Disruption of Mutually Negative Regulatory Feedback Loop between Interferon-Inducible p202 and the E2F Family of Transcription Factors in Lupus-Prone Mice This information is current as of September 29, 2021. Ravichandran Panchanathan, Hong Xin and Divaker Choubey J Immunol 2008; 180:5927-5934; ; doi: 10.4049/jimmunol.180.9.5927 http://www.jimmunol.org/content/180/9/5927 Downloaded from

References This article cites 51 articles, 19 of which you can access for free at:

http://www.jimmunol.org/content/180/9/5927.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

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 © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Disruption of Mutually Negative Regulatory Feedback Loop between Interferon-Inducible p202 Protein and the E2F Family of Transcription Factors in Lupus-Prone Mice1

Ravichandran Panchanathan,*† Hong Xin,† and Divaker Choubey2*†

Studies have identified IFN-inducible Ifi202 gene as a lupus susceptibility gene (encoding p202 protein) in mouse models of lupus disease. However, signaling pathways that regulate the Ifi202 expression in cells remain to be elucidated. We found that steady-state levels of Ifi202 mRNA and protein were high in mouse embryonic fibroblasts (MEFs) from knockout (E2F1؊/؊) and E2F1 and double knockout (E2F1؊/؊E2F2؊/؊) mice than isogenic wild-type MEFs. Moreover, overexpression of E2F1 in mouse fibroblasts decreased expression of p202. Furthermore, expression of E2F1, but not , in mouse fibroblasts repressed the activity of 202-luc-reporter in -reporter assays. Interestingly, the E2F1-mediated transcriptional repression of the 202-luc- Downloaded from reporter was independent of and pRb expression. However, the repression was dependent on the ability of E2F1 to bind DNA. We have identified a potential E2F DNA-binding site in the 5؅-regulatory region of the Ifi202 gene, and mutations in this E2F DNA-binding site reduced the E2F1-mediated transcriptional repression of 202-luc-reporter. Because p202 inhibits the E2F1-mediated transcriptional activation of genes, we compared the expression of E2F1 and its target genes in splenic cells from lupus-prone B6.Nba2 congenic mice, which express increased levels of p202, with age-matched C57BL/6 mice. We found that increased expression of Ifi202 in the congenic

mice was associated with inhibition of E2F1-mediated transcription and decreased expression of E2F1 and its target genes that encode http://www.jimmunol.org/ proapoptotic . Our observations support the idea that increased Ifi202 expression in certain strains of mice contributes to lupus susceptibility in part by inhibiting E2F1-mediated functions. The Journal of Immunology, 2008, 180: 5927–5934.

ystemic lupus erythematosus (SLE)3 is an autoimmune disease, signaling pathways that regulate expression of the Ifi202 disease that predominantly affects women of childbearing gene in immune cells remain to be elucidated. Moreover, it re- S age (1–3). The disease is characterized by the production mains unclear how increased expression of Ifi202 in certain strains of pathogenic autoantibodies, particularly IgG Abs, to nuclear Ags of mice contributes to lupus susceptibility. and development of lupus nephritis (1–3). Based on genetic studies The E2F family of transcription factors comprises at least six in human SLE patients and in mouse models of SLE, there is structurally-related E2Fs (E2F1–6) (10–12). Based on their ability by guest on September 29, 2021 considerable evidence that SLE is a polygenic disease (1, 4, 5). to complex with the pocket family proteins (pRb, p107, and p130) Interestingly, studies have revealed that PBMC from lupus patients and their expression patterns, these transcription factors have been exhibit an IFN signature: expression levels of mRNAs that are grouped into three groups. E2F1, E2F2, and have been encoded by the IFN-inducible genes are up-regulated (6). Consis- grouped together, whereas E2F4 and are grouped together. tent with a role for IFN-inducible genes in human lupus patients, The E2F6 is grouped separately. These E2F factors heterodimerize generation of congenic mice, such as B6.Nba2 (7) and with members of the DP family (DP1 and DP2) (10) of proteins to B10.Yaa.Bxs2/3 (8), coupled with gene expression analyses have form an active transcription factor. The E2F and DP protein het- identified the IFN-inducible Ifi202 gene as a lupus susceptibility erodimer is kept transcriptionally silent and acts as a repressor by gene. Furthermore, a study has revealed that increased expression binding to a pocket protein. /Cdk-mediated of Ifi202 in spleen and kidney cells of MRL/lpr mice is associated of pocket proteins results in the release of “free” E2F/DP dimer, with development of lupus disease (9). Although these studies allowing transcriptional activation of E2F target genes (10–12). have suggested that increased expression of Ifi202 in certain strains Transcriptional activation of genes by E2F in response to mito- of mice is associated with the development of lupus or lupus-like genic stimulation and the identification of E2F DNA-binding sites in a number of genes critical to the regulation of DNA synthesis implicate E2F regulation as an important step in mammalian cell *Department of Environmental Health, University of Cincinnati, Cincinnati, OH cycle progression (10–12). Consistent with this idea, the E2F fam- 45267; and †Loyola University, Maywood, IL 60153 ily of transcription factors contributes to the regulation of G1-to-S Received for publication December 11, 2007. Accepted for publication February phase transition of T and B cells in response to mitogenic stimulus 27, 2008. (11, 12). Importantly, cell growth-inhibitory cytokines, such as The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance IFNs, inhibit progression in part by inhibiting the E2F- with 18 U.S.C. Section 1734 solely to indicate this fact. mediated transcription of genes (13, 14). 1 This work was supported by National Institutes of Health Grant AI066261 (to D.C.). Interestingly, the E2F1 transcription factor autoregulates expres- 2 Address correspondence and reprint requests to Dr. Divaker Choubey, Department sion of the E2F1 gene (15), and the lack of E2F1 expression is of Environmental Health, University of Cincinnati, 3223 Eden Avenue, P.O. Box associated with defects in of cells (16). The E2F1 tran- 670056, Cincinnati, OH 45267. E-mail address: [email protected] scriptional target genes encode proteins with functions in cell cycle 3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; MEF, mouse progression and apoptosis (10–12). The E2F1 target genes encode embryonic fibroblast; Rb, . proteins, such as , DHFR, and E2F1, that function in the

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 G1-to- transition during the cell cycle progression (10, 11, www.jimmunol.org 5928 REGULATION AND ROLE OF p202 IN LUPUS SUSCEPTIBILITY

15). Moreover, E2F1 target genes also encode proteins, such as binding site (indicated as 202-E2F-BS) in the 202-luc-reporter plasmid , PUMA, Noxa, and Bim, that are known to have proapoptotic were introduced using the QuikChange site-directed mutagenesis kit (from functions in immune cells (12, 17). Stratagene) as suggested by the supplier. The sequencing of the mutant reporter plasmid confirmed that the nucleotide sequence in the 202-luc- IFN-inducible Ifi202 gene encodes a 52-kDa phosphoprotein reporter plasmid was changed from AAAGGGCGCGAAA to p202 (18–21). Overexpression of p202 protein in a variety of AAAGGTTTCGAAA. mouse fibroblasts retards cell proliferation and increases cell sur- Reporter assays vival, in part by inhibiting E2F-mediated transcription (20, 21). Furthermore, increased expression of p202 in splenic cells of the For reporter assays, subconfluent cultures of cells (in 6-well plates) were ␮ B6.Nba2 congenic mice (congenic for Nba2 locus derived from transfected with the reporter plasmids 202-luc (2.5 g) and pRL-TK (0.5 ␮g) using a calcium phosphate transfection kit (from Invitrogen), as sug- New Zealand Black (NZB) lupus-prone mice) is associated with gested by the supplier. Unless otherwise indicated, cells were harvested increased production of IgG Abs, splenomegaly, and defects in between 43 and 48 h after transfections. Cells were lysed, and the firefly and apoptosis of B cells in vitro (7, 21). Importantly, generation of Renilla luciferase activities were determined as described previously (28). ␣ ␤ B6.Nba2 mice that are deficient for the IFN- / resulted Isolation of RNA from splenocytes and RT-PCR in more than a 2-fold reduction in Ifi202 mRNA levels in splenic cells (22). Consistent with this observation, others have reported Total single-cell splenocytes were isolated from female B6 or B6.Nba2 Ifi202 mice (8–10 wk of age). After lysis of RBC, splenocytes were resuspended (23) that basal levels of mRNA are 3–4-fold higher in in RPMI 1640 with 10% FBS, 1% penicillin/streptomycin/glutamate, and NZB/W (White) mice than BALB/c or MRL/lpr mice. Although 1ϫ MEM, nonessential amino acids/sodium pyruvate. Splenocytes (5 ϫ these observations are consistent with the regulation of Ifi202 gene by 106 cells) were used to isolate total RNA using TRIzol (Invitrogen). Total

