The PYHIN Protein P205 Regulates the Inflammasome by Controlling Asc Expression

The PYHIN Protein p205 Regulates the Inflammasome by Controlling Asc Expression

This information is current as Sreya Ghosh, Christina Wallerath, Sergio Covarrubias, Veit of September 29, 2021. Hornung, Susan Carpenter and Katherine A. Fitzgerald J Immunol 2017; 199:3249-3260; Prepublished online 20 September 2017; doi: 10.4049/jimmunol.1700823

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Supplementary http://www.jimmunol.org/content/suppl/2017/09/20/jimmunol.170082 Material 3.DCSupplemental http://www.jimmunol.org/ References This article cites 58 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/199/9/3249.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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The PYHIN Protein p205 Regulates the Inﬂammasome by Controlling Asc Expression

Sreya Ghosh,* Christina Wallerath,†,‡ Sergio Covarrubias,x Veit Hornung,†,‡ Susan Carpenter,x and Katherine A. Fitzgerald*

Members of the IFN-inducible PYHIN protein family, such as absent in melanoma-2 and IFN-g–inducible protein (IFI)16, bind dsDNA and form caspase-1–activating inflammasomes that are important in immunity to cytosolic bacteria, DNA viruses, or HIV. IFI16 has also been shown to regulate transcription of type I IFNs during HSV infection. The role of other members of the PYHIN protein family in the regulation of immune responses is much less clear. In this study, we identified an immune-regulatory function for a member of the murine PYHIN protein family, p205 (also called Ifi205). Examination of immune responses induced by dsDNA and other microbial ligands in bone marrow–derived macrophages lacking p205 revealed that inflammasome activation by dsDNA, as well as ligands that engage the NLRP3 inflammasome, was severely compromised in these cells. Further analysis Downloaded from revealed that p205-knockdown cells showed reduced expression of apoptosis-associated speck-like molecule containing CARD domain (Asc) at the protein and RNA levels. p205 knockdown resulted in reduced binding of actively transcribing RNA polymerase II to the endogenous Asc gene, resulting in decreased transcription and processing of Asc pre-mRNA. Deletion of p205 in B16 melanoma cells using CRISPR/Cas9 showed a similar loss of Asc expression. Ectopic expression of p205 induced expression of an Asc promoter–luciferase reporter gene. Together, these findings suggest that p205 controls expression of Asc mRNA to regulate inflammasome responses. These findings expand on our understanding of immune-regulatory roles for the PYHIN protein http://www.jimmunol.org/ family. The Journal of Immunology, 2017, 199: 3249–3260.

