prmt1-mediated arginine methylation of pias1 regulates stat1

10.1101/gad.489409Access the most recent version at doi: 2009 23: 118-132Genes Dev.

Susanne Weber, Florian Maaß, Michael Schuemann, et al. signalingPRMT1-mediated arginine methylation of PIAS1 regulates STAT1

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PRMT1-mediated arginine methylationof PIAS1 regulates STAT1 signaling

Susanne Weber,1 Florian Maaß,1 Michael Schuemann,2 Eberhard Krause,2 Guntram Suske,1

and Uta-Maria Bauer1,3

1Institute of Molecular Biology and Tumor Research (IMT), Philipps-University of Marburg, 35032 Marburg, Germany;2Leibniz Institute of Molecular Pharmacology, 13125 Berlin, Germany

To elucidate the function of the transcriptional coregulator PRMT1 (protein arginine methyltranferase 1) ininterferon (IFN) signaling, we investigated the expression of STAT1 (signal transducer and activator oftranscription) target genes in PRMT1-depleted cells. We show here that PRMT1 represses a subset of IFNg-inducible STAT1 target genes in a methyltransferase-dependent manner. These genes are also regulated by theSTAT1 inhibitor PIAS1 (protein inhibitor of activated STAT1). PIAS1 is arginine methylated by PRMT1 in vitro aswell as in vivo upon IFN treatment. Mutational and mass spectrometric analysis of PIAS1 identifies Arg 303 as thesingle methylation site. Using both methylation-deficient and methylation-mimicking mutants, we find thatarginine methylation of PIAS1 is essential for the repressive function of PRMT1 in IFN-dependent transcriptionand for the recruitment of PIAS1 to STAT1 target gene promoters in the late phase of the IFN response.Methylation-dependent promoter recruitment of PIAS1 results in the release of STAT1 and coincides with thedecline of STAT1-activated transcription. Accordingly, knockdown of PRMT1 or PIAS1 enhances the anti-proliferative effect of IFNg. Our findings identify PRMT1 as a novel and crucial negative regulator of STAT1activation that controls PIAS1-mediated repression by arginine methylation.

[Keywords: Transcriptional repression; interferon signaling; STAT1; PIAS1; PRMT1; arginine methylation]

Supplemental material is available at http://www.genesdev.org.

Received May 26, 2008; revised version accepted October 27, 2008.

Interferons (IFNs) constitute a family of secreted auto-crine and paracrine proteins that regulate innate andadaptive immunity in response to viral and microbial in-fections and modulate proliferation, survival, and apo-ptosis of normal cells as well as tumor cells (Borden et al.2007). IFNs exert their effects by binding to cell surfacereceptors, which triggers receptor dimerization/oligomer-ization, allowing transphosphorylation and activation ofintracellular receptor-associated tyrosine kinases of theJAK (Janus kinase) family. Activated JAKs phosphorylatecritical tyrosine residues on the receptor, which results inthe recruitment of a family of nucleo–cytoplasmic shut-tling transcription factors, the STAT (signal transducerand activator of transcription) proteins (Levy and Darnell2002; Schindler et al. 2007). In the following, receptor-bound STATs are activated by phosphorylation of a singletyrosine residue within the C terminus leading to STATdimerization via a reciprocal phosphotyrosine-SH2 (srchomology 2) interaction. The STAT dimers are retained inthe nucleus, where they bind specific DNA sequences inpromoter regions of target genes and induce gene tran-scription (Meyer and Vinkemeier 2004). Among the seven

mammalian STAT proteins STAT1 and STAT2 are medi-ators of the IFN signaling pathway, whereas the remain-ing STAT family members respond to distinct cytokines.Type I IFNs, such as IFNa and IFNb, activate STAT1/STAT2 heterodimers, and type II IFNg mainly signals viaSTAT1 homodimers (Borden et al. 2007).