IFNs (IFN-␣ or IFN-␤) in immune cells, our studies (24) revealed that RNA was digested with DNase I (to remove any DNA in the preparation), Downloaded from ␮ type I IFN receptor deficiency reduced lupus-like disease in NZB and 0.5 g of RNA was used for RT-PCR reaction using a pair of the Ifi202-specific primers (forward primer: 5Ј-ggtcatctaccaactcagaat-3Ј; re- mice, but did not result in decreased levels of p202 protein. verse primer: 5Ј-ctctaggatg ccactgctgttg-3Ј). For RT-PCR, we used the Our previous studies (25–28) revealed that expression of Ifi202 SuperScript one-step RT-PCR system from Invitrogen. Primers for other is also regulated by IFN-independent signaling pathways. For exam- genes were as follows: mE2F1 (118 bp), forward: 5Ј-GATCGAAGCTT ple, we found that IL-6 up-regulates expression of the Ifi202 gene in TAATGGAGCG-3Ј, reverse: 5Ј-CCCTTGCTTCA GAGAACAG-3Ј; PUMA (211 bp), forward: 5Ј-AGCACTTAGAGT CGCCCGT-3Ј, reverse: mouse fibroblasts and B6.Nba2 splenocytes through activation of 5Ј-GAGGAGT CCCATGAAGAGATTG-3Ј; Bim (244 bp), forward: 5Ј- http://www.jimmunol.org/ transcription factor STAT3 (28). Furthermore, we noted that serum TAAGTTCTGAG TGTGACAGAGA AGG-3Ј, reverse: 5Ј-CAGTTGTA growth factors, such as platelet-derived , basic fibroblast AGATAACCATTTGA GGGTGG-3Ј. growth factor, and TGF-␤, negatively regulate the expression of Immunoblotting Ifi202 (25). However, the molecular mechanisms remain unclear. Because the E2F family of transcription factors contributes to Splenocytes, MEFs, or NIH3T3 cells were collected in PBS and resus- transition from G -to-S phase of cell cycle in response to serum pended in a modified radioimmunoprecipitation assay (RIPA) lysis buffer 1 (50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 1% Nonidet P-40, 0.5% sodium growth factors (10, 11) and because certain E2F family members, deoxycholate, 0.1% SDS), supplemented with protease inhibitors (leupep- such as E2F1 and E2F2, are implicated in the regulation of auto- tin, 50 ␮g/ml; pepstatin A, 50 ␮g/ml; and PMSF, 1 mM) and incubated at immunity (29, 30), we investigated whether the E2F family mem- 4°C for 30 min. Cell lysates were sonicated briefly before centrifugation at by guest on September 29, 2021 bers regulate Ifi202 expression. Herein we report that the E2F fam- 14,000 rpm in a microcentrifuge for 10 min at 4°C. The supernatants were collected, and the protein concentration was measured by Bio-Rad protein ily members differentially regulate expression of the Ifi202 gene. assay kit. Equal amounts of protein were processed for immunoblotting as described previously (28). Materials and Methods EMSAs Cells, cell culture, and reagents Nuclear extract was prepared from mouse fibroblasts as described previ- Ϫ/Ϫ Mouse embryonic fibroblasts (MEFs) from p53-null (p53 ) and isogenic ously (34). The protein concentration was measured with the Bio-Rad pro- wild-type mice (31) were generously provided by Dr. L. Donehower (Bay- tein assay reagents. An oligonucleotide (25 nucleotides long) containing Ϫ/Ϫ lor College of Medicine, Houston, TX). Rb-null (Rb ) and isogenic the potential E2F DNA-binding site (202-E2F-BS) present in the 5Ј-regu- ϩ/ϩ wild-type (Rb ) MEFs (32) were generously provided by Dr. Ed Harlow latory region of Ifi202, or containing an E2F DNA-binding site consensus Ϫ/Ϫ (Harvard Medical School, Boston, MA). MEFs from E2F1-null (E2F1 ) sequence (purchased from Santa Cruz Biotechnology), was end-labeled ϩ/ϩ and wild-type (E2F1 ) mice (16) were generously provided by Dr. L. with [␥-32P]ATP and polynucleotide kinase. Gel-purified labeled oligonu- Yamasaki (Columbia University, New York, NY). MEFs and splenocytes cleotide (probe, 1 ng/assay) was incubated with nuclear extracts containing Ϫ/Ϫ Ϫ/Ϫ from E2F1 and E2F2-double null (E2F1 E2F2 ) and wild-type equal amounts of protein in binding buffer (20 mM Tris-HCl (pH 7.5), 100 ϩ/ϩ ϩ/ϩ (E2F1 E2F2 ) age-matched mice (30) were generously provided by mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 1 mM DTT, Dr. J. DeGregori (University of Colorado, Denver, CO). MEFs and spleno- and 100 ␮g of BSA) for 20 min at room temperature in the presence of 0.5 cytes from age-matched C57BL/6 (B6) and B6.Nba2 congenic mice (7) ␮g of poly(dI:dC). Nuclear extracts were treated with detergent sodium were generously provided by Drs. S. J. Rozzo and B. L. Kotzin (University deoxycholate as described previously (34). In the competition assays, the of Colorado Health Sciences Center, Denver, CO). NIH3T3 fibroblasts nuclear extracts were preincubated with excess unlabeled oligonucleotides stably overexpressing E2F1 (3T3/E2F1 cl3, Ref. 33) were generously pro- at room temperature for 15 min. Samples were analyzed on a 5% native gel vided by Dr. H. Tanaka (Osaka University, Osaka, Japan). Mouse NIH3T3 in 1ϫ TBE (90 mM Tris-borate, 2 mM EDTA (pH 8.0)) buffer. Gel was fibroblasts were purchased from the American Type Culture Collection. dried and exposed to x-ray film at Ϫ80°C. MEFs (between passages 3 and 5) and NIH3T3 fibroblasts were maintained at low density in DMEM containing high glucose, supplemented with 10% Chromatin immunoprecipitation assays calf serum and antibiotics in an incubator with 5% CO . Antiserum to p202 2 ϫ 6 has been described previously (19). National Institutes of Health 3T3 or NIH3T3-E2F1 cells (8 10 ) were cross-linked for 10 min at room temperature by directly adding 1/10th Plasmids and expression vectors medium volume of cross-linking reagent (11% formaldehyde, 100 mM NaCl, 0.5 mM EGTA, 50 mM HEPES (pH 8.0)) to the plate. The cross- Dr. Peggy Farnham (University of California–Davis, Davis, CA) gener- linking reaction was quenched by adding 1/10th volume of 1.25 M glycine ously provided pCMV-mE2F1 expression vector. Dr. J. Nevins (Duke Uni- and incubating cells at room temperature for 5 min. Cells were centrifuged versity, Durham, NC) generously provided plasmids encoding various mu- and washed (twice) with PBS. Cells were resuspended in 500 ␮l of lysis tants of E2F1 (E2F1 (E132), E2F1 ⌬206–220, E2F1 ⌬1–88, and E2F1 buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), supplemented (411/421)). The 202-luc-reporter plasmid (202-luc), containing the 5Ј-reg- with the protease inhibitors) and tubes were incubated on ice for 10 min ulatory sequence (ϳ0.8 kb) of the Ifi202 gene (derived from the EAT with occasional mixing. The cell lysates were sonicated (2 times, 15 s each) cells), has been described previously (28). Mutations in the E2F1 DNA- to break chromatin and processed for the reversal of cross-linking. The cell The Journal of Immunology 5929

