote the (22) and specifically binds to the DNA sequence 59-CACGTG-39 or paper; S.H. and Y.L. designed research, analyzed data, and wrote the paper; H.J. 9 9 and E.M. performed experiments; T.K. designed research and wrote the paper; V.P. 5 -CAGCTG-3 known as an E-box motif to activate its targets (23). and Y.-J.L. designed and supervised research. The N-terminal transactivation domain of MYC is essential for its The microarray and ChIP-sequencing datasets presented in this article have been biological function and directly interacts with components of basal submitted to the Gene Expression Omnibus under accession numbers GSE70276 transcription machinery or chromatin remodeling (the SWItch/sucrose and GSE70275, respectively. nonfermentable [SWI/SNF] chromatin-remodeling complex and his- Address correspondence and reprint requests to Dr. Yong-Jun Liu or Dr. Tae Whan Kim, Sanofi, 640 Memorial Drive, Cambridge, MA 02139 (Y.-J.L.) or tone acetyltransferase) (24–26). Additionally, several recent findings Baylor Institute for Immunology Research, 3434 Live Oak Street, Dallas, TX suggested that MYC represses its target genes by interfering with 75204 (T.W.K.). E-mail addresses: Yong-Jun.liu@sanofi.com (Y.-J.L.) and [email protected] (T.W.K.) transcriptional activators (27) or by recruiting the corepressor com- The online version of this article contains supplemental material. plexes, including histone deacetylases (HDAC) (28, 29) or DNA Abbreviations used in this article: ChIP, chromatin immunoprecipitation; co-IP, protein methyltransferase (30). Many studies have also revealed that MYC complex immunoprecipitation; DEG, differentially expressed gene; HDAC, histone induces its oncogenic activity through transcriptional repression (22, deacetylase; IP, immunoprecipitation; IRF, IFN regulatory factor; NC, nontargeting control; NCOR2, nuclear receptor corepressor 2; pDC, plasmacytoid dendritic cell; 31). Interestingly, MYC activation is reported shown to downregulate qRT-PCR, quantitative RT-PCR; siRNA, small interfering RNA; SLE, systemic lupus many genes involved in the NF-kB response and STAT1, the central erythematosus; SWI/SNF, SWItch/sucrose nonfermentable; VPA, valproic acid. player in type I and II IFN signaling via direct inhibition of tran- Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 scription in Burkitt lymphoma cells (32–34). Less, however, is known www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502385 The Journal of Immunology 3349 about how MYC regulates pDC function, and the direct target genes nmol small interfering RNA (siRNA) for each reaction of 3–5 3 106 cells. of MYC in pDC have not been identified. siMYC, siHDAC3, and siTBK1 were purchased from Dharmacon. The In this study, we describe a previously unrecognized function of program number for electroporation was A033. The NC is a nontargeting pool from Dharmacon. MYC in the TLR-mediated negative regulation of antiviral immune response of pDCs. We demonstrate that transcriptional repression of Quantitative PCR analysis IRF7 by MYC provides an additional layer of tight regulation of IRF7 For quantitative RT-PCR (qRT-PCR) experiments, total RNAwas isolated from that ensures the appropriate level of type I IFN production and prevents cells with Qiagen RNeasy mini kit and reverse transcribed into cDNA using the development of autoimmune disease. Combination studies have iScript cDNA synthesis kit (Bio-Rad). qRT-PCR for IL-6, IL-23A, TNF-a, a b identified the IRF7 gene as a critical target of MYC, although not its IL-8, IFN- 4, IFN- , CXCL10, IRF7, MYC, PLSCR1, IFIT1, TLR7, TLR9, IFNa14, CCND1, p21,TLR7, TBK1, and TLR9 was performed with specific only target. Our results characterize the novel immunomodulatory role of primers using the instructions from the manufacturer (SyBr Green mastermix; transcription factor MYC in human pDCs and may add to our under- Bio-Rad). Primer sequences are available upon request. standing of aberrant pDC function in cancer and autoimmune disease. Measurement of cytokine production from GEN2.2 cells using ELISA Materials and Methods Reagents and Abs GEN2.2 cells were stimulated with the indicated concentrations of CpGA, CpGB, or R848 for 4 h (to detect TNF-a, IL-6, and IL-8) or for 12 h (to CpGA (ODN2116), CpGB (ODN2006), and R848 were purchased from detect IFN-a and IFN-b). Concentrations of IFN-a, IFN-b, TNF-a, IL-6, Invivogen. The following Abs were used for immunoblotting: anti-MYC, and IL-8 in the culture supernatants were measured by ELISA (PBL anti-IRF7, and anti-IRAK1 (Santa Cruz Biotechnology); anti-TLR7 (Abcam); Biomedical Laboratories and R&D Systems). anti-MyD88 and anti-TLR9 (eBioscience); anti-STAT1, anti–nuclear receptor Downloaded from corepressor 2 (NCOR2), anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC4, ChIP-sequencing and ChIP-PCR anti-HDAC6, anti-HDAC7, anti-pIKKa/b, anti-pIkBa, anti-IkBa, anti-pJNK, ChIP assays were performed using ChIP assay kit, according to the anti-pp38, anti-TBK1, anti-pTBK1, and anti-pERK (Cell Signaling Technolo- manufacturer’s instructions (Upstate Biotechnologies). Briefly, unstimu- gies); and anti-GAPDH (Sigma-Aldrich). lated GEN2.2 cells were cross-linked with 1% of formaldehyde for 10 min NE-PER nuclear and cytoplasmic extraction reagents (Pierce) were used at 37˚C, sonicated, and subjected to immunoprecipitation with 2 mg anti- to investigate nuclear translocation of IRF7 as per the manufacturer’s in- MYC Ab (N262X; Santa Cruz Biotechnology), or rabbit IgG control. After