Proper duration and magnitude of IFN-induced geneactivation is crucial, as abnormal and imbalanced JAK–STAT activation is associated with immune disorders andcancer (Darnell 2002; Levy and Darnell 2002). Therefore,cells have evolved mechanisms to allow rapid and tran-sient activation and to prevent excess responses to IFNsand other cytokines. Restriction and inactivation of theSTAT pathway is accomplished by regulators such asprotein tyrosine phosphatases, which inactivate JAKs andSTATs in the cytoplasm or in the nucleus (Levy andDarnell 2002). In addition, SOCS (suppressors of cytokinesignaling) proteins switch off the activity of JAKs by actingas pseudosubstrates or by recruiting the ubiquitinationmachinery (Alexander and Hilton 2004). The PIAS (proteininhibitor of activated STAT) protein family operates inthe nucleus and the family member PIAS1 interacts withactivated STAT1 and suppresses the DNA-binding abilityof the transcription factor (Liu et al. 1998; Shuai and Liu2005). PIAS1 depletion causes increased expression of asubset of STAT1 target genes (Liu et al. 2004). In agreement

3Corresponding author.E-MAIL [email protected]; FAX 0049-6421-2865196.Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.489409.

118 GENES & DEVELOPMENT 23:118–132 � 2009 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/09; www.genesdev.org




with this observation, PIAS1 knockout mice reveal en-hanced protection against pathogenic infections (Liu et al.2004). Whether the Sumo E3 ligase activity of PIAS1 andsumoylation of STAT1 is required for its repressive activityon IFN-inducible gene expression is controversial in theliterature (Rogers et al. 2003; Ungureanu et al. 2003, 2005;Song et al. 2006). Until now, little was known about themolecular mechanism leading to activation of PIAS1,which then restrains STAT1 activity, and how PIAS1interferes with the chromatin binding of STAT1.

Arginine methylation is catalyzed by a family of en-zymes, called PRMTs (protein arginine methyltransferases),consisting of 11 members (Boisvert et al. 2005; Pal and Sif2007). These enzymes share a central highly conservedcatalytic domain and perform mono- and dimethylationof the guanidino group of the arginine residues using S-adenosylmethionine (SAM) as methyl donor (Bedford andRichard 2005). PRMTs regulate various cellular processessuch as RNA processing, nucleo–cytoplasmic transport,DNA repair, transcription, and signal transduction. Sev-eral reports implicate arginine methylation in the regu-lation of IFN signaling: PRMT1 interacts with thecytoplasmic domain of the IFNa receptor, and its de-pletion alters IFNa-induced growth arrest (Abramovichet al. 1997; Altschuler et al. 1999). Tyrosine phosphory-lation, transcriptional activity, and protein stability ofSTAT transcription factors were suggested to be influ-enced by arginine methylation (Mowen et al. 2001; Zhuet al. 2002; Chen et al. 2004). However, arginine methyl-ation of STAT transcription factors itself was not confirmedby other groups, and the nonspecific methyltransferaseinhibitor MTA, which was commonly used to study thein vivo relevance of arginine methylation in this pathway,executes strong side effects and inhibits IFN-inducedphosphorylation and activation of STATs (Meissneret al. 2004; Komyod et al. 2005). Consequently, how ex-actly PRMTs and arginine methylation might regulateIFN signaling, is so far unclear.

Here, we investigated the influence of PRMT1 on IFNsignaling and found that PRMT1 represses a subset ofSTAT1 target genes that overlaps with genes regulated byPIAS1. PRMT1 methylates PIAS1 at Arg 303 (R303) in vitroand in vivo. Methylation-deficient and methylation-mimicking PIAS1 mutants revealed that PIAS1 methylationis essential for the repressive function of PRMT1 in IFN-inducible transcription and allows the recruitment of PIAS1to STAT1 target gene promoters in the late phase of the IFNresponse. Our findings identify PRMT1 as a novel inhibitorof IFN signaling that is involved in turning-off STAT1activation by regulating the repressive function of PIAS1.

