The protozoan parasite, Toxoplasma, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host–pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite Toxoplasma results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such “modifiers.”
References
[1]
Dalton DK, Pitts-Meek S, Keshav S, Figari IS, Bradley A, et al. (1993) Multiple defects of immune cell function in mice with disrupted interferon-gamma genes. Science 259: 1739–1742. pmid:8456300 doi: 10.1126/science.8456300
[2]
Durbin JE, Hackenmiller R, Simon MC, Levy DE (1996) Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 84: 443–450. pmid:8608598 doi: 10.1016/s0092-8674(00)81289-1
[3]
Meraz MA, White JM, Sheehan KC, Bach EA, Rodig SJ, et al. (1996) Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84: 431–442. pmid:8608597 doi: 10.1016/s0092-8674(00)81288-x
[4]
Newport MJ, Huxley CM, Huston S, Hawrylowicz CM, Oostra BA, et al. (1996) A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection. N Engl J Med 335: 1941–1949. pmid:8960473 doi: 10.1056/nejm199612263352602
[5]
Dupuis S, Jouanguy E, Al-Hajjar S, Fieschi C, Al-Mohsen IZ, et al. (2003) Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1 deficiency. Nat Genet 33: 388–391. pmid:12590259 doi: 10.1038/ng1097
[6]
Reich NC, Liu L (2006) Tracking STAT nuclear traffic. Nat Rev Immunol 6: 602–612. pmid:16868551 doi: 10.1038/nri1885
[7]
Muller M, Briscoe J, Laxton C, Guschin D, Ziemiecki A, et al. (1993) The protein tyrosine kinase JAK1 complements defects in interferon-alpha/beta and-gamma signal transduction. Nature 366: 129–135. pmid:8232552 doi: 10.1038/366129a0
[8]
Shuai K, Ziemiecki A, Wilks AF, Harpur AG, Sadowski HB, et al. (1993) Polypeptide signalling to the nucleus through tyrosine phosphorylation of Jak and Stat proteins. Nature 366: 580–583. pmid:7504784 doi: 10.1038/366580a0
[9]
Ehrt S, Schnappinger D, Bekiranov S, Drenkow J, Shi S, et al. (2001) Reprogramming of the macrophage transcriptome in response to interferon-gamma and Mycobacterium tuberculosis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase. J Exp Med 194: 1123–1140. pmid:11602641 doi: 10.1084/jem.194.8.1123
[10]
Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD (1998) How cells respond to interferons. Annu Rev Biochem 67: 227–264. pmid:9759489 doi: 10.1146/annurev.biochem.67.1.227
[11]
Raefsky EL, Platanias LC, Zoumbos NC, Young NS (1985) Studies of interferon as a regulator of hematopoietic cell proliferation. J Immunol 135: 2507–2512. pmid:2411798
[12]
Zoumbos NC, Gascon P, Djeu JY, Young NS (1985) Interferon is a mediator of hematopoietic suppression in aplastic anemia in vitro and possibly in vivo. Proc Natl Acad Sci U S A 82: 188–192. pmid:3918301 doi: 10.1073/pnas.82.1.188
[13]
Baldridge MT, King KY, Boles NC, Weksberg DC, Goodell MA (2010) Quiescent haematopoietic stem cells are activated by IFN-gamma in response to chronic infection. Nature 465: 793–797. doi: 10.1038/nature09135. pmid:20535209
[14]
Kusmartsev S, Gabrilovich DI (2005) STAT1 signaling regulates tumor-associated macrophage-mediated T cell deletion. J Immunol 174: 4880–4891. pmid:15814715 doi: 10.4049/jimmunol.174.8.4880
[15]
Afkarian M, Sedy JR, Yang J, Jacobson NG, Cereb N, et al. (2002) T-bet is a STAT1-induced regulator of IL-12R expression in naive CD4+ T cells. Nat Immunol 3: 549–557. pmid:12006974 doi: 10.1038/ni794
[16]
Sawitzki B, Kingsley CI, Oliveira V, Karim M, Herber M, et al. (2005) IFN-gamma production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo. J Exp Med 201: 1925–1935. pmid:15967822 doi: 10.1084/jem.20050419
[17]