lysates were diluted to a final volume of 1 ml with a mixture of 9 parts of the dilution buffer (1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), plus protease inhibitors) and 1 part of cell lysis buffer. The diluted cell lysates were first precleared by incubating with protein A/G beads (Pierce Biotechnology) for 30 min at 4°C. After preclearing, the cell lysates (containing equal amounts of proteins) were divided into two fractions: one fraction was subjected to immunoprecipitation using mouse mAb to E2F1, and another fraction was immunoprecipitated with the same amount of mouse normal IgG Ab as a control. The immunoprecipitates were collected using protein A/G beads. Beads were centrifuged, washed FIGURE 1. E2F1 negatively regulates expression of Ifi202. A, Total (five times) in 1 ml of washing buffer (1% Triton X-100, 0.1% SDS, 150 RNA was isolated from subconfluent cultures of the wild-type (E2F1ϩ/ϩ, mM NaCl, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0)). The final wash was lane 1) or E2F1-null (E2F1Ϫ/Ϫ, lane 2) MEFs, and steady-state levels for with a buffer containing 1% Triton X-100, 0.1% SDS, 500 mM NaCl, 2 Ifi202 and actin mRNA were analyzed by semiquantitative RT-PCR. B, mM EDTA, 20 mM Tris-HCl (pH 8.0). Immune complexes were eluted ␮ Total cell extracts were prepared from subconfluent cultures of E2F1-null from beads by adding 450 l of elution buffer containing proteinase K and ␮ (lane 1) or wild-type (lane 2) MEFs, and steady-state levels for p202 and RNase A (500 g/ml each), and by incubating tubes at 37°C for 30 min. Tubes were centrifuged and the supernatants containing DNA were col- p68 protein (a protein detected by antiserum to p202 that serves as an lected. The DNA was purified, precipitated, washed, and dissolved in internal control for protein amounts; see Ref. 19) were analyzed by im- water. Equal volumes of DNA were used to perform PCR to detect the munoblotting. C, Total cell extracts were prepared from NIH3T3 cells promoter region of the Ifi202 gene. The following primers were used: re- stably transfected with control vector (lane 1) or plasmid expressing E2F1 gion 383–402 forward primer (5Ј-gtgtctagtggccagtgtac-3Ј) and region (lane 2). The extracts were analyzed by immunoblotting using Abs specific 804–784 reverse primer (5Ј-tctgcagtgatgtacagatcc-3Ј) (see Fig. 5; Ref. 18). Downloaded from to the indicated proteins. The PCR amplification conditions were: incubation at 94°C for 5 min, followed by 94°C for 30 s, 45°C for 30 s, and 72°C for 45 s. The amount

A B p53+/+ p53-/- p53-/- MEFs 1.2 1.2 http://www.jimmunol.org/ 1 202-luc 1 0.8 0.8 202-luc 0.6 0.6 0.4 0.4 0.2 0.2 Relative Luc Relative Act 0

Relative Luc Relative Act 0 1 2 3 4 1 2 3 mE2F-1 mE2F-1 -

- by guest on September 29, 2021 C D 1.6 p53-/- MEFs RbRb-/- MEFs-/- 1.4 1.4 202-luc 1.2 1.2 202-luc 1 202-luc 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 Relative Luc Relative Act. Relative Luc Act. 0 0.2 11 2 2 3 3 0 E2F-1 1 2 3 4 5 6 - ) 8) -8 -220) pCMV -1 (wt -1 (E132) 1 (d1 E2F 1 (411/421) 1 (d206 - E2F - E2F- E2F E2F FIGURE 2. E2F1-mediated transcriptional repression of Ifi202 is independent of p53 and pRb expression, but it is dependent on DNA-binding domain of E2F1. A, Subconfluent cultures of wild-type or p53-null MEFs were transfected with 202-luc-reporter plasmid (2.5 ␮g) and pRL-TK plasmid (0.5 ␮g) along with pCMV (2.5 ␮g, column 1) or increasing amounts (1 or 2 ␮g, columns 2 and 3, respectively) of pCMV-mE2F1 plasmid using a calcium phosphate transfection kit as described in Materials and Methods. Cells were harvested after 44 h to assay for the firefly and Renilla luciferase activities as described in Materials and Methods. Normalized relative luciferase activity is shown. Results from a representative experiment are shown. B, Subconfluent cultures of p53-null MEFs were transfected with 202-luc-reporter plasmid (2.5 ␮g) and pRL-TK plasmid (0.5 ␮g) along with pCMV (2.5 ␮g, column 1)or increasing amounts (1, 2, or 3 ␮g, columns 2, 3, and 4, respectively) of pCMV-mE2F1 plasmid using a calcium phosphate transfection kit. Cells were harvested after 44 h to assay for dual luciferase activities as described in A. Normalized relative luciferase activity is shown. Results from a representative experiment are shown. C, Subconfluent cultures of Rb-null MEFs were transfected with 202-luc-reporter plasmid (2.5 ␮g) and pRL-TK plasmid (0.5 ␮g) along with pCMV (2.5 ␮g, column 1) or increasing amounts (1 or 2 ␮g, columns 2 and 3, respectively) of pCMV-mE2F1 plasmid using a calcium phosphate transfection kit. Cells were harvested after 44 h to assay for dual luciferase activities as described in A. Normalized relative luciferase activity is shown. Results from a representative experiment are shown. D, Subconfluent cultures of p53-null MEFs were transfected with 202-luc-reporter plasmid (2.5 ␮g) and pRL-TK plasmid (0.5 ␮g) along with pCMV plasmid (2.5 ␮g, column 1) or plasmid (2.5 ␮g, column 2) encoding wild type mE2F1 (pCMV-mE2F1), or plasmid (2.5 ␮g) encoding various mutants of E2F1 (E2F1 (E132), column 3; E2F1 ⌬206–220, column 4; E2F1 ⌬1–88, column 5; and E2F1 (411/421) column 6) using a calcium phosphate transfection kit. Cells were harvested after 46 h to assay for dual luciferase activities as described in A. Normalized relative luciferase activity is shown. Results from a representative experiment are shown. 5930 REGULATION AND ROLE OF p202 IN LUPUS SUSCEPTIBILITY