structions. MYC inhibitor (c-Myc inhibitor II, CAS 413611-93-5) was http://www.jimmunol.org/ reverse cross-linking and recovering the chromatin, the isolated chromatin purchased from Calbiochem. HDAC inhibitors, valproic acid (VPA), were was subjected to library preparation using NEXTflex Rapid DNA-Seq Kit purchased from Invivogen. (BIOO Scientific), according to manufacturer’s instruction, with the fol- Online data access lowing exceptions: 1) a bead size selection was performed before the PCR amplification and 2) the libraries were size selected between 200 and 300 The microarray dataset described in this manuscript is deposited in the National bp by 8% PAGE before sequencing. ChIP-sequencing libraries were se- Center for Biotechnology Information Gene Expression Omnibus (http://www. quenced 1 3 50 on a HiSEquation 2500 (Illumina). The resulted sequences ncbi.nlm.nih.gov/geo, Gene Expression Omnibus series accession number were aligned to the hg19 reference genome using Bowtie2. Downstream GSE70276). The chromatin immunoprecipitation (ChIP)-Seq dataset described analysis was performed by the HOMER next-gen sequencing analytical in this manuscript is deposited in Gene Expression Omnibus (GSE70275). suite (http://homer.salk.edu/homer/index.html) (37). Peak finding was performed with the findPeaks command line with the parameters of -style by guest on September 28, 2021 Statistical analysis factor and -i input. Input library was generated from the sonicated chromatin of GEN2.2 cells before the chromatin immunoprecipitation of MYC. Data are presented as mean values 6 1 SD. The p values were calculated , The command line, annotatePeaks.pl, was used for peak annotation and using an unpaired two-tailed Student t test. The p values 0.05 were finding the MYC motif occurrences in peaks. considered statistically significant. Immunoprecipitated DNA was quantified using RT-PCR and qRT-PCR by Cell lines and tissue cultures comparison with input sample signals. The signals were normalized to 1% total chromatin input of the anti-MYC or the control IgG sample. The results pDCs were isolated from healthy donors by sorting as lineage-negative (CD3, were expressed as fold difference between the two samples. Fold enrichment CD14, CD15, CD16, CD19, CD20, CD25, CD56, and CD11c) cells using a was determined from triplicate PCRs at promoter regions of IRF7. pDC isolation kit (Miltenyi Biotec) to .98% purity. Purified pDC were cul- ChIP assays for human primary pDCs were performed, as described above. tured in medium (RPMI 1640; Invitrogen) supplemented with 10% FCS and A total of 10 3 106 human primary pDCs was pooled from three different IL-3 (10 ng/ml; R&D Systems). GEN2.2 cells were provided by J. Plumas healthy donors for one ChIP experiment. Immunoprecipitated DNA was (Universite´ Joseph Fourier, Grenoble, France) and cultured, as described (35). quantified using qRT-PCR by comparing with input sample signals. The Briefly, GEN2.2 cells were cultured in GlutaMax–RPMI 1640 (Life Tech- signals were normalized to 1% total chromatin input of the anti-MYC, anti- nologies) supplemented with 10% FBS (Atlanta Biologicals), MEM-nonessential NCOR2, anti-HDAC3, or the control IgG sample. The results were expressed amino acid solution (Life Technologies), sodium pyruvate, and gentamicin. as fold difference between the two samples. Fold enrichment was determined HEK293T cells were purchased from American Type Culture Collection and from triplicate PCRs at promoter regions of IRF7. cultured in DMEM (Life Technologies) supplemented with 10% FBS (HyClone), L-glutamine, penicillin/streptomycin, and sodium pyruvate (Life Technologies). Luciferase assay RNA extraction, processing, and statistical analysis for Cells transfected with the indicated plasmid were harvested and subjected to luciferase reporter assay using a dual luciferase assay, according to the microarray manufacturer’s instructions (Promega). RNA isolation was performed using Qiagen RNeasy mini kit, according to the manufacturer’s instructions. Amplified RNA was hybridized to Illu- IP and mass spectrometry–based immunoprecipitation analysis mina HT-12 V4 beadchips (47,231 probes) and scanned on an Illumina using nuclear extracts Beadstation 500. Illumina’s GenomeStudio version 2011.1 with the Gene Expression Module v1.9.0 (Illumina, San Diego, CA) was used to generate MYC-binding protein complexes purified from GEN2.2 cells were lysed in signal-intensity values. For the analysis of nontargeting control (NC) lysis buffer (50 mM Tris-HCl [pH 7.4], 250 mM NaCl, 1 mM EDTA, 1% versus MYC in GEN2.2 cells, ANOVAwas used for differentially expressed Triton X-100, 10% glycerol supplemented with a complete protease in- gene (DEG) analysis for CpGB posttreatment, and DEGs were identified by hibitor, and phosphatase inhibitor mixture [Sigma-Aldrich]) and subjected Welch t test p , 0.05 with Benjamini–Hochberg correction and 2-fold to ultracentrifugation. Cleared lysates were incubated overnight with normalized difference to untreated controls (509 DEGs). protein G agarose beads (Pierce) with the indicated Abs. Beads were washed extensively with lysis buffer, separated on a 4–20% gradient polyacryl- Knockdown in GEN2.2 cells amide gel, and stained with Coomassie blue (Sigma-Aldrich). Unstimu- lated GEN2.2 cells were lysed. Nuclear extracts were fractionated, as Transfection of GEN2.2 cells was performed, as described (36). Briefly, shown in Fig. 4A, and then immunoprecipitated with MYC-specific Ab GEN2.2 cells were nucleofected with nucleofector kit V (Amaxa) with 0.4 and rabbit IgG control. Then, immune complexes from MYC- IP were 3350 MYC AND IFN IN pDCs Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. MYC depletion potentiates type I IFN production and signaling in pDC cell line. (A and B) GEN2.2 cells were nucleofected with siRNA against nonspecific control (NC) or MYC and then analyzed for knockdown efficiency by qRT-PCR (A) and by Western blot analysis (B). (C–E) GEN2.2 cell with siRNA against NC or MYC was treated with CpGB prior to hybridizing on Illumina HT-12 V4 beadchips. (C) Hierarchical clustering (Euclidian) of 509 DEGs that changed