Koch MA, Tucker-Heard G, Perdue NR, Killebrew JR, Urdahl KB, et al. (2009) The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat Immunol 10: 595–602. doi: 10.1038/ni.1731. pmid:19412181
[18]
Yap GS, Sher A (1999) Effector cells of both nonhemopoietic and hemopoietic origin are required for interferon (IFN)-gamma- and tumor necrosis factor (TNF)-alpha-dependent host resistance to the intracellular pathogen, Toxoplasma gondii. J Exp Med 189: 1083–1092. pmid:10190899 doi: 10.1084/jem.189.7.1083
[19]
Suzuki Y, Orellana MA, Schreiber RD, Remington JS (1988) Interferon-gamma: the major mediator of resistance against Toxoplasma gondii. Science 240: 516–518. pmid:3128869 doi: 10.1126/science.3128869
[20]
Gavrilescu LC, Butcher BA, Del Rio L, Taylor GA, Denkers EY (2004) STAT1 is essential for antimicrobial effector function but dispensable for gamma interferon production during Toxoplasma gondii infection. Infect Immun 72: 1257–1264. pmid:14977926 doi: 10.1128/iai.72.3.1257-1264.2004
[21]
Lieberman LA, Banica M, Reiner SL, Hunter CA (2004) STAT1 plays a critical role in the regulation of antimicrobial effector mechanisms, but not in the development of Th1-type responses during toxoplasmosis. J Immunol 172: 457–463. pmid:14688355 doi: 10.4049/jimmunol.172.1.457
[22]
Gazzinelli R, Xu Y, Hieny S, Cheever A, Sher A (1992) Simultaneous depletion of CD4+ and CD8+ T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii. J Immunol 149: 175–180. pmid:1351500
[23]
Suzuki Y, Conley FK, Remington JS (1989) Importance of endogenous IFN-gamma for prevention of toxoplasmic encephalitis in mice. J Immunol 143: 2045–2050. pmid:2506275
[24]
Jones TC, Bienz KA, Erb P (1986) In vitro cultivation of Toxoplasma gondii cysts in astrocytes in the presence of gamma interferon. Infect Immun 51: 147–156. pmid:3079728
[25]
Nandan D, Reiner NE (1995) Attenuation of gamma interferon-induced tyrosine phosphorylation in mononuclear phagocytes infected with Leishmania donovani: selective inhibition of signaling through Janus kinases and Stat1. Infect Immun 63: 4495–4500. pmid:7591091
[26]
Ting LM, Kim AC, Cattamanchi A, Ernst JD (1999) Mycobacterium tuberculosis inhibits IFN-gamma transcriptional responses without inhibiting activation of STAT1. J Immunol 163: 3898–3906. pmid:10490990
[27]
Rayamajhi M, Humann J, Penheiter K, Andreasen K, Lenz LL (2010) Induction of IFN-alphabeta enables Listeria monocytogenes to suppress macrophage activation by IFN-gamma. J Exp Med 207: 327–337. doi: 10.1084/jem.20091746. pmid:20123961
[28]
Sakala IG, Chaudhri G, Buller RM, Nuara AA, Bai H, et al. (2007) Poxvirus-encoded gamma interferon binding protein dampens the host immune response to infection. J Virol 81: 3346–3353. pmid:17229697 doi: 10.1128/jvi.01927-06
[29]
Kim SK, Fouts AE, Boothroyd JC (2007) Toxoplasma gondii dysregulates IFN-gamma-inducible gene expression in human fibroblasts: insights from a genome-wide transcriptional profiling. J Immunol 178: 5154–5165. pmid:17404298 doi: 10.4049/jimmunol.178.8.5154
[30]
Luder CG, Walter W, Beuerle B, Maeurer MJ, Gross U (2001) Toxoplasma gondii down-regulates MHC class II gene expression and antigen presentation by murine macrophages via interference with nuclear translocation of STAT1alpha. Eur J Immunol 31: 1475–1484. pmid:11465104 doi: 10.1002/1521-4141(200105)31:5<1475::aid-immu1475>3.0.co;2-c
[31]
Rosowski EE, Saeij JP (2012) Toxoplasma gondii clonal strains all inhibit STAT1 transcriptional activity but polymorphic effectors differentially modulate IFNgamma induced gene expression and STAT1 phosphorylation. PLoS One 7: e51448. doi: 10.1371/journal.pone.0051448. pmid:23240025
[32]