of amplified DNA was compared after 32 or 36 amplification cycles by 1.2 agarose gel electrophoresis to determine whether the amplification of the input DNA was in the linear range. 1 202-Luc (NIH3T3) Results 0.8 Transcription factor E2F1 negatively regulates expression of 0.6 Ifi202 0.4 Our previous studies (25) revealed that serum growth factors, such as platelet-derived growth factor, basic fibroblast growth factor, or 0.2 TGF-␤, negatively regulate expression of Ifi202 in mouse fibro- Relative luciferaseactivity 0 blasts. Moreover, we found (35) that levels of Ifi202 mRNA and 1 2 3 4 5 protein decrease in mouse fibroblasts when cells exit from quies- E2F-1 - + cence (the G0/ of cell cycle) to enter into S phase of cell E2F-2 - + cycle after serum stimulation. Because serum growth factors stim- E2F-3 - + ulate transition of cells from G /G phase of cell cycle to S phase, 0 1 E2F-4 - + in part by activating the E2F1-mediated transcription of S-phase genes (10, 11), we explored whether E2F1 could negatively reg- FIGURE 3. E2Fs differentially regulate expression of Ifi202. Subcon- ulate expression of the Ifi202. For this purpose, we compared basal fluent cultures of NIH3T3 cells were transfected with 202-luc-reporter Ϫ/Ϫ plasmid (2.5 ␮g) and pRL-TK plasmid (0.5 ␮g) along with equal amounts levels of Ifi202 mRNA and protein between E2F1-null (E2F1 ) Downloaded from ␮ and isogenic wild-type (E2Fϩ/ϩ) MEFs. As shown in Fig. 1A, (2.5 g) of pCMV (column 1), pCMV-E2F1 (column 2), pCMV-E2F2 (column 3), pCMV-E2F3 (column 4), or pCMV-E2F4 (column 5) using a basal levels of Ifi202 mRNA were not detectable in the wild-type calcium phosphate transfection kit as described in Materials and Methods. MEFs. However, the mRNA levels were readily detectable in E2F- Cells were harvested after 44 h to assay for the firefly and Renilla luciferase null MEFs. Consistent with detectable mRNA levels in E2F1-null activities as described in Materials and Methods. Normalized relative lu- MEFs, p202 protein levels were detectable in E2F1-null MEFs, but ciferase activity is shown.

not in isogenic wild-type cells (Fig. 1B). Furthermore, overexpression http://www.jimmunol.org/ of E2F1 in NIH3T3 cells resulted in reduced levels of p202 protein (Fig. 1C). Taken together, these observations suggested that expres- sion of E2F1 in MEFs negatively regulated expression of the Ifi202. activity of 202-luc-reporter (compare column 2 with column 1). Interestingly, transfection of cells with a deletion mutant of E2F1 E2F1-mediated transcriptional repression of Ifi202 is p53 and (E132) in which the DNA-binding domain is deleted stimulated pRb independent the activity of the reporter (compare column 3 with column 2). Our previous studies revealed that p53 transcriptionally represses However, expression of deletion mutants of E2F1 in which either expression of Ifi202 (26). This observation made it conceivable the N terminus (⌬1–88 amino acids) or the (⌬206– that E2F1 represses the expression of Ifi202 through stabilization 220) region was deleted repressed the activity of 202-luc-reporter. by guest on September 29, 2021 of p53 (through transcriptional activation of the alternative reading Moreover, expression of the E2F1 double-point mutant 411/421, frame of p19; see Ref. 36). Therefore, we tested whether E2F1- which has previously been shown (37) to be selectively defective in mediated negative regulation of Ifi202 expression is p53 depen- overcoming p107 while maintaining wild-type pRb repressor func- dent. For this purpose, we performed promoter-reporter assays. As tion, inhibited the activity of 202-luc, suggesting that the p107 repres- shown in Fig. 2A, expression of mouse E2F1 (mE2F1) in p53-null sor function is not required for E2F1-medaited repression of Ifi202. (p53Ϫ/Ϫ) or wild-type (p53ϩ/ϩ) MEFs reproducibly decreased the Taken together, these observations suggested that the DNA-binding activity of the 202-luc-reporter ϳ50–60% in three independent domain in E2F1 is required for transcriptional repression of Ifi202. experiments. This observation suggested that the E2F1-mediated transcriptional repression of 202-luc-reporter activity was indepen- Differential regulation of Ifi202 expression by the E2F family dent of p53 expression. Furthermore, we noted that transfection of members p53-null MEFs with increasing amounts of mE2F1 encoding plas- Because the E2F family of transcription factors, such as E2F1,

mid decreased the activity of the 202-luc-reporter in a dose-de- E2F2, or E2F3, but not E2F4, is thought to participate in the G1- pendent manner, and the decrease in the activity of the reporter to-S phase transition of cells (10, 11), we compared the ability of was ϳ70% (Fig. 2B). these transcription factors to repress transcription of Ifi202 gene. Because E2F transcription factors in complex with the pRb (or As shown in Fig. 3, transfection of NIH3T3 cells with a plasmid other pocket proteins, such as p107 and p130) repress transcription encoding either E2F1 or E2F2 transcription factor repressed the ac- of their target genes (10, 11), we also tested whether E2F1-medi- tivity of the 202-luc-reporter to a similar extent. Interestingly, trans- ated transcriptional repression of Ifi202 gene is pRb dependent. fection of plasmid that encodes the E2F3 transcription factor only Our promoter-reporter assays using pRb-null MEFs revealed that partially (40%) inhibited the activity of the reporter. However, ex- E2F1 repressed the activity of 202-luc-reporter in pRb-null MEFs pression of the E2F4 transcription factor did not repress transcription (Fig. 2C). These observations suggested that E2F1-mediated tran- of 202-luc-reporter in several independent experiments. scriptional repression of Ifi202 is pRb independent. Furthermore, consistent with our above observations that both E2F1 and E2F2 repressed transcription of the Ifi202 gene, we E2F1 DNA-binding activity is required for transcriptional found that basal levels of p202 protein were detectable in spleno- repression of Ifi202 cytes from E2F1Ϫ/ϪE2F2Ϫ/Ϫ double knockout mice, but not in The E2F1-mediated transcriptional repression of genes is known to age-matched wild type (E2F1ϩ/ϩE2F2ϩ/ϩ) mice (data not shown). require DNA-binding activity (10, 11). Therefore, we tested Moreover, the basal activity of 202-luc-reporter was consistently whether E2F1-mediated transcriptional repression of Ifi202 gene ϳ2-fold higher in MEFs from the double knockout mice than from requires the DNA-binding domain in E2F1. As shown in Fig. 2D, the wild-type mice in two independent experiments (data not expression of the wild-type E2F1 in p53-null cells decreased the shown). These observations suggested that structurally related The Journal of Immunology 5931

Table I. A comparison of potential E2F1/DP1 DNA binding in the A Ifi202 gene with E2F DNA-binding consensus sequencea 383 402 Ifi202 E2F1-BS E2F1/DP1 DNA-binding TTT CCC GC 784 804 consensus sequence Input IgG1 α-E2F1 202-E2F1-BS TTTCCCTG B NC 202-E2F-BS mutant TTTCCTTT 1 2 3 4 5 6 7 8

a Boldface letters indicate nucleotides that appear in Ͼ90% of recovered E2F1/ 32-cycles DP1 DNA-binding sites (38). Underlined letters indicate variations in the nucleotides from the E2F DNA-binding consensus sequence. 36-cycles

FIGURE 5. Ј E2Fs, such as E2F1, E2F2, and E2F3, repress the transcription of E2F1 binds to potential E2F DNA-binding site in the 5 - regulatory region of Ifi202 gene in chromatin immunoprecipitation assays. the Ifi202 gene, albeit to different extents. A, Schematic representation of the 5Ј-regulatory region of Ifi202 gene con- E2Fs bind to a potential DNA-binding site in the 5Ј-regulatory taining the 202-E2F-BS and relative location of PCR primers that were used to amplify the immunoprecipitated chromatin. B, Soluble chromatin region of the Ifi202 gene was prepared from NIH3T3 (lanes 3, 5, and 7) or NIH3T3-E2F1 (lanes 4, Because E2F-mediated transcriptional repression of 202-luc-re- 6, and 8) cells. Chromatin was incubated with Abs to E2F1 (lanes 7 and 8) porter was dependent on the ability of E2F to bind DNA (Fig. 2D), or, as a negative control, with isotype IgG1 Abs (lanes 5 and 6). DNA was we analyzed the 5Ј-regulatory region of Ifi202 gene for the pres- extracted from immunoprecipitates and PCR amplified (upper panel,32 Downloaded from cycles; lower panel, 36 cycles) using a pair of primers that covered the E2F1 DNA-binding site in the 5Ј-regulatory region of the Ifi202 gene. As a positive control for PCR, we also amplified the input chromatin DNA A from NIH3T3 (lane 3) and NIH3T3-E2F1 (lane 4) cells. As a negative -817 -274 CAGGGAAA -267 -1 control for PCR, we did not include any template DNA in the PCR reaction (lane 2). 202-E2F-BS http://www.jimmunol.org/

ence of potential E2F DNA-binding sites using the Matinspector B C 2.0 software program. The sequence analyses revealed a potential 202-E2F-BS E2F DNA-binding site (5Ј-TTTCCCTG-3Ј; 202-E2F-BS). A com- Ј

obe parison of the 202-E2F-BS in the Ifi202 5 -regulatory region with

+50X Probe NE DOC

Pr NE 20X Ј Ј

50X (E2F CS)