oreign nucleic acids (NAs) play a critical role in initiating form of cell death called pyroptosis (10). Although most inflam- innate and adaptive immune responses. NA sensors expressed masomes are composed of members of the NLR family, the dsDNA- F in distinct cellular compartments survey the extracellular and activated inflammasome is formed following dsDNA binding to a intracellular environment for signs of infection and initiate immune PYHIN protein, AIM2. Work from several laboratories, including defenses against bacterial, viral, and eukaryotic pathogens (1). NA our own, has defined AIM2 as a cytosolic DNA- binding innate sensors include RNA sensors, such as TLR3, TLR7/8, and RIG-I/ immune receptor (7, 11–13). AIM2 binds pathogen-derived dsDNA by guest on September 29, 2021 MDA5 (2–4), as well as DNA sensors, such as TLR9, cGAS, absent that accumulates in the cytosol during infection with DNA viruses or in melanoma-2 (AIM2), and IFN-g–inducible protein (IFI)16 (5–8). cytosolic bacterial pathogens (14, 15). In some instances, AIM2 can In addition, recognition of foreign NAs, especially dsDNA, leads also recognize host dsDNA that gains access to the cytosol leading to assembly of an inflammasome, a large caspase-1–activating multi- to autoinflammation (16). The related PYHIN protein IFI16 also protein complex that controls the proteolytic processing and release forms an inflammasome during infection with Kaposi’s sarcoma of IL-1b (9). Inflammasome activation also results in a proinflammatory herpesvirus and HIV-1 (17, 18). The PYHIN proteins were first characterized as a family of IFN- inducible proteins that are predominantly nuclear localized (19). *Program in Innate Immunity, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605; †Gene Center, Ludwig Maximilian University of PYHINs are constitutively expressed in different hematopoietic Munich, Munich 81377, Germany; ‡Department of Biochemistry, Ludwig Maximilian cell types, although most members of this family are also induc- University of Munich, Munich 81377, Germany; and xDepartment of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064 ible by type I IFN in nonhematopoietic cells (19). Phylogenetic Received for publication June 7, 2017. Accepted for publication August 22, 2017. analysis of the mammalian PYHIN proteins, also called the AIM2- like receptors, show strong evolutionary and functional diversity (20, This work was supported by National Institutes of Health Grants AI093752, AI083713, and AI067497. 21). The murine PYHIN locus has undergone extensive gene du- Address correspondence and reprint requests to Dr. Katherine A. Fitzgerald, Program plication, with 13 members encoded in the murine genome in contrast in Innate Immunity, Department of Medicine, University of Massachusetts Medical to the human gene locus with 5 genes, including MNDA, PYHIN1, School, LRB, Room 308, 364 Plantation Street, Worcester, MA 01605. E-mail address: kate.fi[email protected] POP3, IFI16, and AIM2 (19, 22). PYHIN proteins have been impli- The online version of this article contains supplemental material. cated in a wide range of cellular processes, including transcription, tumor suppression, cell cycle, cell growth, differentiation, and cell Abbreviations used in this article: AIM2, absent in melanoma-2; Asc, apoptosis-associated speck-like molecule containing CARD domain; BMDM, bone marrow–derived death (19). The majority of the PYHIN proteins share the same macrophage; CDS, coding DNA sequence; ChIP, chromatin immunoprecipitation; structural domains. They contain an N-terminal a-helical domain, CTD, C-terminal domain; GFP shRNA, shRNA sequence targeting GFP; HA, hemagglutinin; iBMDM, immortalized BMDM; IFI, IFN-g–inducible protein; ISD, IFN known as the pyrin domain, which is capable of homotypic stimulatory DNA; KD, knockdown; KO, knockout; LDH, lactate dehydrogenase; protein–protein interactions, and one or more HIN200 domains in NA, nucleic acid; pdAdT, poly-dAdT; poly I:C, polyinosinic-polycytidylic acid; their C terminus, with the exception of murine p208 and human qPCR, quantitative PCR; RNA Pol II, RNA polymerase II; serine-2P RNA Pol II, RNA Pol II phosphorylated at serine 2; serine-5P RNA Pol II, RNA Pol II phos- POP3, which contain only a pyrin domain, and murine p202, phorylated at serine 5; shRNA, short hairpin RNA; SV, Sendai virus; 39UTR, 39 which contains only two HIN200 domains (19, 20). Most PYHIN untranslated region. proteins contain a nuclear localization signal in their N terminus Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 that can be monopartite, bipartite, or both. Some family members www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700823 3250 p205 REGULATES INFLAMMASOME ACTIVATION also contain a nuclear export sequence that enables them to shuttle dehydrogenase (LDH), which is released into the supernatant upon cell lysis, between the nucleus and the cytosol. AIM2 is the most conserved using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) kit. family member. Unlike the other PYHINs, AIM2 is localized in Triton-X (10%) was added to the cells as a representation of 100% cell death. the cytosol. Phylogenetic analysis indicates that, with the exception Short hairpin RNA–mediated silencing of AIM2, there is a complete lack of orthology among mammalian The short hairpin RNA (shRNA) sequences targeting p205 were cloned into AIM2-like receptors (20, 21). a lentiviral pLKO.1 TRC cloning vector. Two separate shRNA sequences Although the role of AIM2 and IFI16 in dsDNA recognition and used for knocking down p205 targeted the coding DNA sequence (CDS) immune activation has been well established, the role of other (TRCN0000095887) or the 39 untranslated region (39UTR) (TRCN0000095884). members of the PYHIN protein family, especially those in the HEK 293T cells were transfected with 4 mg of shRNA along with 3 mgof pSPAX (gag/pol) and 1 mg of pMD2 (VSV-G) plasmids for production of mouse, remains unclear. Recently, genetic studies in mice lacking lentiviral particles. Viral particles were collected at 48 h, filtered, and added the entire PYHIN locus and analysis of type I IFN induction to iBMDMs. As controls, BMDMs were transduced with an empty pLKO.1 following dsDNA treatment in cells from these animals demon- vector or pLKO.1 containing an shRNA sequence targeting GFP (GFP shRNA). strated no clear link to dsDNA recognition and induction of type I The cells were selected for effective transduction using puromycin (5 mg/ml). IFN in murine myeloid cells (23). Therefore, it remains to be defined Quantitative RT-PCR and NanoString whether the PYHINs have other immune-regulatory functions. In this study, we examined the contribution of the murine PYHIN RNA was extracted using an RNeasy Kit (QIAGEN). cDNA was synthesized from 1 mg of total RNA using an iScript cDNA Synthesis Kit (Bio-Rad) protein p205 (also known as Ifi205) in innate immunity. p205 is or a QuantiTect Reverse Transcription Kit (QIAGEN). Quantitative RT-PCR primarily expressed in macrophages and granulocytes (24). p205 was performed using iQ SYBR Green Supermix (Bio-Rad) or a QuantiNova shares the highest homology with p204/Ifi204 and the human SYBR Green RT-PCR Kit (QIAGEN). Primers were constructed to respec- Downloaded from PYHIN protein, MNDA (25). p205 has been implicated as a positive tive genes (Supplemental Table I). Target gene expressions are relative to housekeeping gene expression and were normalized to respective controls. regulator of cell growth and differentiation during hematopoiesis, The levels of p204, p205, Mnda, Mndal, and Aim2 were measured using an adipogenesis, and osteogenesis (26–28). To evaluate a possible im- nCounter (NanoString Technologies). Briefly, endogenous RNA transcript mune function for p205, we investigated the ability of p205 to counting was performed on total RNA hybridized to a custom gene expression control immune gene expression. Using a series of loss-of-function CodeSet and analyzed on an nCounter Digital Analyzer (29). The counts were normalized to internal spike-in and endogenous controls per NanoString and gain-of-function approaches combined with functional studies, http://www.jimmunol.org/ Technologies’ specifications. The heat map was generated using Morpheus we demonstrate that p205 controls expression of the inflammasome software. adapter molecule apoptosis-associated speck-like molecule containing CARD domain (Asc) and, as such, regulates inflammasome- Western blot and coimmunoprecipitation dependent processing of IL-1b. This effect of p205 was not related For detecting caspase-1 and IL-1b in the supernatants and lysates, 20% vol to the prior work linking AIM2 and IFI16 to sensing of foreign RIPA buffer and 30% vol SDS-loading dye were added directly to the wells DNA. Rather, we find that p205 works in the nucleus to control Asc containing cells and media. The samples were boiled at 100˚C for 15–30 min gene expression. These findings add to our understanding of PYHIN and were run on 13% polyacrylamide gels. Cytosolic and nuclear fractions were prepared for protein detection using an Active Motif Nuclear Extract proteins in innate immunity, expanding their functions beyond dsDNA Kit. Coimmunoprecipitation assays were carried out with 5 3 106 cells sensing to innate immune gene regulation. (treated or untreated) in ice-cold RIPA buffer (without SDS and DOC) using by guest on September 29, 2021 Dynabeads Protein A (catalog number 10001D; Novex/Life Technologies) conjugated with specific Abs and immunoblotted for proteins of interest. Abs Materials and Methods used were against Asc (catalog number sc-22514-R; Santa Cruz Biotech- Reagents and plasmids nology or catalog number 67824; Cell Signaling Technology), IL-1b (catalog number AF-401-NA; R&D Systems), caspase-1 (catalog number AG-20B- LPS and poly-dAdT (pdAdT) were obtained from Sigma, and immuno- 0042; Adipogen), Aim2 (catalog number 14-6008; eBioscience), Nlrp3 stimulatory DNA oligonucleotides were synthesized as described (8). Nigericin (Enzo Life Sciences), Histone 3 (catalog number ab1791; Abcam), c/EBPb and ATP were from InvivoGen and Sigma, respectively. Polyinosinic- (catalog number sc-150; Santa Cruz Biotechnology), Usf2 (catalog number polycytidylic acid (poly I:C) was obtained from InvivoGen. Sendai virus sc-862; Santa Cruz Biotechnology), Gapdh (catalog number G9295; Sigma), (SV; Cantrell strain) was purchased from Charles River Laboratories b-actin (catalog number A3854; Sigma), HA-tag (Anti-HA-Peroxidase; catalog (Wilmington, MA). Lipofectamine 2000 Transfection Reagent was from number 12 013 819 001; Roche), and FLAG-tag (catalog number A8592; Invitrogen. GeneJuice was from Novagen (Madison, WI). Universal type I Sigma). An affinity-purified polyclonal Ab against p205 was generated IFN and IFN-g were from PBL Interferon Source (Piscataway, NJ) and using the following peptide: AGLDRLINFCERVPTL-amide (21st Century PeproTech (catalog number 315-05), respectively. Salmonella typhimurium Biochemicals). (SL1344 laboratory strain) was from M. O’Riordan. The plasmids used were p65-pCMV4, c/EBPb-pcDNA (Addgene), and pGL3-enhancer lu- Confocal microscopy ciferase reporter (Promega). Other plasmids, such as Asc in pMSCVneo HEK 293T cells were transfected with p205 tagged with CFP; 24 h (Clontech), p205-hemagglutinin (HA) in pRZ-retro, Aim2-FLAG, p204- posttransfection, the cells were washed with PBS, stained with Acridine HA, p205-HA in pEF-BOS, or HA-tagged p205 mutants that lacked the orange and/or cholera toxin B (Thermo Fisher), and visualized by confocal pyrin domain, the HIN domain, or both domains in pMSCV-PIG (Addgene), were made in the laboratory. microscopy (Leica 8000) for localization. Luciferase assays Cell culture, stimulations, ELISA, and cell death assays The Asc gene promoter from 22000 to +10 bp was cloned into the pGL3- Primary bone marrow–derived macrophages (BMDMs) from C57BL/6J enhancer reporter vector upstream of the firefly luciferase gene. The promoter- mice, cultured with L929 supernatant as a source for M-CSF, were reporter construct was transfected alone or cotransfected with plasmids transformed using CreJ2 virus to make immortalized BMDM (iBMDMs). expressing p205, p204, Aim2, or p205 deletion mutants, p65/RelA, c/EBPb, The transformation process does not change their ability to induce type I or a combination thereof in HEK 293T cells. A plasmid expressing Renilla IFNs or other immune genes. The cells were cultured in DMEM with 10% luciferase gene under thymidine kinase (pGL4-TK Renilla) promoter was in- FCS and penicillin streptomycin. Antibiotics for selection were used as cluded as a transfection efficiency control. Data are presented as fold change required. Cells were primed with repurified LPS at 100 ng/ml for 2–3 h and over the Asc reporter construct alone and relative to transfection efficiency. stimulated with Salmonella sp., nigericin, or ATP for 1 h, pdAdT and IFN stimulatory DNA (ISD) for 6 h, and poly I:C and SV overnight. Cells in- Chromatin immunoprecipitation fected with S. typhimurium and media containing gentamicin (100 mg/ml) were added to kill the extracellular bacteria. Supernatants from the stim- Cells were fixed with 1% formaldehyde, lysed, and sheared. The DNA was ulated cells were analyzed for the cytokines IL-1b (eBioscience) and IFN-b quantified, and 5 mg of total chromatin was immunoprecipitated with by ELISA. Cell death was measured by quantitating the amount of lactate specific Abs and Dynabeads Protein G (catalog number 10009D; Novex/Life The Journal of Immunology 3251