in MYC-ablated GEN2.2 cells versus control at 0, 2, or 4 h after CpGB treatment (Benjamini–Hochberg correction p value 0.01). The IFN-stimulated genes are highlighted in the dashed line, and representative genes are listed. Data are representative of at least three independent experiments. (D) Cytoscape network displaying the differentially upregulated genes (false discovery rate 0.01 and fold change .2) in MYC-depleted pDC cells connected to the time point after CpGB treatment. Genes that were upregulated at more than one time point were connected to a multiple time point hub and are represented as a darker color. (E and F) Microarray results shown in (C) were confirmed with qRT-PCR (E) and Western blot analysis (F). mRNA levels of IFN-b, CXCL10, IFN-a14, IRF7, TLR7, and STAT1 in MYC- and NC-siRNA–nucleofected GEN2.2 cells are represented at the time points after stimulation with CpGB. Abs against IRF7, STAT1, TLR7, and GAPDH were used for Western blot analysis. Data are representative of two independent experiments (average of triplicates 6 SD). (G) Enhanced proinflammatory cytokine production in MYC-depleted (Figure legend continues) The Journal of Immunology 3351 separated on a 4–20% gradient SDS-PAGE gel (Thermo Scientific) and analysis using SABiosciences’ proprietary database DECODE stained with Coomassie blue for visualization. All bands were in-gel (DECipherment of DNA Elements) revealed 123 potential tran- digested with trypsin, and the proteins were identified by nanoflow liquid scriptional targets of MYC, including IFITM3, IRF7, IRF9, chromatography–tandam mass spectrometry analysis with a nano-LC1000 (Thermo Scientific) coupled to a Thermo ORbitrap Velos (Thermo Scien- ISG15, ISG20, SOCS1, and STAT1 (Fig. 2B, Supplemental tific) mass spectrometer. Acquired mass spectrometry spectra were processed Table II). Interestingly, IRF7 was one of the most significantly by BioWorks software to convert data into peptide and protein composition enriched compared with nonspecific input in ChIP-Seq. IRF7 is information. All bands were analyzed by liquid chromatography-mass spec- the key regulator of type I IFN production in pDC and contains trometry at the Proteomics Center, Baylor College of Medicine (Houston, TX). E-box motif in the promoter region (59-CACGTG-39) (39). The occupancy of MYC in the IRF7 promoter region was confirmed by Results qRT-PCR using specific primers against the IRF7 promoter (Fig. MYC ablation in pDC cell lines revealed an inhibitory effect of 2C, 2D). Occupancy of MYC on IRF7 promoter region was fur- MYC in antiviral immune response ther confirmed with ChIP after knockdown of MYC (Fig. 2E). As To study the effects of MYC in the transcriptional signature of human expected, in the absence of MYC (MYC KD), MYC occupancy on pDC, MYC was ablated in the pDC cell line GEN2.2. GEN2.2 IRF7 promoter was dramatically reduced (Fig. 2F). Next, to val- displays a similar phenotype and function to human primary pDCs idate these results, we conducted a luciferase reporter assay using (35). Knockdown experiments were performed in GEN2.2 cells to the IRF7 promoter conjugated with luciferase. The 293T cells deplete Myc mRNA by Nucleofection, and their mRNA levels were stably expressing human TLR9 (Invivogen) were transiently compared with that of nonspecific siRNA control (NC). The transfected with an IRF7 promoter construct and siRNA against knockdown efficiency of Myc mRNA and MYC protein was eval- MYC. Interestingly, IRF7 promoter-Luc activity was significantly Downloaded from uated by qRT-PCR and by Western blot analysis, respectively induced when MYC was ablated, indicating that MYC occupies (Fig. 1A, 1B). Then, MYC-ablated GEN2.2 cells were treated with the IRF7 promoter and represses its transcription (Fig. 2G). The CpGB (0, 2, and 4 h) for pDC maturation and activation. Cell ly- increase of IRF7 promoter activity in the absence of MYC was sates were hybridized on Illumina human beadchips, and analysis further augmented when stimulated with CpGB. These results (Benjamini–Hochberg correction with p value: 0.01) revealed a total demonstrate direct interactions between MYC and the IRF7 pro-

of 509 DEG in Myc siRNA-transfected cells after CpGB treatment moter region. Therefore, we hypothesized that MYC regulates http://www.jimmunol.org/ compared with untreated cells (Fig. 1C, Supplemental Table I). We pDC antiviral immune response through its transcriptional re- observed significantly increased transcription of ISGs (IFIT1, IFIT2, pression of IRF7. IFIT3, OAS1, OASL, ISG20, etc.) in MYC-knockdown pDCs MYC, NCOR2, and HDAC3 form a repressor complex to compared with NC, indicating that MYC functions as a negative downregulate IRF7 expression through histone deacetylation regulator of the IFN signaling pathway. Moreover, stimulation of MYC-depleted pDCs with CpGB significantly increased the tran- It is well established that MYC can inhibit the expression of its scription of pre-existing subset of ISGs and additional ISGs in a target genes through the recruitment of corepressors such as time-dependent manner (Fig. 1C, marked with a dashed line). HDACs or histone methyl transferases (30, 40). To understand the Moreover, the genes that were significantly upregulated at every molecular mechanisms by which MYC represses IRF7, we per- by guest on September 28, 2021 time point in MYC-ablated cells (false discovery rate 0.01, fold formed mass spectrometry–based immunoprecipitation analysis change $2) are represented as a gene symbol/time point connec- using a specific Ab against MYC. As seen in Fig. 3A, MYC tivity network using Cytoscape 3.1.1 (38) (Fig. 1D). It is noteworthy predominantly exists in the nuclear fraction of GEN2.2, in which a that most of the upregulated genes are ISGs, including genes that nuclear fraction marker HDAC1 exists. GAPDH