100X 50X other E2F DNA-binding consensus sites (5 -TTTCCCGC-3 ; E2F- 1 2 3 4 1 2 3 4 5 6 CS), which are bound by most (90%) E2F1/DP1 complexes (38), revealed at least two variations or mismatches between the 202- E2F-BS and E2F-CS sequence (see Table I). by guest on September 29, 2021 Next, we performed gel-mobility shift assays to determine * * whether 202-E2F-BS (Fig. 4A)inIfi202 gene can bind to E2F transcription factors. As shown in Fig. 4B, in nuclear extracts from NIH3T3 cells, we could detect binding of proteins to an oligonu- cleotide (probe) containing the 202-E2F-BS sequence (compare lane 2 with lane 1). Importantly, treatment of nuclear extracts with detergent deoxycholate, which has been previously known to dis- sociate E2F proteins from the pocket family proteins in NIH3T3 Probe: 202-E2F-BS cells (34), resulted in “free” E2F (a dimmer between E2F and DP Probe: DHFR-E2F-CS proteins). Moreover, competition with 50-fold excess of unlabeled FIGURE 4. Identification of a potential E2F DNA-binding site in the oligonucleotide containing the E2F-CS greatly reduced the binding 5Ј-regulatory region of Ifi202 gene in gel-mobility shift assays. A, Sche- of proteins to the oligonucleotide with the 202-E2F-BS sequence matic representation of a potential E2F DNA-binding site (202-E2F-BS) in (compare lane 4 with lanes 2 or 3). Ј the 5 -regulatory region of Ifi202 gene. B, Nuclear extracts from NIH3T3 Because the 202-E2F-BS sequence contains at least two varia- cells were subjected to gel-mobility shift assays using radiolabeled oligo- tions (or mismatches) in nucleotides from the E2F-CS (see Table nucleotide containing the 202-E2F DNA-binding sequence (probe) as de- scribed in Materials and Methods. Shown are the nuclear extract without I), we sought to compare the relative affinity of 202-E2F-BS with any treatment (lane 2), after treatment with deoxycholate (lane 3), or after E2F-CS. For this purpose, we performed gel-mobility shift assays competition with 50-fold excess of cold oligonucleotide containing the E2F using nuclear extracts from NIH3T3 cells and labeled oligonucle- DNA-binding consensus sequence (lane 4). As a control, we also analyzed otide with E2F-CS from the DHFR, a well-known E2F target gene the probe without incubation with the extracts (lane 1). C, Nuclear extracts (10). As shown in Fig. 4C, proteins in nuclear extracts bound to from NIH3T3 cells were subjected to gel-mobility shift assays using ra- E2F-CS, and a competition with 50-fold excess of cold E2F-CS diolabeled oligonucleotide containing the E2F DNA-binding consensus se- reduced the binding of proteins to the probe (compare lane 3 with quence from the DHFR gene (probe) as described in Materials and Meth- lane 2). Furthermore, as expected from the presence of two vari- ods. Shown are the nuclear extract without any treatment (lane 2), after ations in the 202-E2F-BS sequence, competition with 20- or 50- competition with 50-fold excess of cold oligonucleotide containing the fold excess of cold oligonucleotide with 202-E2F-BS did not re- E2F-CS (lane 3), and after competition with 20-fold (lane 4), 50-fold (lane 5), or 100-fold (lane 6) excess of cold oligonucleotide containing the 202- duce binding of labeled E2F-CS with proteins (compare lane 4 or E2F-BS. As a control, we also analyzed the probe without incubation with 5 with lane 2). However, competition with 100-fold excess cold the extracts (lane 1). The arrow indicates the location of E2F transcription oligonucleotide containing the 202-E2F-BS sequence reduced the factors in complex with the pocket family of proteins, and the asterisk binding of the probe Ͼ50%. These observations suggested that indicates the location of “free” E2F transcription factors. potential E2F-BS in the 5Ј-regulator region of Ifi202 gene can bind 5932 REGULATION AND ROLE OF p202 IN LUPUS SUSCEPTIBILITY

1.2 in the 202-luc-reporter plasmid by changing a critical nucleotide 202-luc (wt) 1 from C to T (see Table I) by site-directed mutagenesis and com- 202-luc (mut) pared the activity of the wild-type reporter with the mutant re- 0.8 porter. As shown in Fig. 6, expression of mE2F1 repressed the 0.6 activity of wild-type 202-luc-reporter. Interestingly, introduction 0.4 of a point mutation in the 202-E2F biding sequence resulted in a 0.2 60% decrease in the E2F1-mediated transcriptional repression in

Relative Luc Act Relative 0 two independent experiments. These observations suggested that Vector mE2F1 E2F1 represses the expression of Ifi202 in part through the 202- Ј FIGURE 6. The E2F1 transcriptionally represses Ifi202 expression E2F-BS in the 5 -regulatory region of the Ifi202 gene. through the E2F1 DNA-binding site. Subconfluent cultures of p53-null MEFs were transfected with wild-type or mutant 202-luc-reporter plasmid Increased expression of p202 in B6.Nba2 mice is associated (2.5 ␮g) and pRL-TK plasmid (0.5 ␮g) along with pCMV (2.5 ␮g, column with inhibition of E2F1 expression and down-regulation of the 1) or pCMV-mE2F1 (2.5 ␮g, column 2) plasmid using a calcium phosphate E2F1 target genes transfection kit as described in Materials and Methods. Cells were har- vested after 44 h to assay for the firefly and Renilla luciferase activities as Our previous studies (34, 39) revealed that increased levels of described in Materials and Methods. Normalized relative luciferase activ- p202 in cells inhibit E2F1- and E2F4-mediated transcription of ity is shown. Results from a representative experiment are shown. target genes. These observations raised the possibility that in- creased expression of p202 in B6.Nba2 mice contributes to lupus Downloaded from susceptibility, in part by down-regulating the expression of E2F1 to E2F protein complexes in gel-mobility shift assays with - and its target genes. Therefore, we tested whether increased ex- tively low affinity relative to the E2F DNA-binding sites found in pression of p202 in MEFs derived from the B6.Nba2 mice affects most E2F-responsive genes. the transcriptional activity of E2F. For this purpose, we compared To confirm binding of E2F1 to 202-E2F-BS in vivo, we per- the activity of a well-known E2F-responsive reporter (E2F-luc) in formed chromatin immunoprecipitation assays. As shown in Fig. MEFs derived from B6 and B6.Nba2 mice. As shown in Fig. 7A, 5B, some binding of E2F1 to Ifi202 regulatory region was detected basal activity of the E2F-responsive reporter was 70% lower in http://www.jimmunol.org/ in asynchronous cultures of NIH3T3 cells (compare lane 7 with unsynchronized B6.Nba2 than in B6 MEFs. This observation lane 5). However, overexpression of E2F1 in NIH3T3 cells, which prompted us to compare the expression of E2F1 mRNA and pro- decreased the expression of p202 (see Fig. 1C), increased binding tein between B6 and B6.Nba2 splenocytes in vivo. As shown in of E2F1 to the regulatory region of the Ifi202 (compare lane 8 with Fig. 7B, levels of E2F1 mRNA were ϳ80% lower in B6.Nba2 lane 7). This observation is consistent with our observation that the splenocytes than in B6 splenocytes. Additionally, we found that E2F1 DNA-binding activity is required to repress transcription of mRNA levels of known E2F1 target genes, such as PUMA and 202-luc-reporter (Fig. 2D) and that E2Fs can bind to 202-E2F-BS Bim, were also lower in splenocytes derived from the B6.Nba2