Zimmermann S, Murray PJ, Heeg K, Dalpke AH (2006) Induction of suppressor of cytokine signaling-1 by Toxoplasma gondii contributes to immune evasion in macrophages by blocking IFN-gamma signaling. J Immunol 176: 1840–1847. pmid:16424215 doi: 10.4049/jimmunol.176.3.1840
[33]
Lang C, Hildebrandt A, Brand F, Opitz L, Dihazi H, et al. (2012) Impaired chromatin remodelling at STAT1-regulated promoters leads to global unresponsiveness of Toxoplasma gondii-Infected macrophages to IFN-gamma. PLoS Pathog 8: e1002483. doi: 10.1371/journal.ppat.1002483. pmid:22275866
[34]
Zhang JH, Chung TD, Oldenburg KR (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4: 67–73. pmid:10838414 doi: 10.1177/108705719900400206
[35]
Temple G, Gerhard DS, Rasooly R, Feingold EA, Good PJ, et al. (2009) The completion of the Mammalian Gene Collection (MGC). Genome Res 19: 2324–2333. doi: 10.1101/gr.095976.109. pmid:19767417
[36]
Liu B, Liao J, Rao X, Kushner SA, Chung CD, et al. (1998) Inhibition of Stat1-mediated gene activation by PIAS1. Proc Natl Acad Sci U S A 95: 10626–10631. pmid:9724754 doi: 10.1073/pnas.95.18.10626
[37]
Zhang JJ, Vinkemeier U, Gu W, Chakravarti D, Horvath CM, et al. (1996) Two contact regions between Stat1 and CBP/p300 in interferon gamma signaling. Proc Natl Acad Sci U S A 93: 15092–15096. pmid:8986769 doi: 10.1073/pnas.93.26.15092
[38]
Gaji RY, Huynh MH, Carruthers VB (2013) A novel high throughput invasion screen identifies host actin regulators required for efficient cell entry by Toxoplasma gondii. PLoS One 8: e64693. doi: 10.1371/journal.pone.0064693. pmid:23741372
[39]
Saeij JP, Boyle JP, Grigg ME, Arrizabalaga G, Boothroyd JC (2005) Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains. Infect Immun 73: 695–702. pmid:15664907 doi: 10.1128/iai.73.2.695-702.2005
[40]
Kroger A, Koster M, Schroeder K, Hauser H, Mueller PP (2002) Activities of IRF-1. J Interferon Cytokine Res 22: 5–14. pmid:11846971 doi: 10.1089/107999002753452610
[41]
Shi Y, Chichung Lie D, Taupin P, Nakashima K, Ray J, et al. (2004) Expression and function of orphan nuclear receptor TLX in adult neural stem cells. Nature 427: 78–83. pmid:14702088 doi: 10.1038/nature02211
[42]
Xu Z, Yu S, Hsu CH, Eguchi J, Rosen ED (2008) The orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II is a critical regulator of adipogenesis. Proc Natl Acad Sci U S A 105: 2421–2426. doi: 10.1073/pnas.0707082105. pmid:18250317
[43]
You LR, Lin FJ, Lee CT, DeMayo FJ, Tsai MJ, et al. (2005) Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity. Nature 435: 98–104. pmid:15875024 doi: 10.1038/nature03511
[44]
Joseph B, Wallen-Mackenzie A, Benoit G, Murata T, Joodmardi E, et al. (2003) p57(Kip2) cooperates with Nurr1 in developing dopamine cells. Proc Natl Acad Sci U S A 100: 15619–15624. pmid:14671317 doi: 10.1073/pnas.2635658100
[45]
Ross DT, Scherf U, Eisen MB, Perou CM, Rees C, et al. (2000) Systematic variation in gene expression patterns in human cancer cell lines. Nat Genet 24: 227–235. pmid:10700174 doi: 10.1038/73432
[46]
Holbeck S, Chang J, Best AM, Bookout AL, Mangelsdorf DJ, et al. (2010) Expression profiling of nuclear receptors in the NCI60 cancer cell panel reveals receptor-drug and receptor-gene interactions. Mol Endocrinol 24: 1287–1296. doi: 10.1210/me.2010-0040. pmid:20375240
[47]
Zhang CL, Zou Y, He W, Gage FH, Evans RM (2008) A role for adult TLX-positive neural stem cells in learning and behaviour. Nature 451: 1004–1007. doi: 10.1038/nature06562. pmid:18235445
[48]
Wang X, Zhao Y, Zhang X, Badie H, Zhou Y, et al. (2013) Loss of sorting nexin 27 contributes to excitatory synaptic dysfunction by modulating glutamate receptor recycling in Down's syndrome. Nat Med 19: 473–480. doi: 10.1038/nm.3117. pmid:23524343