was used as a were upregulated at 0 and 2 h (IFI27, IFIT1, MX2, OAS1, OAS2, control to show the purity of cytoplasmic and nuclear fractions. To andSTAT1)andonesthatwereupregulatedat0,2,and4h(IRF7, purify the MYC-bound protein complexes, nuclear extracts from IFI6, and IFI35). In addition, type I IFNs such as IFNA2, IFNA4, GEN2.2 were prepared, and the polypeptides bound to the MYC IFNA10, IFNA13, and IFNA14 were stimulated at 2–4 h post- Ab or control IgG were separated by gradient PAGE and visual- treatment. The induced expression of these ISGs was validated by ized by Coomassie staining (Fig. 3B). Several polypeptide bands qRT-PCR and Western blot analysis (Fig. 1E, 1F). Subsequently, were observed distinctly in the nuclear extracts pulled down with type I IFN protein and proinflammatory cytokine productions were MYC Ab, but not with control IgG. The polypeptide bands spe- measured using IFN-a,IFN-b, IL-6, and TNF-a ELISA (Fig. 1G), cific for anti-MYC pulldown were excised from the gel and ana- and greatly enhanced type I IFN and cytokine productions were lyzed by liquid chromatography mass spectrometry. Among many observed when MYC was ablated in the pDC cell line. interesting candidates that were identified by mass spectrometry– based immunoprecipitation analysis to interact with MYC, those MYC directly occupies IRF7 promoter to repress its expression proteins with chromosome-remodeling activities are listed in MYC is a well-known transcription factor that regulates the ex- Table I. NCOR2 (shown at 274 kDa in MYC pulldowns) was pression of many genes either as an enhancer or repressor by especially interesting because this transcriptional corepressor is binding to consensus sequences (E-boxes). To identify direct tar- known to recruit HDAC to specific DNA promoter regions gets of MYC, we performed chromatin immunoprecipitation/DNA (41, 42). The interaction between NCOR2 and MYC was further sequencing (ChIP-Seq) in the GEN2.2 cell line. The ChIP-Seq confirmed by independent protein complex immunoprecipita- result revealed that 46.2% of MYC-bound motif have enriched tion (co-IP) experiments using nuclear extracts from GEN2.2 in E-box motif (59-CACGTG-39, p value; 1e-847) as shown in (Fig. 3C). There have been several reports suggesting possible Homer de novo motif analysis (Fig. 2A). Intersection of the gene interactions of MYC with HDAC3 or with NCOR2/HDAC3 (42– list from microarray (Fig. 1A), ChIP-Seq results, and a promoter 45). Thus, we hypothesized that MYC and/or NCOR2 recruit (or

GEN2.2 cells. GEN2.2 cells treated with siRNA against MYC or NC for 48 h were further stimulated with CpGB or a control for the indicated time. IFN-b production was measured by ELISA using the supernatant from each experiment. All PCR results were normalized to the GAPDH and are presented as relative- fold changes with respect to NC siRNA-nucleofected GEN2.2 cells. Data are representative of three independent experiments (average of triplicates 6 SD). 3352 MYC AND IFN IN pDCs Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. IRF7 is a direct transcriptional target of MYC in pDC. Direct target genes of MYC were identified using ChIP-Seq. (A–D) GEN2.2 (unstimulated or CpGB-treated) cells were cross-linked and immunoprecipitated with anti-MYC Ab or rabbit IgG control. In parallel, cross-linked input (without Ab) was put aside for the comparison. The bound DNAs were sequenced using HiSEquation 2000 (Illumina). (A) The most highly enriched motif (46.2% of targets, with p value 1 e-847) was analyzed and presented using Homer de novo motif analysis. (B) The Venn diagram shows the intersections of ChIP-Seq, promoter analysis, and microarray. Putative direct target genes of MYC (123 genes listed in Supplemental Table II) include IRF7. Venn diagrams were constructed using Venny. (C) The immunoprecipitated chromatin was isolated and used for semiquantitative RT-PCR with IRF7 promoter primers. MYC directly binds to the promoter region of IRF7. SpiB, another important transcription factor for pDC development, was used as a negative control. Data are representative of three independent experiments. (D) Amplification of the IRF7 promoter was visualized using qRT-PCR by comparing with input sample signals (1% of total crude). The levels of IRF7 promoter in MYC-bound or control IgG-bound DNA were normalized to input and expressed as fold difference between the two samples. Data are representative of three independent experiments (average of triplicates 6 SD). (E) GEN2.2 cells were nucleofected with siRNA against NC or MYC and then analyzed for knockdown efficiency by Western blot analysis. (F) GEN2.2 cells nucleofected with siRNA against NC or MYC were cross-linked and immunoprecipitated with anti-MYC Ab or rabbit IgG control. Amplification of the IRF7 promoter was visualized using qRT-PCR by comparing with input sample signals (1% of total crude). The levels of IRF7 promoter in MYC-bound or control IgG-bound DNA were normalized to input and expressed as fold difference between the two samples. Data are representative of three independent experiments (average of triplicates 6 SD). (G) Depletion of MYC showed an enhanced basal promoter activity of IRF7. HEK 293T cells stably expressing TLR9 were transiently cotransfected with luciferase reporter plasmid conjugated with control or human IRF7 promoter and transfected with MYC or NC siRNA. Twenty-four hours after transfection, cells were further stimulated with or without CpGB. Dual luciferase assays were performed 8 h after CpGB stimulation. All luciferase activity was normalized to the expression of the Renilla luciferase activity. The results were visualized as a fold of induction over the unstimulated NC siRNA-transfected GEN2.2 cells. Data are representative of three independent experiments (average of triplicates 6 SD).