sequence in gel-mobility shift assays (Fig. 4). mice. We also compared the protein levels of E2F1 and its tran- by guest on September 29, 2021 scriptional target, cyclin E, between B6 and B6.Nba2 splenocytes E2Fs represses transcription of Ifi202 gene through the E2F-BS (Fig. 7C). We noted that levels of both E2F1 and cyclin E were in the regulatory region lower in B6.Nba2 splenocytes than in B6. Additionally, we also Our observation that E2F1 expression repressed expression of the compared the expression levels of p73, a transcriptional target of Ifi202 and E2F1 bound to a potential E2F DNA-binding site in the the E2F1, between age-matched B6 and B6.Nba2 female mice. As 5Ј-regulatory region of Ifi202 gene prompted us to determine shown in Fig. 7D, levels of p73 were lower in splenocytes derived whether this E2F DNA-binding site is needed for E2F1-mediated from two B6.Nba2 female mice than in B6 female mice. These transcriptional repression. Therefore, we mutated the 202-E2F-BS observations provide support to the idea that increased levels of

FIGURE 7. Increased expression of Ifi202 in B6.Nba2 mice is associated with reduced expression levels of E2F1 and inhibition of E2F1-mediated transcription of target genes. A, Subconfluent cultures of B6 or B6.Nba2 MEFs were transfected with E2F-luc-reporter plasmid (2.5 ␮g) and pRL-TK plasmid (0.5 ␮g) using a calcium phosphate transfection kit as described in Materials and Methods. Cells were harvested after 44 h to assay for the firefly and Renilla luciferase activities as described in Materials and Methods. Normalized relative luciferase activity is shown. B, Total RNA was isolated from splenocytes from age-matched (10 wk) B6 (lane 1) or B6.Nba2 (lane 2) female mice, and steady-state levels of mRNAs were analyzed by semiquantitative RT-PCR using a pair of primers specific for the indicated individual genes. C, Total cell extracts were prepared from splenocytes isolated from age-matched (10 wk) B6 (lane 1) or B6.Nba2 (lane 2) female mice, and steady-state levels of proteins were analyzed by immunoblotting using Abs specific for the indicated proteins. D, Total cell extracts were prepared from splenocytes isolated from age-matched (10 wk) B6 (lanes 1 and 2) or B6.Nba2 (lanes 3 and 4) female mice, and steady-state levels of p73 and actin proteins were analyzed by immunoblotting using specific Abs. The Journal of Immunology 5933

p202 in B6.Nba2 mice contribute to lupus susceptibility by de- We found that E2F-mediated transcriptional repression of Ifi202 creasing the expression of E2F1 transcription factor and inhibition gene was independent of p53 or pRb expression (Fig. 2). Because of E2F1-mediated transcription of target genes. the p53 protein family includes p63 and p73 proteins (48) and the pRb protein family includes p107 and p130 proteins (11), our ob- Discussion servations do not rule out the possibility that E2F1-mediated tran- Studies involving SLE patients (6) and mouse models of lupus scriptional repression of Ifi202 gene depends on other p53 or pRb disease (9, 23) have revealed a role for IFN-inducible genes in the family proteins. Further work will be needed to test these development of lupus disease. Because IFN-inducible proteins that possibilities. regulate cell proliferation and survival are likely to contribute to The IFN-inducible p202 protein binds to E2F1 and inhibits the development of autoimmunity, identification of such IFN-in- E2F1-mediated transcription of target genes (34). Binding of p202 ducible proteins will allow us to elucidate their role in immune to E2F1 results in inhibition of specific DNA-binding activity of regulation. Furthermore, it is important to understand how cellular E2F complexes in gel-mobility shift assays (34). Moreover, p202 levels of IFN-inducible proteins are regulated by other signaling inhibits E2F1-mediated apoptosis (49). Taken together, these ob- pathways in immune cells. servations support the idea that increased levels of p202 in immune Through regulation of a set of genes that regulate transition from cells (B and T cells) increase the threshold for apoptosis by inhib- G1-to-S phase of cell cycle, the transcription factor E2F1 regulates iting E2F-mediated transcription of its target genes, which encode cell proliferation, differentiation, and apoptosis of B and T cells proapoptotic proteins. Consistent with the above idea we found (29, 30, 36, 40–47). Moreover, defects in cell cycle progression that increased expression of Ifi202 in B6.Nba2 was associated with and apoptosis of immune cells that result from the lack of E2F1 or increases in the numbers of T and B cells and splenomegaly (7). Downloaded from E2F1 and E2F2 function contribute to the development of lupus- Stimulation of the TCR on cycling peripheral T cells causes like disease (29, 30). Consistent with this idea, the transcription their apoptosis by TCR activation-induced cell death (44). Fur- factor E2F1 is important for activation-induced cell death in T thermore, E2F1-null or p73-null primary T cells do not undergo cells (44). Furthermore, E2F1 and E2F2 transcription factors de- TCR-mediated apoptosis (44). Similarly, E2F1-mediated expres- termine the threshold for Ag-induced T cell proliferation (30), and sion of proapoptotic proteins, such as Bim and Puma, contributes the activity of E2F2 is needed for suppression of T cell prolifer- to apoptosis of cells (50). Therefore, our observations that in- http://www.jimmunol.org/ ation and immunologic self-tolerance (29). Consequently, disrup- creased expression of p202 in B6.Nba2 splenic cells is associated tion of the gene for the E2F1 transcription factor causes significant with reduced expression of E2F1, Bim, and p73 provide support increases in T cell number and splenomegaly (16). Similarly, mice for the idea that inhibition of TCR activation-induced cell death by that are null for E2F2 transcription factor develop a lupus-like p202 contributes to accumulation of T cells. disease (29). Importantly, the E2F2 transcription factor represses Studies have suggested that E2F1 plays a role in the induction of the transcription of the E2F1 gene, whose activity is required in ARF, p53, and apoptosis during thymic negative selection (36). cells for normal S phase entry during cell cycle progression (29). Because increased expression of p202 in cells inhibits p53- and Because serum growth factors down-regulate the expression of