[49]
Aranda A, Pascual A (2001) Nuclear hormone receptors and gene expression. Physiol Rev 81: 1269–1304. pmid:11427696
[50]
Benod C, Villagomez R, Filgueira CS, Hwang PK, Leonard PG, et al. (2014) The human orphan nuclear receptor tailless (TLX, NR2E1) is druggable. PLoS One 9: e99440. doi: 10.1371/journal.pone.0099440. pmid:24936658
[51]
Robertson G, Hirst M, Bainbridge M, Bilenky M, Zhao Y, et al. (2007) Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods 4: 651–657. pmid:17558387 doi: 10.1038/nmeth1068
[52]
Mashayekhi M, Sandau MM, Dunay IR, Frickel EM, Khan A, et al. (2011) CD8alpha(+) dendritic cells are the critical source of interleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites. Immunity 35: 249–259. doi: 10.1016/j.immuni.2011.08.008. pmid:21867928
[53]
Rosas LE, Keiser T, Pyles R, Durbin J, Satoskar AR (2003) Development of protective immunity against cutaneous leishmaniasis is dependent on STAT1-mediated IFN signaling pathway. Eur J Immunol 33: 1799–1805. pmid:12811839 doi: 10.1002/eji.200323163
[54]
Natoli G (2009) Control of NF-kappaB-dependent transcriptional responses by chromatin organization. Cold Spring Harb Perspect Biol 1: a000224. doi: 10.1101/cshperspect.a000224. pmid:20066094
[55]
Murphy LO, Smith S, Chen RH, Fingar DC, Blenis J (2002) Molecular interpretation of ERK signal duration by immediate early gene products. Nat Cell Biol 4: 556–564. pmid:12134156 doi: 10.1038/ncb822
[56]
Metivier R, Penot G, Hubner MR, Reid G, Brand H, et al. (2003) Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115: 751–763. pmid:14675539 doi: 10.1016/s0092-8674(03)00934-6
[57]
Mullen AC, Orlando DA, Newman JJ, Loven J, Kumar RM, et al. (2011) Master transcription factors determine cell-type-specific responses to TGF-beta signaling. Cell 147: 565–576. doi: 10.1016/j.cell.2011.08.050. pmid:22036565
[58]
Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P (2003) Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med 9: 213–219. pmid:12524534 doi: 10.1038/nm820
[59]
Pascual-Garcia M, Rue L, Leon T, Julve J, Carbo JM, et al. (2013) Reciprocal negative cross-talk between liver X receptors (LXRs) and STAT1: effects on IFN-gamma-induced inflammatory responses and LXR-dependent gene expression. J Immunol 190: 6520–6532. doi: 10.4049/jimmunol.1201393. pmid:23686490
[60]
Lee JH, Park SM, Kim OS, Lee CS, Woo JH, et al. (2009) Differential SUMOylation of LXRalpha and LXRbeta mediates transrepression of STAT1 inflammatory signaling in IFN-gamma-stimulated brain astrocytes. Mol Cell 35: 806–817. doi: 10.1016/j.molcel.2009.07.021. pmid:19782030
[61]
Li S, Sun G, Murai K, Ye P, Shi Y (2012) Characterization of TLX expression in neural stem cells and progenitor cells in adult brains. PLoS One 7: e43324. doi: 10.1371/journal.pone.0043324. pmid:22952666
[62]
Schote AB, Turner JD, Schiltz J, Muller CP (2007) Nuclear receptors in human immune cells: expression and correlations. Mol Immunol 44: 1436–1445. pmid:16837048 doi: 10.1016/j.molimm.2006.04.021
[63]
Muhlethaler-Mottet A, Di Berardino W, Otten LA, Mach B (1998) Activation of the MHC class II transactivator CIITA by interferon-gamma requires cooperative interaction between Stat1 and USF-1. Immunity 8: 157–166. pmid:9491997 doi: 10.1016/s1074-7613(00)80468-9
[64]
Al-Kandari W, Koneni R, Navalgund V, Aleksandrova A, Jambunathan S, et al. (2007) The zinc finger proteins ZXDA and ZXDC form a complex that binds CIITA and regulates MHC II gene transcription. J Mol Biol 369: 1175–1187. pmid:17493635 doi: 10.1016/j.jmb.2007.04.033
[65]