form a multiprotein complex with) specific HDAC(s) to the endogenously assemble. Additionally, physical interaction of three promoter region of their target genes and repress their expres- protein MYC/NCOR2/HDAC3 was confirmed by co-IP for sion. Therefore, another co-IP experiment was performed to see NCOR2 (Fig. 3E). These findings further emphasize the epige- whether specific HDAC(s) could interact with MYC. As shown in netic regulation of IRF7, especially supporting chromatin deace- Fig. 3D, HDAC3 could be specifically recruited to the MYC im- tylation by a MYC/NCOR2/HDAC3 complex to regulate the mune complexes, revealing that MYC and HDAC3 physically and expression of IRF7 protein. To determine whether chromatin The Journal of Immunology 3353 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. MYC/NCOR2/HDAC3 complex represses type I IFN production and signaling in pDC. (A) Nuclear fraction of pDC cell line revealed the nuclear localization of MYC. Cytosolic and nuclear fractions from unstimulated GEN2.2 cells were immunoblotted with Ab against MYC, HDAC1, and GAPDH. HDAC1 and GAPDH were used as nuclear and cytosolic markers, respectively. Data are representative of three independent experiments. (B) MYC-bound protein complexes were identified by mass spectrometry–based immunoprecipitation analysis. Unstimulated GEN2.2 cells were lysed, and nuclear extracts were fractionated as in (A) and immunoprecipitated with MYC-specific Ab and rabbit IgG control. Then, immune complexes from MYC-IP were separated on a 4–20% gradient SDS-PAGE gel (Thermo Scientific) and stained with Coomassie blue (Sigma-Aldrich). All bands were in-gel digested with trypsin, and proteins were identified by nanoflow liquid chromatography–tandam mass spectrometry analysis with a nano-LC1000 (Thermo Scientific) coupled to Thermo ORbitrap Velos (Thermo Scientific) mass spectrometer. Acquired mass spectrometry spectra were processed by BioWorks software to convert data into peptide and protein composition information. (C) Coimmunoprecipitation experiments showed direct interaction of MYC and NCOR2 in unstimulated pDC cell lines. Unstimulated GEN2.2 cells were fractionated for nuclear extracts and co-IPed with anti-MYC Ab and immunoblotted with anti-NCOR2. IP with IgG was used as a control. Data are representative of three independent experiments. (D) MYC interacts with HDAC3. Co-IP experiments showed direct interaction of MYC and HDAC3 in unstimulated pDC cell lines. Unstimulated GEN2.2 cells were fractionated for nuclear extracts, co-IPed with anti-MYC Ab, and immunoblotted with anti-HDACs. IP with IgG was used as a control. Data are representative of three independent experiments. (E) Reciprocal co-IP showing endogenous binding of NCOR2, HDAC3, and MYC. Nuclear extracts of GEN2.2 cells were subjected to IP with anti-NCOR2 Ab, followed by Western blot analysis for NCOR2, MYC, and HDAC3. Data are representative of three independent experiments. (F) Pharmaceutical inhibition of HDACs recovered IRF7 expression. GEN2.2 cells were treated with 100 mM HDAC inhibitor, VPA, and mRNA was measured for IRF7. Primers specific for GAPDH mRNA were used to normalize samples. These data are representative of three independent experiments performed in duplicate. (G) HDAC3 depletion enhanced IRF7 expression in GEN2.2 cells. Cell lysates from GEN2.2 cells transfected with control and HDAC3 siRNAs were analyzed by Western blot analysis with Abs against IRF7, HDAC3, and GAPDH. Data are representative of three independent experiments. (H) HDAC3 depletion enhanced IRF7 expression in GEN2.2 cells. GEN2.2 cells were transfected with control and HDAC3 siRNAs for 48 h, followed by stimulation with CpGB (0.5 mM) for 4 h. IRF7, IFN-a4, and IFN-b mRNA were analyzed by qRT-PCR. Primers specific for (Figure legend continues) 3354 MYC AND IFN IN pDCs

Table I. Selected putative MYC-interacting proteins from IP–mass spectrometry

National Center for Biotechnology Information GenInfo Identifier No. Protein Name Size (kDa) 331284178 Nuclear receptor corepressor 2 isoform 1 2525 192807320 Transcription activator BRG1 isoform F 1647 21237808 SWI/SNF complex subunit SMARCC2 isoform b 1214 325651836 SWI/SNF-related matrix-associated actin-dependent 1052 regulator of chromatin subfamily A member 5 156523968 Poly[ADP-ribose] polymerase 1 1014 5032179 Transcription intermediary factor 1-b 835 14141170 Metastasis-associated protein MTA2 668 106879206 Bifunctional lysine-specific demethylase and histidyl-hydroxylase 641 NO66 223556010 Protein arginine N-methyltransferase 3 isoform 3 361 structure plays a critical role in the regulation of IRF7, GEN2.2 (47), we treated primary pDC with IL-3 to promote viability and cells were treated with HDAC inhibitor, VPA. The expression of activation while treating with MYC inhibitor. The dose of MYC IRF7 was significantly increased after the time course