E2F1-mediated transcription (20, 21), our observations are consis- by guest on September 29, 2021 Ifi202 (25) and p202 inhibits the E2F (E2F1 and E2F4)-mediated tent with the idea that increased levels of p202 in B6.Nba2 mice transcription (34, 39), we tested whether E2F transcription factors contribute to defects in apoptosis during thymic negative selection. could regulate expression of the Ifi202. Moreover, our observations suggest that transcriptional repression Our experiments revealed that: 1) steady-sate levels of Ifi202 Ifi202 mRNA and protein were higher in E2F-null than in wild-type of gene by both p53 (26) and E2F1 (this study) may be MEFs and overexpression of E2F1 decreased p202 levels (Fig. 1); required for normal thymic negative selection. 2) splenic cells from E2F1 and E2F2 double knockout mice ex- Increased expression of the p202 in cell lines inhibits the tran- ␬ pressed higher levels of p202 than did isogenic wild-type cells scriptional activity of factors such as p53, NF- B, and AP-1 (20, (Fig. 3); 3) in promoter-reporter assays, expression of E2F1, E2F2, 21). These transcription factors, by modulating transcription of and E2F3, but not E2F4, repressed transcription of the 202-luc- target genes, are known to regulate cell proliferation and survival reporter (Fig. 3); 4) the 5Ј-regulatory region of the Ifi202 gene (20, 21). Consistent with inhibition of the transcriptional activity contains a potential E2F DNA-binding site (202-E2F-BS) that of the above factors by p202, the increased expression of p202 in bound to E2F protein complexes in gel-mobility shift assays (Fig. cell lines, depending on the cell context, either sensitizes cells to 4) and in chromatin immunoprecipitation assays (Fig. 5), and a apoptosis or decrease the susceptibility to apoptosis (21). Impor- point mutation in this E2F DNA-binding site decreased the E2F- tantly, we found that increased expression of p202 in splenic mediated transcriptional repression of 202-luc-reporter (Fig. 6); B6.Nba2 cells was associated with inhibition of p53-mediated and 5) increased expression of Ifi202 in splenic cells from B6.Nba2 transcription of proapoptotic genes and defects in apoptosis of congenic mice was associated with reduced expression of E2F1 and cells (51). Therefore, our earlier observations that certain E2Fs, its target genes (Fig. 7). Taken together, these observations demon- such as E2F1 and E2F2, repress the transcription of the Ifi202 gene strated that: 1) E2Fs differentially regulate expression of the Ifi202 are consistent with our above observations. gene, and 2) increased expression of p202 in splenic cells from In summary, our observations provide support for our model B6.Nba2 mice negatively regulates E2F-medaited transcription. (Fig. 8). The model predicts that mitogenic signaling pathways that The 202-luc-reporter construct that is used in our promoter-re- activate the E2F1-mediated transcription negatively regulate Ifi202 porter assays (Figs. 2 and 3) contains only ϳ800 bp from the expression. In contrast, signaling pathways (and host factors) that 5Ј-regulatory region of the Ifi202 gene. Therefore, it is conceivable contribute to increased expression of Ifi202 inhibit E2F1-mediated that E2F-responsive elements upstream to this 800-bp region in transcription of proapoptotic genes in cells. Therefore, defects in Ifi202 gene also contribute to the regulation of the gene. This could mutual regulation of Ifi202 and E2F1 expression and functions are account for moderate decreases in the activity of the 202-luc-re- likely to contribute to defects in cell proliferation and/or apoptosis, porter in our assays after expression of E2F (E2F1, E2F2, or E2F3) resulting in autoimmunity. Our observations will serve as a basis transcription factors and significantly increased steady-state levels to identify signaling pathways and molecules that contribute to the of Ifi202 mRNA and protein in E2F1-null cells. development of autoimmune diseases. 5934 REGULATION AND ROLE OF p202 IN LUPUS SUSCEPTIBILITY

Promoter polymorphisms 22. Jorgensesn, T. N., E. Roper, J. M. Thurman, P. Marrack, and B. L. Kotzin. 2007. Host factors Type I interferon signaling is involved in the spontaneous development of lupus-like Cellular stresses ϫ disease in B6.Nba2 and (B6.Nba2 NZW)F1 mice. Genes Immun. 8: 653–662. Cytokines (e.g., IL-6) 23. Lu, Q., N. Shen, X. M. Li, and S. L. Chen. 2007. Genomic view of IFN-␣ response Viruses in pre-autoimmune NZB/W and MRL/lpr mice. Genes Immun. 8: 590–603. Type I IFNs 24. Santiago-Raber, M. L., R. Baccala, K. M. Haraldsson, D. Choubey, T. A. Stewart, D. H. Kono, and A. N. Theofilopoulos. 2003. Type-I interferon receptor defi- E2F-mediated ciency reduces lupus-like disease in NZB mice. J. Exp. Med. 197: 777–788. transcriptional Expression 25. Geng, Y., S. D’Souza, H. Xin, S. Walter, and D. Choubey. 2000. p202 levels are of Ifi202 activation of Apoptosis pro-apoptotic negatively regulated by serum growth factors. Cell Growth Differ. 11: 475–483. genes 26. D’Souza, S., H. Xin, S. Walter, and D. Choubey. 2001. The gene encoding p202, an interferon-inducible negative regulator of the p53 tumor suppressor, is a target Growth of p53-mediated transcriptional repression. J. Biol. Chem. 276: 298–305. factors E2Fs (E2F1 and E2F2) 27. Xin, H., Y. Geng, R. Pramanik, and D. Choubey. 2003. Induction of p202, a mod- ulator of apoptosis, during oncogenic transformation of NIH 3T3 cells by activated FIGURE 8. Regulation of Ifi202 gene by E2F1 transcription factor and H-Ras (Q61L) contributes to cell survival. J. Cell. Biochem. 88: 191–204. the role of p202 in lupus susceptibility through inhibition of E2F-mediated 28. Pramanik, R., T. N. Jorgensen, H. Xin, B. L. Kotzin, and D. Choubey. 2004. transcription of target genes. Interleukin-6 induces expression of Ifi202, an interferon-inducible candidate gene for lupus susceptibility. J. Biol. Chem. 279: 16121–16127. 29. Murga, M., O. Fernandez,-Capetillo, S. J. Field, B. Moreno, L. R. Borlado, Y. Fujiwara, D. Balomenos, A. Vicario, A. C. Carrera, S. H. Orkin, et al. 2001. Mutation of E2F2 in mice causes enhanced T lymphocyte proliferation, leading Acknowledgments to the development of autoimmunity. Immunity 15: 959–970. We thank Drs. L. Donehower, Ed Harlow, L. Yamasaki, J. DeGregori, 30. Zhu, J. W., S. J. Field, L. Gore, M. Thomson, H. Yang, Y. Fujiwara, R. D. Cardiff, M. Greenberg, S. H. Orkin, and J. DeGregori. 2001. E2F1 and

S. J. Rozzo, B. L. Kotzin, H. Tanaka, P. Farnham, and J. Nevins for gen- Downloaded from E2F2 determine thresholds for antigen-induced T-cell proliferation and suppress erously providing cells and reagents. We also thank Drs. tumorigenesis. Mol. Cell. Biol. 21: 8547–8564. P. Lengyel and B. L. Kotzin for thoughtful suggestions. 31. Donehower, L. A., M. Harvey, B. L. Slagle, M. J. McArthur, C. A. Montgomery, J. S. Butel, and A. Bradley. 1992. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356: 215–221. Disclosures 32. Classon, M., S. Salama, C. Gorka, R. Mulloy, P. Braun, and E. Harlow. 2000. The authors have no financial conflicts of interest. Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control. Proc. Natl. Acad. Sci. USA 97: 10820–10825.

33. Tanaka, H., I. Matsumura, S. Ezoe, Y. Satoh, T. Sakamaki, C. Albanese, http://www.jimmunol.org/ References T. Machii, R. G. Pestell, and Y. Kanakura. 2002. E2F1 and c- potentiate 1. Kotzin, B. L. 1996. Systemic lupus erythematosus. Cell 85: 303–306. apoptosis through inhibition of NF-␬B activity that facilitates MnSOD-mediated 2. Tsokos, G. C., and G. M. Kammer. 2000. Molecular aberrations in human sys- ROS elimination. Mol. Cell. 9: 1017–1029. temic lupus erythematosus. Mol. Med. Today 6: 418–424. 34. Choubey, D., S. J. Li, B. Datta, J. U. Gutterman, and P. Lengyel. 1996. Inhibition 3. Theofilopoulos, A. N., R. Baccala, B. Beutler, and D. H. Kono. 2005. Type I inter- of E2F-mediated transcription by p202. EMBO J. 15: 5688–5678. feron (␣/␤) in immunity and autoimmunity. Annu. Rev. Immunol. 23: 307–336. 35. Xin, H., R. Pramanik, and D. Choubey. 2003. Retinoblastoma (Rb) protein up- 4. Jorgensen, T. N., M. R. Gubbels, and B. L. Kotzin. 2004. New insights into disease regulates expression of the Ifi202 gene encoding an interferon-inducible negative pathogenesis from mouse lupus genetics. Curr. Opin. Immunol. 16: 787–793. regulator of cell growth. Oncogene 22: 4775–4785. 5. Zhu, J., and C. Mohan. 2007. SLE 1, 2, 3. . . genetic dissection of lupus. Adv. Exp. 36. Zhu, J. W., D. DeRyckere, F. X. Li, Y. Y. Wan, and J. DeGregori. 1999. A role Med. Biol. 601: 85–95. for E2F1 in the induction of ARF, p53, and apoptosis during thymic negative 6. Banchereau, J., and V. Pascual. 2006. Type I interferon in systemic lupus ery- selection. Cell Growth Differ. 10: 829–838.