Technologies). The DNA was then reverse cross-linked, purified, and quan- (Fig. 2A) and the coding region (KD CDS) (Fig. 2B) of the mRNA. titated by quantitative PCR (qPCR) amplification with primers at the Asc and The level of p205 was evaluated by qPCR. The shRNA targeting the Gapdh genes (Supplemental Table I). Abs used were against total RNA pol II coding region specifically knocked down p205 mRNA with no ap- (catalog number 39097; Active Motif), phosphoserine-2 RNA polymerase II (product code ab5095; Abcam), phosphoserine-5 RNA polymerase II (product parent effect on the two most closely related murine PYHINs, code ab5131; Abcam), or IgG isotype (product code ab37415; Abcam and murine Mnda or p204 mRNA. This effect was also evaluated using product number 5415; Cell Signaling Technology). NanoString in which p205 mRNA was reduced with little impact on CRISPR/Cas9-mediated gene knockout four other related PYHIN genes (p204, Mnda, Mndal,andAim2) (Fig. 2C). B16 mouse melanoma cells were cultured in DMEM containing 10% FBS, Both of these macrophage cell lines with p205 KD were then 0.5% ciprofloxacin, and 0.0075% 2-ME and transfected with 200 ng of plasmid containing mCherry-Cas9 and a U6 promoter-driven guide RNA tested for their ability to respond to different immune stimuli and against p205 (target sequence: 59-ATGAAGCCGAAGATGAGACCTGG- induce cytokine gene expression. We first evaluated the inducible 39) using Lipofectamine 2000, according to the manufacturer’s protocol expression of type I IFN because prior work had identified other (30). Two days after transfection, cells were sorted for mCherry expression. PYHIN proteins, such as IFI16 and p204, as regulators of type I IFN Positive cells were plated at limiting dilution to obtain single-cell clones. gene expression. iBMDMs expressing shRNA targeting p205 or Genotyping of the B16 clones was conducted by deep sequencing (Illumina, MiSeq), as previously described (31), using the following primer sequences: GFP were stimulated with LPS, pdAdT, ISD, SV, or poly I:C, all of 59-ACACTCTTTCCCTACACGACGctcttccgatctCGTGAAGAAGATCAA- which induced the type I IFN, IFN-b, and the levels of which were GGCATCTG-39 and 59-TGACTGGAGTTCAGACGTGTGctcttccgatctAAA- measured by ELISA. For most of these ligands, the inducible TCTCAGGGAGAAGTGGGGGA-39 (uppercase letters: first PCR adapter expression of IFN-b was largely unaffected by the absence of sequences; lowercase letters: linker sequences; bold letters: target site-specific primer sequences). Cell clones harboring all-allelic frameshift mutations were p205 (Fig. 2D). However, stimulation of the p205-KD macro- Downloaded from then selected as p205-knockout (KO) cell clones. phages with pdAdT, a synthetic dsDNA mimetic, showed an enhanced IFN-b response compared with the control cells. p205-KD Statistics cells were also tested for IL-1b production, because PYHINs, ELISA and luciferase assays are presented as mean 6 SD from three inde- such as AIM2 and IFI16, form inflammasome complexes that pendent biological replicates and are representative of at least three separate regulate IL-1b production. The cell lines were treated for 2 h with experiments. Data were analyzed using two-way ANOVA by Prism 6 200 ng/ml LPS, which acts as signal 1 to prime cells, allowing http://www.jimmunol.org/ Software (GraphPad, San Diego, CA). The p values ,0.05 were considered significant (*p , 0.05, **p , 0.001, ***p , 0.005, ****p , 0.0001). transcriptional induction of pro–IL-1b. These cells were then challenged with transfected pdAdT (or ISD) or nigericin, which activate the Aim2 and Nlrp3 inflammasomes, respectively. Mac- Results rophages lacking p205 had reduced levels of IL-1b in response to LPS and IFN-inducible p205 localizes to the nucleus in all of these stimuli (Fig. 2E). macrophages To understand whether the diminished levels of p205 impacted The p205 gene is encoded on chromosome 1q, the last of the 13 the priming (signal 1) or processing of pro–IL-1b (signal 2), the consecutive genes of the mouse PYHIN locus. p205 is encoded on supernatants and lysates from the stimulated macrophages were the reverse strand and is flanked by p202b at the 59 end and several tested for levels of the inactive and proteolytically processed by guest on September 29, 2021 olfactory receptor genes, as well as the Spta1 gene, at its 39 end. forms of caspase-1 and IL-1b. Cells lacking p205 expressed p205 is expressed in murine primary BMDMs, and treatment of similar levels of pro–IL-1b following stimulation with LPS, ruling these cells with LPS, type I IFN, and IFN-g further unregulated its out an effect of p205 on TLR4 signaling and transcription of expression, similar to other PYHIN genes, such as Mnda and p204 pro–IL-1b (Fig. 2F, top panel). In contrast, the levels of mature (Fig. 1A–C). The inducible expression of p205 was as robust as processed IL-1b were significantly reduced in the KD cells the well-characterized immune genes, such as Il6 or Cxcl10, that (Fig. 2F, upper middle panel). Moreover, the activated form are used as positive controls. of caspase-1 was also undetectable in cells with shRNA KD To evaluate the localization of p205 in macrophages, we over- for p205 (Fig. 2F, bottom panel), despite comparable amounts expressed HA-tagged p205 in iBMDMs and performed subcellular of procaspase-1 between the control and the KD cell lines fractionation to purify nuclear and cytosolic extracts. Analysis of (Fig. 2F, lower middle panel). Collectively, these observations these fractions by immunoblotting for HA-tagged p205 in two indicate that KD of p205 impacted inflammasome-dependent independently transduced BMDMs showed that p205 is primarily activation of caspase-1, leading to reduced processing of pro– localized in the nucleus (Fig. 1D). We also confirmed the nuclear IL-1b. localization of p205 using confocal microscopy by imaging a Reduced expression of p205 leads to reduced expression of Asc CFP-tagged p205 construct expressed in HEK 293T cells (Fig. 1E). Treatment of primary BMDMs with LPS, type I IFN, and To understand the mechanistic basis for the defect in inflammasome IFN-g increased p205 protein expression in the nucleus (Fig. 1F). activation in cells expressing p205-shRNA, we first measured the Further, we stimulated primary BMDMs with type I IFN at dif- expression of Nlrp3 and Aim2, as well as the adapter protein Asc, ferent time points and separated the cell lysates into nuclear and in these LPS-stimulated macrophages. Although there was no cytosolic fractions, which were immunoblotted for endogenous difference in the protein levels of Nlrp3 or Aim2, expression of the p205. Upon IFN treatment, p205 expression was robustly increased Asc protein was completely absent in the p205-KD cell lines over time in the nucleus (Fig. 1G). (Fig. 3A). We further confirmed the loss of p205 protein in these cell lines using a new Ab generated against p205, and the LPS- Knockdown of p205 in BMDMs results in impaired stimulated macrophages lacking p205 showed no Asc protein ex- inflammasome activation pression (Fig. 3B). The expression of Asc was further investigated To determine the potential immune function of p205, we generated by measuring its transcript levels. These studies indicated that murine iBMDMs expressing shRNA targeting p205. BMDMs were cells with shRNA KD of p205 had decreased levels of Asc mRNA transduced with pLKO.1 lentiviral particles containing shRNA (Fig. 3C). Collectively, these findings indicate that p205 regulates targeting p205 or GFP as a control. Two shRNA sequences for p205 Asc mRNA levels. To discern whether the defective inflamma- were used that targeted the 39UTR (knockdown [KD] 39UTR) some responses that we observed in the p205-shRNA iBMDMs was 3252 p205 REGULATES INFLAMMASOME ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. p205 is induced by LPS and types I and II IFN and localizes to the nucleus. Primary BMDMs stimulated with LPS (200 ng/ml) (A), type I IFN (100 U/ml) (B), or IFN-g (20 ng/ml) (C) at different time points (0, 2, 4, 8, 16, and 24 h) were tested for p205 mRNA expression, as well as other PYHIN genes (Mnda, p204, and Aim2). Levels of IL-6 or Cxcl10 mRNA were included as positive controls. Gene expression is reported relative to a combination of three housekeeping genes (Gapdh, Hprt, and b-actin). (D) Immunoblot analysis of the nuclear and cytoplasmic fractions of wild-type (WT) BMDMs transduced with empty vector (EV) and WT BMDMs overexpressing HA-tagged p205 using anti-HA Ab. Histone 3 and Aim2 were used as controls for nuclear and cytosolic extracts, respectively. (E) Confocal microscopy of CFP-tagged p205 (green) in HEK 293T cells stained for nucleus using Acridine orange (upper panels; scale bar, 25 mm) and cholera toxin B (Ctx-B) for staining plasma membrane (lower panels; scale bar, 10 mm). Data are representative of two independent experiments. (F) Primary BMDMs that were left untreated (Ctl) or were treated with LPS for 6 h, type I IFN for 16 h, or IFN-g for 16 h were separated into nuclear and cytosolic fractions and immunoblotted for endogenous p205. (G) Western blot analysis of endogenous p205 expression in the nuclear and cytosolic extracts of primary macrophages treated with type I IFN, as indicated. Usf2 and Gapdh were used as controls for nuclear and cytosolic fractions, respectively. n.s., nonspecific band. specifically due to the absence of Asc, we restored Asc expression in these cells (Fig. 3E). S. typhimurium also activates the inflammasome. these cells using retroviral transduction. The Asc cDNA was cloned When Salmonella is in its log phase of growth (i.e., the bacteria are into a retroviral vector, pMSCV, and viral particles were transduced actively dividing), they are recognized by Nlrc4, whereas Salmonella in p205-KD macrophages to renew Asc expression. p205-shRNA is primarily recognized by Nlrp3 when in the nondividing or stationary cells transduced with the retrovirus had restored Asc levels comparable growth phase. Unlike Nlrp3 or Aim2, Nlrc4 itself contains a CARD to those seen in control cell lines (Fig. 3D). The reconstituted stable domain and can directly recruit procaspase-1, as well as engage Asc to cell lines were then tested for IL-1b release by ELISA. IL-1b do so. Thus, Nlrc4 can activate the inflammasome in a manner that is release by the macrophages following LPS priming and pdAdT or only partially dependent on Asc (32). The p205-KD cells were chal- nigericin treatment was fully restored upon re-expression of Asc in lenged with Salmonella in their stationary phase and log phase after The Journal of Immunology 3253 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. p205 KD using shRNA results in impaired inﬂammasome activation. BMDMs transduced with shRNA targeting the 39UTR (A)ortheCDS(B)of the p205 gene were inspected for expression of p205, Mnda, and p204 mRNA relative to b-actin mRNA and normalized to the expression in GFP shRNA BMDMs. (C) Heat map of PYHIN gene expression in p205-KD 39UTR and KD CDS BMDMs compared with control (Ctl) BMDMs. The p205-KD BMDMs were primed with LPS (200 ng/ml) for 3 h and then stimulated with transfected pdAdT (1 mg/ml for 6 h), transfected ISD (3 mM for 6 h), nigericin (10 mMfor 1 h), or ATP (5 mM) or were stimulated alone with SV (overnight) or poly I:C (overnight). Secreted IFN-b (D)andIL-1b (E) levels were assessed by ELISA. (F) GFP shRNA CTL, p205-KD 39UTR, and KD CDS were primed with LPS (200 ng/ml) for 3 h and then stimulated with pdAdT (1 mg/ml for 6 h) or nigericin (10 mM for 1 h); supernatants and lysates from the macrophages were immunoblotted for pro–IL-1b (35 kDa), the cleaved form of IL-1b (p17), procaspase-1 (45 kDa), and the active subunit of caspase-1 (p20). *p , 0.05, ***p , 0.005, ****p , 0.0001. CTL, control; n.s., not signiﬁcant.