of VPA inhibitor was titrated and optimized for primary pDC (data not treatment compared with that of DMSO-treated GEN2.2 (Fig. 3F), shown). In agreement with an observation in pDC cell line, type I Downloaded from suggesting an essential role of epigenetic regulation when MYC/ IFN (IFN-a/b) productions were increased when MYC was NCOR2/HDAC3 represses IRF7 expression in pDC. Importantly, inhibited in human primary pDC after CpGA and CpGB treatment consistent with our hypothesis that the MYC/NCOR2/HDAC3 ter- (Fig. 4E, 4F, respectively). These data describe a molecular basis nary complex inhibits IRF7 transcription in pDC, the knockdown of for the therapeutic activity of anti-MYC agents. HDAC3 in GEN2.2 cells showed a significantly higher level of IRF7 Next, binding of MYC on IRF promoter region shown in pDC cell protein expression (Fig. 3G) and type I IFN (IFN-a and -b)pro- line GEN2.2 was further validated in human primary pDC by ChIP. http://www.jimmunol.org/ duction (Fig. 3H, 3I, measurements of mRNA and protein, respec- The occupancy of MYC in the IRF7 promoter region was confirmed tively). Next, the effect of TLR9 ligand stimulation on the MYC/ by qRT-PCR using primers against the IRF7 promoter (Fig. 4G). NCOR2/HDAC3 ternary complex formation was tested using co-IP Consistent with an observation in GEN2.2 cells, IRF7 promoter was experiments with MYC-specific Ab, followed by CpGB stimulation occupied by MYC after CpGB treatment. Moreover, NCOR2- and on GEN2.2 cells (Fig. 3J). CpGB stimulation did not affect and/or HDAC3-ChIP showed similar results as MYC-ChIP in human pri- alter the interaction of ternary complex. These data directly suggest mary pDC, further supporting our hypothesis (Fig. 4H). that HDAC3 contributes to MYC-dependent repression of IRF7 MYC inhibits TLR9-mediated NF-kB/MAPK activation as well transcription and demonstrate that epigenetic changes on the IRF7 as IRF7 nuclear translocation in pDC by guest on September 28, 2021 promoter by HDAC3 might serve as a mechanism to fine-tune the hyperactivation of pDC. Activation of TLR9 in pDC stimulates a complex network of signal transduction pathways (48). To pursue the hypothesis Pharmaceutical inhibition of MYC reveals the inhibitory that MYC plays a central role in TLR9-mediated proin- function of MYC on IRF7 expression flammatory cytokine/chemokine production in pDCs, the im- To assess the therapeutic potential of reducing type I IFN pro- pact of depletion of MYC on TLR9 downstream signaling duction in pDC by blocking MYC, we employed a pharmaceutical pathways was assessed. Interestingly, consistent with the ro- inhibitor of MYC (c-Myc Inhibitor II; CalBiochem). Interestingly, bust production of type I IFN and proinflammatory cytokines/ MYC inhibition caused by the pharmaceutical inhibitor dramati- chemokines (Fig. 1C–E), silencing of MYC expression greatly cally induced IRF7 mRNA expression (Fig. 4A). As a control, enhances TLR9-mediated IKKa/b phosphorylation and IkBa CyclinD1 (CCND1, which is known to be positively regulated by phosphorylation and degradation, hallmarks of substantial MYC) and p21 (negatively regulated by MYC) are differently NF-kB activation (Fig. 5A). Moreover, MAPKs, including regulated by the MYC inhibitor, as expected (Fig. 4B) (46). The JNK, p38 MAPKs (p38), and ERKs, are shown to be activated, as MYC inhibitor induced IFN-a/b and proinflammatory cytokine evidenced by their phosphorylation status in MYC-depleted IL-8 and IL-6 (Fig. 4C, 4D, measurements of mRNA and protein, GEN2.2 cells. respectively), further supporting the negative role of MYC in pDC IRF7 translocation to the nucleus from the cytoplasm after viral function. infection is the prerequisite for robust type I IFN production in pDC We next sought to investigate whether pharmaceutical inhibition (49, 50). Therefore, to investigate the requirement of MYC for of MYC in GEN2.2 pDC cell line could be represented in human CpG-triggered signaling that culminates in the activation of IRF7, primary pDC. To this end, we measured type I IFN productions we monitored nuclear localization of IRF7 in GEN2.2 cells after after pharmaceutical inhibition of MYC using the same inhibitor. MYC knockdown (Fig. 5B). Interestingly, IRF7 nuclear translo- Because survival of isolated primary pDC is strictly IL-3 dependent cation was greatly induced by knockdown of MYC and further