thematosus and other autoimmune diseases. Immunity 25: 383–392. 37. Cress, W. D., D. G. Johnson, and J. R. Nevins. 1993. A genetic analysis of the by guest on September 29, 2021 7. Rozzo, S. J., J. D. Allard, D. Choubey, T. J. Vyse, S. Izui, G. Peltz, and E2F1 gene distinguishes regulation by Rb, p107, and adenovirus E4. Mol. Cell. B. L. Kotzin. 2001. Evidence for an interferon-inducible gene, Ifi202,inthe Biol. 13: 6314–6325. susceptibility to systemic lupus. Immunity 15: 435–443. 38. Tao, Y., R. F. Kassatly, W. D. Cress, and J. M. Horowitz. 1997. Subunit composition 8. Haywood, M. E. K., S. J. Rose, S. Horswell, M. J. Lees, G. Fu, M. J. Walport, determines E2F DNA-binding site specificity. Mol. Cell. Biol. 17: 6994–7007. and B. J. Morley. 2006. Overlapping BXSB congenic intervals, in combination 39. Choubey, D., and J. U. Gutterman. 1997. Inhibition of E2F-4/DP-1-stimulated with microarray gene expression, reveal novel lupus candidate genes. Genes Im- transcription by p202. Oncogene 15: 291–301. mun. 7: 250–263. 40. Brennam, P., J. W. Babbage, B. M. Burgering, B. Groner, K. Reif, and 9. Liu, J., G. Karypis, A. L. Vegoe, P. Ruiz, G. S. Gilkeson, and T. W. Behrens. D. A. Cantrell. 1997. Phosphatidylinositol 3-kinase couples the interleukin-2 re- 2006. Genomic view of systemic autoimmunity in MRL/lpr mice. Genes Immun. ceptor to the cell cycle regulator E2F. Immunity 7: 679–689. 7: 156–168. 41. Lam, E. W., J. Glassford, J. van der Sman, L. Banerji, A. R. Pizzey, N. Shaun, 10. Slansky, J. E., and P. J. Farnham. 1996. Introduction to the E2F family: protein B. Thomas, and G. G. Klaus. 1999. Modulation of E2F activity in primary mouse structure and gene regulation. Curr. Top. Microbiol. Immunol. 208: 1–30. B cells following stimulation via surface IgM and CD40 receptors. Eur. J. Im- 11. Nevins, J. R. 1998. Toward an understanding of the functional complexity of the munol. 29: 3380–3389. E2F and retinoblastoma families. Cell Growth Differ. 9: 585–593. 42. van der Sman, J., N. S. Thomas, and E. W. Lam. 1999. Modulation of E2F 12. DeGregori, J. 2002. The genetics of the E2F family of transcription factors: complexes during G0 to S phase transition in human primary B-lymphocytes. shared functions and unique roles. Biochem. Biophys. Acta 1602: 131–150. J. Biol. Chem. 274: 12009–12016. 13. Iwase, S., Y. Furukawa, J. Kikuchi, M. Nagai, Y. Terui, M. Nakamura, and 43. Lam, E. W., M. S. Choi, J. van der Sman, S. A. Burbidge, and G. G. Klaus. 1998. H. Yamada. 1997. Modulation of E2F activity is linked to interferon-induced Modulation of E2F activity via signaling through surface IgM and CD40 recep- growth suppression of hematopoietic cells. J. Biol. Chem. 272: 12406–12414. tors in WEHI-231 B lymphoma cells. J. Biol. Chem. 273: 10051–10057. 14. Thomas, N. S., A. R. Pizzey, S. Tiwari, C. D. Williams, and J. Yang. 1998. p130, 44. Lissy, N. A., P. K. Davis, M. Irwin, W. G. Kaelin, and S. F. Dowdy. 2000. A p107, and pRb are differentially regulated in proliferating cells and during cell common E2F-1 and p73 pathway mediates cell death induced by TCR activation. cycle arrest by ␣-interferon. J. Biol. Chem. 273: 23659–23667. Nature 407: 642–645. 15. Johnson, D. G., K. Ohtani, and J. R. Nevins. 1994. Auto-regulatory control of 45. Garcia, I., M. Murga, A. Vicario, S. J. Field, and A. M. Zubiaga. 2000. A role for E2F1 expression in response to positive and negative regulators of cell cycle E2F1 in the induction of apoptosis during thymic negative selection. Cell Growth progression. Genes Dev. 8: 1514–1525. Differ. 11: 91–98. 16. Yamasaki, L., T. Jacks, R. Bronson, E. Goillot, E. Harlow, and N. J. Dyson. 1996. 46. Cao, Q., Y. Xia, M. Azadniv, and I. N. Crispe. 2004. The E2F-1 transcription Tumor induction and tissue atrophy in mice lacking E2F-1. Cell 85: 537–548. factor promotes -8 and bid expression, and enhances Fas signaling in T 17. Phillips, A. C., and K. H. Vousden. 2001. E2F-1 and apoptosis. Apoptosis 6: cells. J. Immunol. 173: 1111–1117. 173–182. 47. DeRyckere, D., and J. DeGregori. 2005. E2F1 and E2F2 are differentially re- 18. Choubey, D., J. Snoddy, V. Chaturvedi, E. Toniato, G. Opdenakker, A. Thakur, quired for homeostasis-driven and antigen-induced T cell proliferation in vivo. H. Samanta, D. A. Engel, and P. Lengyel. 1989. Interferons as gene activators. J. Immunol. 175: 647–655. Indications for repeated gene duplication during the evolution of a cluster of 48. Urist, M., and C. Prives. 2002. p53 leans on its siblings. Cancer Cell. 1: 311–313. interferon-activatable genes on murine chromosome 1. J. Biol. Chem. 264: 49. Yan, D. H., A. Abramina, Z. Li, Y. Ding, Y. Wen, T. J. Liu, and K. Hunt. 2003. 17182–17189. P202, an interferon-inducible protein, inhibits E2F1-mediated apoptosis in pros- 19. Choubey, D., and P. Lengyel. 1993. Interferon action: cytoplasmic and nuclear tate cancer cells. Biochem. Biophys. Res. Commun. 303: 219–222. localization of the interferon-inducible 52-kD protein that is encoded by the 50. Hershko, T., and D. Ginsberg. 2003. Up-regulation of Bcl-2 homology 3 (BH3)- Ifi202 gene from the gene 200 cluster. J. Interferon Res. 13: 43–52. only proteins by E2F1 mediates apoptosis. J. Biol. Chem. 279: 8627–8634. 20. Choubey, D. 2000. p202: an interferon-inducible negative regulator of cell 51. Xin, H., S. D’Souza, T. N. Jorgensen, A. T. Vaughan, P. Lengyel, B. L. Kotzin, growth. J. Biol. Regul. Homeostatic Agents 14: 187–192. and D. Choubey. 2006. Increased expression of Ifi202, an IFN-activatable gene, 21. Choubey, D., and B. L. Kotzin. 2002. Interferon-inducible p202 in the suscepti- in B6.Nba2 lupus susceptible mice inhibits p53-mediated apoptosis. J. Immunol. bility to systemic lupus. Front. Biosci. 7: e252–e262. 176: 5863–5870.