LPS stimulation for 1 h. Salmonella in the stationary phase (overnight Inflammasome activation also leads to pyroptotic cell death (10), culture), which primarily engages Nlrp3 to activate the inflamma- which can be monitored by examining the release of LDH enzyme. some, showed minimal IL-1b production by ELISA. However, when pdAdT treatment of macrophages leads to Aim2 inflammasome- the same cells were infected with the actively dividing Salmonella (4– dependent release of LDH. Consistent with the other effects on 6 h culture), the cells retained part of their inflammasome-activating inflammasome function, the release of LDH was also significantly capability that was again rescued to its full potential upon recon- affected in cells with p205 KD. Restoration of Asc in these KD cell stitution with Asc (Fig. 3F). Further, stimulation with LPS and lines rendered these cells susceptible to cell death, similar to the pdAdT in the Asc-reconstituted macrophages showed processing levels seen in the control cell line (Fig. 3H). Altogether, these of caspase-1 and IL-1b into their active forms, signifying a func- findings attribute the loss of inflammasome responses in p205-KD tional inflammasome (Fig. 3G). cells directly to p205-dependent effects on Asc expression. 3254 p205 REGULATES INFLAMMASOME ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Loss of p205 leads to a defect in Asc expression. (A) Levels of Nlrp3, Aim2, Asc, and b-actin proteins were elucidated by Western blot in LPS-stimulated (200 ng/ml) GFP shRNA CTL, p205-KD 39UTR, and KD CDS macrophages. (B) Immunoblot of p205, Asc, and b-actin proteins in LPS- stimulated (200 ng/ml for 6 h) p205-KD macrophages. (C) Levels of p205 and Asc mRNA (relative to b-actin and normalized to GFP shRNA BMDMs) were detected by qPCR in the same cell lines. (D) Western blot analysis of Asc overexpression in the p205-KD BMDMs that were left untreated or treated with LPS (200 ng/ml for 3 h). Asc-reconstituted cell lines tested for IL-1b production on stimulation with LPS (200 ng/ml for 3 h) and pdAdT (1 mg/ml for 6 h) or ATP (5 mM for 1 h) (E) and with overnight culture (stationary phase) or log-phase culture of Salmonella sp. by ELISA (F). Levels of caspase-1 and IL-1b processing in the Asc-reconstituted cells were detected by Western blot (G), and cell death was measured by the amounts of LDH released with LPS (200 ng/ml for 3 h) and pdAdT (1, 2, or 3 mg/ml for 6 h) and/or ATP (5 mM for 1 h) stimulation (H). *p , 0.05, **p , 0.001, ***p , 0.005, ****p , 0.0001. CTL, control; n.s., not signiﬁcant.