Results

PRMT1 coregulates PIAS1-dependent STAT1target genes

Since the role of arginine methylation in IFN signaling iscontroversial (Abramovich et al. 1997; Mowen et al. 2001;Meissner et al. 2004; Komyod et al. 2005), we investigatedthe potential role of PRMT1 in this pathway. We depleted

PRMT1 in HeLa cells using transient transfection of twodifferent siRNAs directed against the PRMT1 transcript(siPRMT1_1 and siPRMT1_2). The efficient depletion ofendogenous PRMT1 by the two alternative siRNAs incomparison with the control siRNA (siScr) was deter-mined on RNA (Fig. 1A) and protein level (Fig. 1B). Theexpression of STAT1 or PIAS1 was not altered by knock-down of PRMT1 (Fig. 1A,B). To examine the effect ofPRMT1 on IFNg-inducible STAT1-dependent transcrip-tion we performed RT-QPCR of known STAT1 targetgenes (Ramana et al. 2002) in control (siScr) and PRMT1-depleted HeLa cells either unstimulated (0 h) or stimu-lated with IFNg for 2, 4, and 6 h. As shown in Figure 1C,the IFNg-inducible transcription of several STAT1 tar-gets, including CXCL9 (Cxc chemokine ligand 9),CXCL10 (Cxc chemokine ligand 10), and GBP1 (guany-late-binding protein 1), was enhanced threefold to 10-foldupon knockdown of PRMT1 using either of the twosiRNAs against PRMT1. In contrast, the basal transcriptlevel of these genes was not affected by PRMT1 depletion.IFN induction of other STAT1 target genes, like IRF1 (IFNresponse factor 1) and TAP1 (transporter associated withanti-gene presentation 1), was not significantly influ-enced by PRMT1 knockdown, suggesting that tran-scriptional activation of only a subset of STAT1 targetgenes is repressed by PRMT1 (Fig. 1C). The PRMT1-dependent STAT1 targets identified here appeared tomatch the subset of IFN-induced genes selectively con-trolled by the inhibitor of STAT1 signaling, PIAS1 (Liuet al. 2004). We asked whether siRNA-mediated depletionof the PIAS1 protein would affect the same subset ofIFNg-dependent STAT1 target genes as PRMT1 depletiondoes. Indeed, we found that PIAS1 knockdown, whichwas verified by Western blot (Fig. 1D), enhanced the IFN-inducible transcription of the PRMT1-dependent STAT1targets CXCL9, CXCL10, and GBP1, whereas PRMT1-independent genes, like IRF1 and TAP1, were not im-paired by suppression of PIAS1 (Fig. 1E). We obtainedsimilar results for the IFN-responsive A375 melanoma(Supplemental Fig. S1) and HEK293 cells (data not shown).Taken together, these data identify PRMT1 and PIAS1 asnegative regulators of the same STAT1 target genes.

PRMT1 acts upstream of PIAS1 in STAT1 signaling

In the following, we studied whether PRMT1 and PIAS1synergize in their repressive function on STAT1-medi-ated transcription. We performed single and doubleknockdown of PRMT1 and PIAS1 in HeLa cells (Supple-mental Fig. S2A). Using RT-QPCR we found that thesingle and double depletion results in a similar increasein IFN-induced transcription of the PRMT1/PIAS1-dependent STAT1 targets. Double depletion of PRMT1and PIAS1 did not lead to an additive effect comparedwith the effects of single depletion of PRMT1 or PIAS1(Supplemental Fig. S2B). As expected, the PRMT1/PIAS1-independent targets were not influenced by the doubledepletion (Supplemental Fig. S2B). These observationssuggest that PRMT1 and PIAS1 functionally depend oneach other in the STAT1 pathway.

Arginine methylation of PIAS1

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To further dissect the relationship between PRMT1and PIAS1 we expressed Myc-tagged PRMT1 by transienttransfection in HEK293 cells (Fig. 1F) and investigatedIFN-induced transcription. Overexpression of PRMT1diminished transcriptional induction of specifically the

PRMT1/PIAS1-dependent subgroup of STAT1 targetgenes (Fig. 1G), reinforcing our findings in PRMT1-de-pleted cells (Fig. 1C). When we depleted PIAS1 in cellsoverexpressing PRMT1 (Fig. 1F), the repressive effectof exogenous PRMT1 was abrogated and transcriptional