Al-Kandari W, Jambunathan S, Navalgund V, Koneni R, Freer M, et al. (2007) ZXDC, a novel zinc finger protein that binds CIITA and activates MHC gene transcription. Mol Immunol 44: 311–321. pmid:16600381 doi: 10.1016/j.molimm.2006.02.029
[66]
Zhong G, Fan T, Liu L (1999) Chlamydia inhibits interferon gamma-inducible major histocompatibility complex class II expression by degradation of upstream stimulatory factor 1. J Exp Med 189: 1931–1938. pmid:10377188 doi: 10.1084/jem.189.12.1931
[67]
Zhong G, Fan P, Ji H, Dong F, Huang Y (2001) Identification of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J Exp Med 193: 935–942. pmid:11304554 doi: 10.1084/jem.193.8.935
[68]
Jayaraman G, Srinivas R, Duggan C, Ferreira E, Swaminathan S, et al. (1999) p300/cAMP-responsive element-binding protein interactions with ets-1 and ets-2 in the transcriptional activation of the human stromelysin promoter. J Biol Chem 274: 17342–17352. pmid:10358095 doi: 10.1074/jbc.274.24.17342
[69]
Donald RG, Roos DS (1994) Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii. Mol Biochem Parasitol 63: 243–253. pmid:8008022 doi: 10.1016/0166-6851(94)90060-4
[70]
Chanda SK, White S, Orth AP, Reisdorph R, Miraglia L, et al. (2003) Genome-scale functional profiling of the mammalian AP-1 signaling pathway. Proc Natl Acad Sci U S A 100: 12153–12158. pmid:14514886 doi: 10.1073/pnas.1934839100
[71]
Du P, Kibbe WA, Lin SM (2008) lumi: a pipeline for processing Illumina microarray. Bioinformatics 24: 1547–1548. doi: 10.1093/bioinformatics/btn224. pmid:18467348
[72]
Smyth GK (2005) limma: Linear Models for Microarray Data. In: Gentleman R, Carey V, Huber W, Irizarry R, Dudoit S, editors. Bioinformatics and Computational Biology Solutions Using R and Bioconductor: Springer New York. pp. 397–420.
[73]
Smyth GK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3: Article3. doi: 10.2202/1544-6115.1027
[74]
Reiner A, Yekutieli D, Benjamini Y (2003) Identifying differentially expressed genes using false discovery rate controlling procedures. Bioinformatics 19: 368–375. pmid:12584122 doi: 10.1093/bioinformatics/btf877
[75]
Benjamini Y, Hochberg Y (1995) Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society Series B (Methodological) 57: 289–300.
[76]
Dennis G Jr., Sherman BT, Hosack DA, Yang J, Gao W, et al. (2003) DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 4: P3. pmid:12734009 doi: 10.1186/gb-2003-4-5-p3
[77]
Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4: 44–57. doi: 10.1038/nprot.2008.211. pmid:19131956
[78]
Finn RD, Mistry J, Tate J, Coggill P, Heger A, et al. (2010) The Pfam protein families database. Nucleic Acids Res 38: D211–222. doi: 10.1093/nar/gkp985. pmid:19920124
[79]
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, et al. (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13: 2498–2504. pmid:14597658 doi: 10.1101/gr.1239303
[80]
Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, et al. (2007) Integration of biological networks and gene expression data using Cytoscape. Nat Protoc 2: 2366–2382. pmid:17947979 doi: 10.1038/nprot.2007.324
[81]
Gao J, Ade AS, Tarcea VG, Weymouth TE, Mirel BR, et al. (2009) Integrating and annotating the interactome using the MiMI plugin for cytoscape. Bioinformatics 25: 137–138. doi: 10.1093/bioinformatics/btn501. pmid:18812364
[82]
Birmingham A, Selfors LM, Forster T, Wrobel D, Kennedy CJ, et al. (2009) Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 6: 569–575. doi: 10.1038/nmeth.1351. pmid:19644458
[83]
Wilson EH, Wille-Reece U, Dzierszinski F, Hunter CA (2005) A critical role for IL-10 in limiting inflammation during toxoplasmic encephalitis. J Neuroimmunol 165: 63–74. pmid:16005735 doi: 10.1016/j.jneuroim.2005.04.018