GAPDH mRNA were used to normalize samples. Data are representative of three independent experiments (average of triplicates 6 SD). (I)HDAC3 depletion enhanced type I IFN expression in GEN2.2 cells. A human pDC cell line was transfected with siRNAs against HDAC3 for 48 h, followed by stimulation with CpGB (0.5 mM) for 4 h in 96-well culture plates. IFN-a and IFN-b production was measured by ELISA. These data are representative of three independent experiments performed in duplicate. (J) MYC interacts with NCOR2 and HDAC3 in GEN2.2 cells stimulated with CpGB. Co-IP experiments showed direct interaction of MYC, NCOR2, and HDAC3 in GEN2.2 cells unstimulated and stimulated with CpGB (0.5 mM) for 2 h. GEN2.2 cells were fractionated for nuclear extracts, co-IPed with anti-MYC Ab, and immunoblotted with anti-HDAC3 and NCOR2. IP with IgG was used as a control. Data are representative of three independent experiments. The Journal of Immunology 3355 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Pharmaceutical inhibition of MYC enhanced IRF7 expression and type I IFN production in pDC. Pharmaceutical inhibition of MYC (MYC inhibitor) represses MYC and enhances IRF7 expression. (A) GEN2.2 cells were treated with 100 mM MYC inhibitor (c-Myc inhibitor II, CAS 413611-93-5; Calbiochem), and their mRNA was measured for MYC and IRF7. Data are representative of three independent experiments (average of triplicates 6 SD). (B) Effect of pharmaceutical inhibition of MYC was tested for CCND1. p21 was used as a control. Data are representative of three independent experiments (average of triplicates 6 SD). (C and D) Pharmaceutical inhibition of MYC enhanced CpGB-mediated type I IFN production, as shown in the level of IFN- a4, IFN-b, IL-8, and IL-6 in mRNA (C) and protein levels (D). Data are representative of three independent experiments (average of triplicates 6 SD). (E and F) Human primary pDC from six different healthy donors were pretreated with 1 mM MYC inhibitor for 0.5 h, followed by stimulation with CpGA (1 mM, E) and CpGB (0.5 mM, F) for 6 h. IFN-a and IFN-b production was measured by ELISA. Data are representative of six independent experiments (average of triplicates 6 SD). (G and H) Pools of 10 3 106 human primary pDC from 10 different healthy donors stimulated with CpGB (0.5 mM) for 12 h were cross-linked and immunoprecipitated with anti-MYC (G) Ab, anti-NCOR2 and anti-HDAC3 (H) Abs, or rabbit IgG control. Ampliﬁcation of the IRF7 promoter was visualized using qRT-PCR by comparing with input sample signals (1% of total crude). The levels of IRF7 promoter in MYC-bound or control IgG-bound DNA were normalized to input and expressed as fold difference between the two samples. Data are representative of three independent experiments (average of triplicates 6 SD). enhanced by CpGB treatment, whereas NC had no effect. This Taken together, these data further support that MYC directly oc- result further supports the inhibitory role of MYC in nuclear cupies the promoter region of IRF7 to repress the type I IFN translocation of IRF7 and resulting type I IFN production in pDC. production in pDC. 3356 MYC AND IFN IN pDCs Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. MYC depletion enhances TLR9 downstream signaling. (A) GEN2.2 cells nucleofected with either NC or MYC-speciﬁc siRNA were analyzed for TLR9 downstream signaling pathway. Cell lysates from siRNA-transfected GEN2.2 cells, with or without 0.5 mM CpGB for the indicated times, were analyzed by Western blot analysis with Abs against IRAK1, phospho-IKKa/b, phospho-IkBa,IkBa, phospho-JNK, phospho-p38, phospho-ERK, and GAPDH. All results are representative of three independent experiments. (B) MYC depletion enhances TLR9-mediated IRF7 nuclear translocation. Nuclear fractions from GEN2.2 cells transfected with NC and MYC siRNAs for 48 h were stimulated with or without 0.5 mM CpGB for the indicated times and immunoblotted with anti-IRF7. Anti- HDAC1 and anti-GAPDH were used as nuclear and cytosolic markers, respectively. All results are representative of three independent experiments. (C)MYC depletion enhances TLR9-mediated TBK1 activation. Cell lysates from NC and MYC siRNA-transfected GEN2.2 cells, with or without 0.5 mMCpGBforthe indicated times, were analyzed by Western blot analysis with Abs against pTBK1, TBK1, and GAPDH. All results are representative of three independent experiments. (D) MYC depletion enhances TLR7-mediated TBK1 activation. Cell lysates from NC and MYC siRNA-transfected GEN2.2 cells, with or without 1 mg/ml R848 for the indicated times, were analyzed by Western blot analysis with Abs against pTBK1, TBK1, and GAPDH. All results are representative of three independent experiments. (E and F) GEN2.2 cells were nucleofected with siRNA against NC or TBK1andthenanalyzedforknockdownefﬁciencybyqRT-PCR(E) and by Western blot analysis (F). (G)mRNAlevelsofIFN-a4andIFN-b in TBK1- and NC-siRNA–nucleofected GEN2.2 cells are represented at the time points after stimulation with CpGB. Data are representative of two independent experiments (average of triplicates 6 SD). (H) GEN2.2 cells treated with siRNA against TBK1 or NC for 48 h were further stimulated with CpGB or a control for the indicated time. IFN-a and IFN-b production was measured by ELISA using the supernatant from each experiment. Data are representative of two independent experiments (average of triplicates 6 SD).