Figure 1. PRMT1 and PIAS1 regulate the same subset of STAT1 target genes. (A–C) HeLa cells were transfected with control siRNA(siScramble, siScr) or with two alternative siRNAs against PRMT1 (siPRMT1_1 and siPRMT1_2). After 48 h, cells were induced with 5ng/mL IFNg for the indicated time points. Subsequently cells were harvested and total RNA or protein extracts were prepared. (A) RT-QPCR was performed for detection of transcript levels of PRMT1 or PIAS1, respectively. Results were normalized using GAPDHmRNA level as a reference. Transcript levels in uninduced control cells were set to 1. (B) For detection of knockdown efficiency, proteinlevels of PRMT1, STAT1, and b-tubulin were analyzed by Western blot from 20 mg of protein extract (not induced with IFNg). Fordetection of PIAS1, 80 mg of extract were used. (C) Total RNA was analyzed for transcript levels of CXCL9, CXCL10, GBP1, IRF1, andTAP1 normalized to GAPDH. Transcript levels in uninduced control cells were set to 1. (D,E) siRNA-mediated control (siScr) and PIAS1knockdown (siPIAS1) was performed in HeLa cells. IFNg induction was conducted as in A–C. RNA and protein extracts were prepared.(D) PIAS1 knockdown was determined by Western blot from uninduced extracts. (E) Transcript levels of STAT1 target genes wereanalyzed by RT-QPCR as described in C. (F,G) HEK293 cells were transfected with control siRNA (siScr) or siRNA against PIAS1, and asindicated, after 24 h cells were transfected with Myc-tagged PRMT1. After 48 h, cells were induced with 5 ng/mL IFNg for the indicatedtime points. Subsequently, cells were harvested for the preparation of total RNA or protein extracts. (F) Efficiency of PRMT1overexpression and PIAS1 knockdown was analyzed by Western blot for Myc-tag, PIAS1, and b-tubulin in uninduced extracts. (G)Transcript levels of STAT1 target genes were analyzed by RT-QPCR as described in C.

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induction of the PRMT1/PIAS1-dependent STAT1 tar-get genes was enhanced to a similar extent as with thesole depletion of PIAS1 (Fig. 1G). These results indicatethat PRMT1 operates upstream of PIAS1 in this path-way and requires PIAS1 to inhibit IFNg-dependent tran-scription.

Endogenous PRMT1 and PIAS1 proteins interactand are recruited to STAT1 target genes in the latephase of the IFN response

Next, we investigated whether the functional coopera-tion between PRMT1 and PIAS1 could reflect interactionof both proteins. We performed GST pulldown experi-ments of Flag-EGFP- (control) or Flag-PIAS1-overexpress-ing HEK293 cell extracts with GST alone and GST-PRMT1 (Fig. 2A). Pulldowns were analyzed by anti-FlagWestern blot and revealed a specific interaction betweenPRMT1 and PIAS1 in vitro (Fig. 2A). Using recombinantHis-tagged PIAS1 protein we demonstrated in furtherpulldown assays with GST-PRMT1 that the two proteinsinteract directly (Fig. 2B).

We then determined whether the two proteins bindalso endogenously to each other and how such an in-teraction might be influenced in the course of the IFNresponse. HeLa cells were left untreated or treated for 3

and 6 h with IFNg. Nuclear extracts of these cells weresubjected to anti-PIAS1 immunoprecipitation (IP). Immu-noprecipitates were probed for the presence of PRMT1 byWestern blot analysis. PRMT1 und PIAS1 associated veryweakly in the uninduced state and at the 3-h inductiontime point, whereas the interaction of the two proteinswas enhanced after 6 h of IFNg treatment (Fig. 2C). Whenwe examined the PIAS1 precipitates for the presence ofSTAT1, PIAS1 was found to associate with STAT1 after6 h of IFNg treatment in agreement with other reports,which also showed an IFN-dependent interaction (Liuet al. 1998). Essentially the same results were obtained,when we included ethidium bromide in the IP (Laiand Herr 1992) indicating that the interaction betweenPIAS1, PRMT1, and STAT1 was not DNA-mediated (datanot shown). IFNg stimulation did not affect the proteinlevels of the three proteins, as shown by Western blotanalysis of the corresponding nuclear extracts (Fig. 2D).These data strongly suggest that PIAS1 preferentially in-teracts with PRMT1 and STAT1 in the late phase of theIFN response.