Reconstitution of p205 expression in macrophages restores Asc p205 restored Asc protein expression in these KD cell lines (Fig. expression and inflammasome activation 4C). p205 protein expression was detected using an anti-HA Ab. We next restored expression of p205 in the KD cells using an HA- Cell lysates from GFP shRNA p205-KD 39UTR cell lines, along tagged retroviral vector. The levels of p205 gene expression were with the p205-KD 39UTR cell line reconstituted with p205-HA, measured by qPCR (Fig. 4A). The reconstitution of p205 restored were separated into nuclear and cytoplasmic fractions. The reconstituted Asc mRNA expression (Fig. 4B). Moreover, reconstitution with p205 was found to be primarily nuclear. Levels of Asc increased in the The Journal of Immunology 3255 nucleus and cytoplasm in cells expressing the p205-HA. Levels of primers, as indicated (Fig. 5B). We also expanded this analysis Aim2 were largely unchanged upon p205 reconstitution. We also using Abs to RNA Pol II phosphorylated at position serine 5 ectopically expressed the HA-tagged p205 in wild-type iBMDMs, (serine-5P RNA Pol II) or RNA Pol II phosphorylated at serine 2 which resulted in increased Asc mRNA levels compared with those (serine-2P RNA Pol II) in its C-terminal domain (CTD). The RNA found in control cell lines (Fig. 4D). Analysis of IL-1b release and Pol II CTD contains multiple repeats of a heptapeptide sequence, cell death in these cells further demonstrated that reconstitution of YSPTSPS, which is differentially phosphorylated to control the p205 in the KD cells restored inflammasome-dependent responses function of the polymerase (33). When preferentially phosphory- (Fig. 4E, 4F). Altogether, these findings provide further evidence for lated at serine 5 alone, there is only abortive transcription where a role for p205 as a regulator of Asc expression and inflammasome the RNA Pol II can initiate, but not elongate or process functional responses. mRNA (33). The change from an initiating to an actively elon- gating RNA Pol II occurs with the increase in phosphorylation of RNA polymerase II occupancy at the Asc locus is reduced in the serine 2 residue on the CTD repeats (33). Consistent with the macrophages expressing p205-shRNA findings with Abs to total RNA Pol II, the distributions of serine- The reduced mRNA levels of Asc in cells lacking p205 could be 5P RNA Pol II (Fig. 5C) and serine-2P RNA Pol II (Fig. 5D) on the due to effects on Asc gene transcription or Asc mRNA stability. To Asc gene were considerably lower in p205-shRNA cells compared further explore how p205 impacted Asc mRNA levels, we per- with the GFP shRNA BMDMs. The recruitment of total RNA Pol II, formed chromatin immunoprecipitation (ChIP) assays to explore serine-5P RNA Pol II, and serine-2P RNA Pol II to the house- the binding of RNA polymerase II (RNA Pol II) to the endogenous keeping gene Gapdh was equivalent between the GFP shRNA and