TBK1 has been implicated in TLR-mediated IRF3 phosphorylation/ blot analysis in MYC knockdown cells. Because human pDC pre- activation in many tissues by cell- and ligand-speciﬁc manner (51, 52). dominantly expresses TLR7 and TLR9 (53–55), TLR7- and TLR9- However,theroleofTBK1inIRF7activationinpDChasbeen speciﬁc ligands (CpGB and R848, respectively) were treated after poorly studied. To test whether TBK1 plays any roles in the IFN MYC knockdown. Interestingly, the phosphorylation of TBK1, a production in pDCs, expression of TBK1 was measured in Western hallmark of TBK1 activation, was enhanced in the absence of MYC The Journal of Immunology 3357

(Fig. 5C, 5D). We then knocked down TBK1 in pDC cell line two classes of important noncoding RNAs in eukaryotes (61–63). (Fig. 5E, 5F); however, TLR7/9-mediated type I IFN production These noncoding RNAs have fundamental roles in development, showed no difference compared with NC siRNA, indicating that immunity, and tumorigenesis (64, 65). Therefore, it would be in- TBK1 may not have a massive role in TLR7/9-mediated type I IFN teresting to look at the epigenetic regulation of noncoding RNAs in production (Fig. 5G, 5H, and data not shown). the transcriptional regulation of pDC by MYC. IRF7 and its target genes have been implicated as strong in- Discussion hibitors of metastasis of certain cancers such as breast cancer pDC comprise a unique cell type that is dedicated to the production (66). Several studies suggested that IRF7 could be a funda- of type I IFN (IFN-a/b) in response to viruses and bacteria. In the mental molecule for type I IFN-based antitumor therapies (67–69); current study, we identified a novel central regulatory node that however, 70% of cancers have aberrant expression of MYC (70– controls maintenance of the pDC response and IFN secretion: 72). Therefore, we could hypothesize that the balance between MYC. Ablation of MYC in pDC cell lines (GEN2.2) resulted in an type I IFN production and MYC may be a potential indicator of enhanced antiviral immune response with remarkable ISG pro- cancer and autoimmune disease. In fact, very recent research from duction, including IRF7, a key player in pDC function. ChIP- the international multisite SLE cohort study revealed that breast, sequencing analysis identified that MYC directly bound at the endometrial, and ovarian cancer risks were significantly decreased promoter region of IRF7. in a SLE group compared with a non-SLE group (73). These Although both CpGA and B are known to induce the secretion of cancers are sex hormone–dependent cancers that affect only IFN-a, their downstream genes and mechanism somewhat differ women. However, SLE is also known to predominantly affect from each other (56). CpGA upregulates genes that are associated women of childbearing age and above. As such, we suspect that Downloaded from with metabolic functions, but CpGB uniquely upregulates genes that the interrelationship between MYC/IRF7 plays a role in tumori- support antibacterial responses such as the NF-kB–dependent path- genesis of these types of cancers in SLE patients. It is intriguing way. It has been suggested that MYC suppresses NF-kB and its that rates of sex hormone–independent cancers were not signifi- target genes (32–34). Consistent with previous reports, known, direct cantly altered in SLE patients. Moreover, it is well established targets of NF-kB transcription factor were upregulated in MYC that type I IFN-based anticancer therapies often resulted in

knockdown cells in our study; however, detailed mechanism of how exacerbated phenotypes, especially for those with an autoimmune http://www.jimmunol.org/ MYC suppresses NF-kB activation cannot be fully explained with disease such as SLE or rheumatoid arthritis (74, 75). our present study because of the complexity of NF-kB system and In summary, our findings highlight a new physiological role for the possibility of many positive or negative feedback regulation. MYC and identify MYC as an important brake for uncontrollable Therefore, further in-depth study is warranted. We used CpGB to type I IFN production in pDC. Because excessive expression of address global downstream pathways of antiviral activity in pDC. type I IFN initiates autoimmune disease development and type I Consistent with the increased production of type I IFN and proin- IFN is the most potent mediator of antitumor activity, we propose flammatory cytokine/chemokine in MYC-depleted cells, activation that new approaches to manipulate MYC expression in pDC for of TLR9-mediated NF-kB and MAPK as well as IRF7 nuclear early therapeutic intervention in autoimmune disease as well as translocation were greatly enhanced with MYC depletion. Accord- potential anticancer therapeutics might be promising. by guest on September 28, 2021 ingly, a MYC inhibitor dramatically induced IRF7, IFN-a/b, and IL- 8 expression, further highlighting the regulatory role of MYC in Acknowledgments antiviral innate immune responses in pDC. It is important to note We acknowledge Harrod Carson for editing the manuscript. that, like IRF7, other crucial transcription factors for type I IFN production, such as STAT1 and IRF9, which form the ISGF3 com- Disclosures plex with STAT2 (57), are also transcriptionally repressed by MYC. The authors have no financial conflicts of interest. Additionally, two important ISGs, ISG15 and ISG20, are also shown as direct targets of MYC. ISG15 is, to our knowledge, the first ISG identified and is a key regulator of ISGylation, a newly characterized References ubiquitinylation-like posttranslational modification in immune re- 1. Cella, M., D. Jarrossay, F. Facchetti, O. Alebardi, H. Nakajima, A. Lanzavecchia, sponses (58). ISG20 is another important ISG, which inhibits RNA and M. Colonna. 1999. Plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type I interferon. Nat. Med. 5: 919–923. and DNA virus replications through its exonuclease activity (59, 60). 2. 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