Subsequently, we explored whether PRMT1 and PIAS1are recruited to STAT1 target gene promoters. We per-formed chromatin IP (ChIP) analysis in either uninducedcells or 3, 6, and 9 h after addition of IFNg usingantibodies against STAT1, PIAS1, and PRMT1 and assayed

Figure 2. PRMT1 interacts with PIAS1and binds simultaneously with PIAS1 toSTAT1 target gene promoters in the latephase of the IFNg response. (A) Proteinlysates of Flag-EGFP- (control) or Flag-PIAS1-overexpressing HEK293 cells weresubjected to GST pulldown experiments.(Left panel) Equal amounts of GST aloneand GST-PRMT1 protein, as indicatedin the Coomassie-stained SDS gel (full-length protein bands marked with aster-isks), coupled to glutathione beads wereincubated with 250 mg of protein lysates.(Right panel) Bound PIAS1 protein wasvisualized by Western blot using Flagantibody. (Middle panel) One percent in-put (2.5 mg) of HEK293 cell extract isshown. (B) GST pulldown with recom-binant His-tagged PIAS1 purified fromSf9 cells were performed using GSTand GST-PRMT1 protein. Bound PIAS1protein was detected by anti-His-tag West-ern blot. Five percent input of recom-binant PIAS1 is shown. (C,D) Co-IPs ofendogenous PIAS1, PRMT1, and STAT1were performed. HeLa cells were inducedwith IFNg for the indicated time points

and nuclear extracts were prepared. (C) IP of PIAS1 (anti-PIAS1) or control IP (IgG) was carried out from 1 mg of nuclear extract andexamined by Western blot using antibodies against PRMT1, STAT1, and PIAS1. (D) Input Western blots corresponding to CoIP in Cwere stained for PIAS1, PRMT1, P-STAT1, and STAT1. (E–G) Recruitment of STAT1, PIAS1, and PRMT1 to STAT1 target genepromoters was investigated by ChIP. HeLa cells were treated in a time course experiment with IFNg (5 ng/mL). Subsequently, cells wereharvested and subjected to ChIP analysis using control antibodies (IgG) and antibodies against STAT1, PIAS1, and PRMT1.Immunoprecipitated DNA was analyzed in triplicates by QPCR with primers for the CXCL9 (E), CXCL10 (F), and IRF1 (G) genepromoters and displayed as percentage input. Standard deviation was calculated from the triplicates, and error bars are indicatedaccordingly. Representative figure legend is show in E.






for the enrichment of the PRMT1/PIAS1-dependentSTAT1 target genes CXCL9 and CXCL10 and the PRMT1/PIAS1-independent target IRF1. STAT1 binding to theCXCL9 and CXCL10 promoter peaked at 3 h IFNg treat-ment, gradually declined between 6 and 9 h and reachedthe basal (uninduced) level of recruitment at the 9 h timepoint (Fig. 2E,F). Consistent with this detachment ofSTAT1 from the CXCL9 and CXCL10 promoter and thedecrease in transcriptional induction in the late phase ofIFN treatment (Supplemental Fig. S3), PIAS1 and PRMT1bound to both promoters after 6 and 9 h of IFN induction(Fig. 2E,F). In contrast, the promoter of the PRMT1/PIAS1-independent STAT1 target gene IRF1 was neveroccupied by PIAS1 or PRMT1 throughout the wholeIFN time course, but STAT1 was strongly enriched after3 and 6 h of IFNg stimulation (Fig. 2G). These resultsindicate that both PRMT1 and PIAS1 are simultaneouslyrecruited to the PRMT1/PIAS1-dependent genes in thelate phase of IFNg signaling, which coincides with the

detachment of STAT1 and with the gradual decline oftranscriptional activation.