Asc promoter in the GFP shRNA and p205-KD BMDMs. Fig. 5A p205-KD macrophages (Fig. 5E). Downloaded from shows a schematic diagram of the Asc gene locus and the location Although p205-KD BMDMs showed reduced levels of RNA of the primers used to evaluate RNA Pol II binding. Using Abs to Pol II recruited at the endogenous loci, the levels of Asc mRNA RNA Pol II and an isotype-control IgG, we evaluated RNA Pol II appeared to be affected more severely than the ChIP data indicated. binding to the endogenous Asc promoter. RNA Pol II binding was Recent studies have shown that RNA Pol II is also a major player considerably lower in the p205-KD cell lines using a series of in successful mRNA processing and splicing. Transcription and http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Reconstitution of p205 rescues Asc expression and inﬂammasome activation. p205 (A) and Asc (B) mRNA expression levels relative to b-actin and normalized to GFP shRNA BMDMs were measured in BMDMs reconstituted with p205. (C) Western blot analysis of nuclear and cytoplasmic fractions of p205-reconstituted cell lines to detect p205-HA, histone H3, Asc, and Aim2. (D) p205 and Asc mRNA levels by qPCR relative to b-actin in p205-overexpressing BMDMs. The rescued cells were tested for IL-1b production (E) and cell death (F) with LPS (200 ng/ml for 3 h) and pdAdT (1, 2, or 3 mg/ml for 6 h) or ATP (5 mM for 1 h) by ELISA and by measuring the amount of LDH released, respectively. *p , 0.05, **p , 0.001, ***p , 0.005, ****p , 0.0001. n.s., not signiﬁcant. 3256 p205 REGULATES INFLAMMASOME ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. p205 modulates transcription and splicing from the endogenous Asc gene. (A) Schematic diagram of the Asc gene locus and ChIP primer locations on the gene. (B) Recruitment of total RNA Pol II to the endogenous Asc gene in GFP shRNA CTL, p205-KD 39UTR, and KD CDS macrophages. Recruitment of serine-5P RNA Pol II (C) and serine-2P RNA Pol II (D) on the endogenous Asc gene. (E) Recruitment of total RNA Pol II, serine-5P RNA Pol II, and serine-2P RNA Pol II to the Gapdh transcription start site (TSS). All values are represented as the percentage fraction of total input DNA. Data were calculated against the IgG isotype control and are representative of three independent experiments. (F) Quantitative RT-PCR analysis of p205, nascent, or mature Asc mRNA expression (relative to Hprt and Gapdh and normalized to EV CTL BMDMs) in p205-KD macrophages. *p , 0.05, **p , 0.001, ***p , 0.005, ****p , 0.0001. CTL, control; EV, empty vector; n.s., not significant. splicing are dependent on each other, a phenomenon that is called reporter gene. This effect was specific to p205, because trans- cotranscriptional splicing (34). Hence, to understand whether p205 fection of two related PYHIN proteins, p204 and Aim2, had no was regulating transcription and mRNA processing of the Asc effect (Fig. 6A). We also determined the role of specific domains transcript, we measured the levels of Asc pre-mRNA, as well as of p205 on reporter gene activity by generating and testing deletion that of the processed and mature mRNA, in cells with and without mutants of p205 that lacked the pyrin domain, the HIN domain, or p205. By designing primers that specifically measure pre-mRNA both domains. p205 mutants lacking the HIN or PYD domain failed and mature mRNA (Supplemental Table I), we found that very to induce expression from the Asc promoter (Fig. 6B), indicating low levels of Asc pre-mRNA and little mature Asc mRNA were that the HIN and PYD domains were important. being made in p205-KD BMDMs (Fig. 5F). We conclude from Analysis of the Asc promoter for binding sites for transcription these data that p205 controls Asc expression at the level of gene factors revealed putative binding sites for c/EBPb and NF-kB among transcription, as well as processing of mRNA. others. Parsons and Vertino (35) had shown that TNF-a treatment could enhance Asc expression through p65/RelA in MCF7 cell lines. p205 drives expression from an Asc promoter-reporter Furthermore, p205 has been shown to colocalize and bind directly construct in a dose-dependent manner with the c/EBPb in mouse adipose-derived stem cells (27). Fur- To gain further insight into the mechanisms involved in the tran- thermore, c/EBPb has also been implicated in the induction of scription of Asc, we cloned the Asc promoter (22000 to +10 Asc several proinflammatory genes in macrophages (36). Therefore, gene) as a reporter gene upstream of a firefly luciferase gene. This we tested the effect of ectopic expression of c/EBPb or p65/RelA promoter-reporter construct was transfected into HEK 293T cells on Asc reporter gene expression. In both cases, these transcription together with increasing concentrations of p205. Ectopic expres- factors increased Asc luciferase reporter gene expression in a dose- sion of p205 led to a dose-dependent increase in the Asc-luciferase dependent manner (Fig. 6C, left panel). In addition, coexpressing The Journal of Immunology 3257 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. p205 drives expression from an Asc promoter-luciferase reporter construct. HEK 293T cells were transfected with a mixture containing 10 ng of TK-Renilla luciferase with 1 ng of Asc promoter firefly luciferase reporter and increasing amounts of p205-HA, p204-HA, or Aim2-FLAG (A) or with full-length p205 or p205-deletion mutants (B), as indicated. (C) Transfection of increasing concentrations of c/EBPb, p65/RelA, and p205 alone, or cotransfection of increasing concentrations of p205-HA with c/EBPb or p65/RelA with Asc promoter-reporter or, transfection of the Asc promoter-reporter with increasing concentrations of p205 with c/EBPb and p65/RelA, as indicated. All luciferase values were measured and normalized to Renilla values. Values are displayed as fold change over the Asc reporter construct alone. Data are representative of three independent experiments. (D) Coimmuno- precipitation (I.P.) and immunoblot (I.B.) of overexpressed HA-tagged p205 and c/EBPb in HEK 293T cells using anti-HA and anti-c/EBPb Abs. (E) Coimmunoprecipitation and Western blot of endogenous p205 and c/EBPb in LPS-stimulated BMDMs using Abs against p205 and c/EBPb. Data are representative of two independent experiments (n.s., nonspecific band). *p , 0.05, **p , 0.001, ****p , 0.0001. n.s., not significant. p205 with c/EBPb or p65/RelA further enhanced Asc reporter gene of p205 were transfected into B16 melanoma cells to generate p205- expression (Fig. 6C, middle panel). Transfection of c/EBPb and KO cells. A clone of these cells was produced and evaluated for p65/RelA, together with increasing concentrations of p205, further p205 deficiency using sequencing, qPCR, and Western blot. Levels synergized to drive more robust expression from the Asc gene re- of p205 mRNA following overnight IFN-g stimulation were unde- porter (Fig. 6C, right panel). Furthermore, transiently transfected tectable in these cells (Fig. 7A). In addition, there was no discernible HA-tagged p205 was found to interact with overexpressed c/EBPb p205 protein expression in p205-KO cells stimulated with SV (Fig. in HEK 293T cells (Fig. 6D), as well as endogenously, in resting 7B). Similar to what we had observed in our macrophage system, we and LPS-stimulated (200 ng/ml for 3 or 6 h) wild-type macrophages found that cells lacking p205 had a loss of mature Asc mRNA and (Fig. 6E) by coimmunoprecipitation assays. Together, these obser- protein (Fig. 7C, 7D). These observations further support our shRNA- vations indicate that p205 can increase Asc reporter gene activation based studies and underscore the importance of p205 as a regulator of and can collaborate with c/EBPb or p65/RelA to do so. Asc expression.

KO of p205 using CRIPSR/Cas9 impairs Asc expression Discussion Having defined p205 as a regulator of Asc gene expression using Studies over the last decade have defined the importance of AIM2, shRNA–based KD approaches, we also wanted to evaluate the impact a cytosolic DNA-binding protein, as a regulator of caspase-1 activity of p205 on Asc gene expression using an independent strategy and in and proteolytic processing of IL-1b and IL-18 (37). The role of the a different cell type that expresses p205. We used B16 melanoma PYHINs in sensing dsDNA was further highlighted by the identifi- cell lines to generate CRISPR/Cas9-mediated KO of p205. Plasmids cation of IFI16 as a regulator of type I IFN gene transcription expressing Cas9 and the short guide RNA targeting specific regions following HSV-1 infection (8). 3258 p205 REGULATES INFLAMMASOME ACTIVATION

Early studies had also linked members of the murine PYHIN inflammasome. The abrogated inflammasome activation in p205-KD family to type I IFN gene regulation. KD of mouse Ifi203 and macrophages upon pdAdT stimulation also likely explained the en- mouse Ifi204 have been shown to dampen the IFN response to hanced IFN-b levels detected in these cells, because prior studies infection with multiple pathogens, including HSV-1, HIV, murine from our laboratory (14) and other investigators (48) have shown that leukemia virus, Francisella tularensis, and Mycobacterium tu- activation of the Aim2 inflammasome by intracellular DNA antag- berculosis (8, 38, 39). Despite these studies, a recent genetic study onizes the type I IFN pathway. In cells with defects in inflammasome by Gray et al. (23) using mice lacking the entire PYHIN locus responses, there is a more robust dsDNA-driven induction of type I (lacking all 13 PYHIN genes) found that there was no change in IFNs. the IFN signature when challenged with various immunostimu- By carefully measuring expression levels of key components of latory DNA ligands, DNA virus infection, and lentivirus infection. the Aim2 and Nlrp3 pathways, we found that cells lacking p205 had This body of work indicates that the murine PYHIN proteins play reduced expression of Asc. This effect was observed at the protein limited roles in DNA ligand recognition, at least in the myeloid and mRNA levels. The compromised inflammasome-dependent cells and fibroblasts tested in these studies, raising the possibility responses observed in these cells could be fully rescued by ec- that the PYHINs have alternative functions in these cells. topically expressing either p205 or Asc itself. Cells lacking p205 Prior to the discovery of AIM2 and IFI16 as sensors of microbial had reduced RNA Pol II binding to the Asc gene, indicating that DNA, PYHIN proteins were shown to regulate cell growth, dif- p205 functioned in part to control Asc gene transcription. In ad- ferentiation, tumor suppression, and DNA damage responses (25). dition, by comparing the levels of Asc pre-mRNA with those of IFI16, IFIX, p202, and p204 regulate cell cycle transcription factors, the mature transcript, we could observe additional effects on Asc such as p53, p21, pRb, and E2F, resulting in cell cycle arrest (40). mRNA processing. It is broadly accepted that the CTD of RNA Downloaded from p202 acts as a transcriptional repressor targeting NF-kB (41), AP-1 Pol II is involved in efficient transcription, as well as mRNA (42, 43), MYOD1 (44), and myogenin (19, 43). p204 also regulates processing (34), and the serine 2 phosphorylated polymerase de- gene expression during monocyte/macrophage differentiation and termines the rate of mRNA elongation, spliceosome assembly, and osteoblast differentiation (45–47). splicing efficiency (49). Thus, we deduced from our ChIP exper- In this article, we expand upon these studies and define p205, a iments and qPCR analysis of Asc pre-mRNA and mature tran-

murine PYHIN protein, as an additional regulator of gene ex- scripts that the absence of p205 affects Asc gene transcription, as http://www.jimmunol.org/ pression in innate immunity. Using a series of loss-of-function well as the processing of the immature Asc mRNA. approaches (shRNA and CRISPR/Cas9-mediated gene editing), Further, we show that, in regulating gene expression, p205 co- we report that p205 regulates expression of the inflammasome adapter operates with c/EBPb and p65/RelA to drive Asc expression. The protein Asc and in doing so controls inflammasome-activation path- transcription factor c/EBPb is important in controlling macrophage ways broadly. Cells lacking p205 failed to activate caspase-1 and differentiation. c/EBPb remains fully active in resting macrophages inflammasome-dependent processing of IL-1b in response to multiple and stays positioned on target genes, ready to stimulate transcription ligands that engage the Aim2 inflammasome, as well as the Nlrp3 with other inducible transcription factors (36). p205 has previously by guest on September 29, 2021

FIGURE 7. CRISPR KO of p205 leads to reduced Asc expression in B16 melanoma. (A) Expression of p205 mRNA in IFN-stimulated wild-type (WT) and p205-KO B16 melanoma cell lines relative to Hprt and normalized to nontreated (NT). (B) p205 and b-actin protein expression in WT and p205-KO cell lines stimulated with SV (n.s., nonspecific band). Mature mRNA and pre-mRNA profile of Asc expression (relative to Hprt and normalized to WT) (C) and Western blot analysis of Asc and b-actin (D) in WT and p205-KO B16 melanoma cell lines. **p , 0.001, ***p , 0.005, ****p , 0.0001. n.s., not significant. The Journal of Immunology 3259 been shown to interact with c/EBPb in adipocytes. p205 also in- 11. Burckst€ ummer,€ T., C. Baumann, S. Bluml,€ E. Dixit, G. Durnberger,€ H. Jahn, b M. Planyavsky, M. Bilban, J. Colinge, K. L. Bennett, and G. Superti-Furga. teracts with c/EBP in unstimulated macrophages and maintains 2009. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplas- basal expression of Asc, whereas under stimulated conditions, LPS- mic DNA sensor for the inflammasome. Nat. Immunol. 10: 266–272. inducible p205 may interact with c/EBPb and activated NF-kB 12. Fernandes-Alnemri, T., J. W. Yu, P. Datta, J. Wu, and E. S. Alnemri. 2009. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. transcription factors to further enhance expression. Nature 458: 509–513. Previous studies have shown that ASC, originally identified as 13. Roberts, T. L., A. Idris, J. A. Dunn, G. M. Kelly, C. M. Burnton, S. Hodgson, target of methylation induced silencing-1, is influenced by DNA L. L. Hardy, V. Garceau, M. J. Sweet, I. L. Ross, et al. 2009. HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA. Science methylation. The ASC gene contains a 600-bp-long CpG island 323: 1057–1060. located near the transcription start site, and the methylation status 14. Rathinam, V. A., Z. Jiang, S. N. Waggoner, S. Sharma, L. E. Cole, L. Waggoner, of this CpG island correlates with the expression level of ASC/ S. K. Vanaja, B. G. Monks, S. Ganesan, E. Latz, et al. 2010. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA target of methylation induced silencing-1. The methylation status viruses. Nat. Immunol. 11: 395–402. of the CpG island, as well as promoter proximal pausing of RNA 15. Fernandes-Alnemri, T., J. W. Yu, C. Juliana, L. Solorzano, S. Kang, J. Wu, Pol II in multiple cancer cell lines and tissues, results in reduced P. Datta, M. McCormick, L. Huang, E. McDermott, et al. 2010. The AIM2 inflammasome is critical for innate immunity to Francisella tularensis. Nat. expression of ASC (50–58). Our studies indicate that Asc levels Immunol. 11: 385–393. are influenced by p205 in murine macrophages, adding additional 16. Baum, R., S. Sharma, S. Carpenter, Q. Z. Li, P. Busto, K. A. Fitzgerald, A. Marshak-Rothstein, and E. M. Gravallese. 2015. Cutting edge: AIM2 and understanding to the regulation of this important immune gene. endosomal TLRs differentially regulate arthritis and autoantibody production in Our findings expand on our understanding of transcriptional DNase II-deficient mice. J. Immunol. 194: 873–877. regulatory roles for the PYHIN proteins. p205 has previously 17. Kerur, N., M. V. Veettil, N. Sharma-Walia, V. Bottero, S. Sadagopan, P. Otageri, and B. Chandran. 2011. IFI16 acts as a nuclear pathogen sensor to induce the

CIP/WAF Downloaded from been shown to control p21 gene expression via p53 in inflammasome in response to Kaposi sarcoma-associated herpesvirus infection. Saos2 cell lines and to impact gene expression in adipogenesis Cell Host Microbe 9: 363–375. and osteogenesis via transcriptional mechanisms. p205 acts as a 18. Monroe, K. M., Z. Yang, J. R. Johnson, X. Geng, G. Doitsh, N. J. Krogan, and W. C. Greene. 2014. IFI16 DNA sensor is required for death of lymphoid CD4 transactivator synergizing with p53 to induce expression of the T cells abortively infected with HIV. Science 343: 428–432. cell cycle inhibitor p21 to inhibit cell growth. In adipocytes, p205 19. Schattgen, S. A., and K. A. Fitzgerald. 2011. The PYHIN protein family as interacts with c/EBPb and c/EBPa to further activate the tran- mediators of host defenses. Immunol. Rev. 243: 109–118. 20. Cridland, J. A., E. Z. Curley, M. N. Wykes, K. Schroder, M. J. Sweet, scriptional activity of c/EBPa and PPARg. All of these studies T. L. 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the AIM2 inﬂammasome by intracellular DNA. J. Immunol. 196: 3191–3198. Int. J. Cancer 106: 198–204. http://www.jimmunol.org/ by guest on September 29, 2021