PRMT1 is required for promoter recruitment of PIAS1and promoter detachment of STAT1

To test whether PRMT1 influences the promoter recruit-ment of PIAS1 we conducted ChIP analysis in PRMT1-depleted cells and control (siScr) cells, which were eitheruntreated or IFN-treated. PRMT1 depletion in the corre-sponding chromatin samples was verified by Western blot(Fig. 3A). In control cells STAT1, PIAS1, and PRMT1binding to the CXCL9 and CXCL10 promoter overlappedafter 6 h of IFNg stimulation (Fig. 3B, top and middlepanels), in agreement with Figure 2, E and F. Depletionof PRMT1 resulted in a loss of PIAS1 recruitment and inan enhanced and prolonged recruitment of STAT1 to theCXCL9 and CXCL10 promoter during the IFN response(Fig. 3B, top and middle panels). In contrast association of

Figure 3. PIAS1 and STAT1 promoter recruitment depends on PRMT1. (A–C) HeLa cells were transfected with control siRNA orsiRNA against PRMT1 and PIAS1, respectively. After 48 h, cells were induced with IFNg (5 ng/mL) for the indicated time points andsubjected to lysis for ChIP analysis. Depletion of PRMT1 and PIAS1 was determined by Western blot of the corresponding chromatinsamples (A). Promoter recruitment of STAT1, PIAS1, and PRMT1 to STAT1 target gene promoters (CXCL9, CXCL10, and IRF1) wasdetermined by ChIP-QPCR in PRMT1 or PIAS1 knockdown cells (B,C). Immunoprecipitated DNA was analyzed in triplicates by QPCRand results were displayed as percentage input.

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STAT1 with the PRMT1/PIAS1-independent IRF1 pro-moter was not influenced by PRMT1 suppression (Fig. 3B,bottom panel). A similar effect on STAT1 binding wasalso observed in PIAS1-depleted cells, in which IFN-induced STAT1 binding of the CXCL9 and CXCL10promoter was increased (Fig. 3A,B, top and middle pan-els), but binding of the IRF1 promoter was unchanged(Fig. 3B, bottom panel). PIAS1 knockdown did not affectthe recruitment of PRMT1 to the CXCL9 and CXCL10gene promoter (Fig. 3B, top and middle panels) corrobo-rating our observation that PRMT1 acts upstream ofPIAS1 in this pathway (Fig. 1G). However, PRMT1 pro-moter recruitment requires IFNg-induced binding ofSTAT1 to its target genes, as ChIP analysis in STAT1-deficient U3A cells revealed (Supplemental Fig. S4). Tostrengthen our observation that PRMT1 and PIAS1 reg-ulate the magnitude and kinetic of STAT1 promoterbinding we performed ChIP analysis for STAT1 in a moredetailed IFN induction time course including additionaltime points. As shown in Figure 3C, depletion of PRMT1or PIAS1 resulted in an increased and prolonged STAT1binding with a clearly delayed peak of STAT1 recruitmentto a subset of target genes. These results indicate thatPRMT1 regulates PIAS1-mediated repression of STAT1target genes at the chromatin level by allowing promoterrecruitment of PIAS1, which subsequently diminishespromoter association of STAT1.

Recent studies suggested a role of PIAS1-mediatedsumoylation of STAT1 in the repression of STAT1 signal-ing (Ungureanu et al. 2003, 2005). We asked whetherPRMT1 has an impact on the sumoylation of STAT1.PRMT1 depletion did not change the levels of sumoylatedSTAT1 (Supplemental Fig. S5) indicating that PRMT1 doesnot control the sumoylation activity of PIAS1. Moreover,overexpression of the sumoylation-deficient PIAS1 mu-tant W372A (Liu et al. 2007) revealed a similar repressiveeffect on the IFN induction of the same STAT1 targetgenes as wild-type PIAS1 (Supplemental Fig. S6). Theseresults imply that sumoylation activity by PIAS1 is notessential for transcriptional inhibition of the STAT1target genes investigated here.

The methyltransferase activity of PRMT1 is requiredfor its repressive function in STAT1 signaling

As PRMT1 possesses arginine methyltransferase activitytoward histones and other nuclear proteins (Wang et al.2001; Kwak et al. 2003; Barrero and Malik 2006) westudied in the following whether the catalytic activityof PRMT1 is required for its repressive function in STAT1-dependent transcription. Therefore, we established tran-sient overexpression of wild-type PRMT1 (WT) and thecatalytically inactive PRMT1 mutant XGX (Balint et al.2005), which were equally well expressed (Fig. 4A). Asexpected IFNg-induced expression of CXCL9, CXCL10,and GBP1 was diminished in the presence of exogenouswild-type PRMT1 compared with control cells (Fig. 4B). Incontrast, the catalytically inactive PRMT1 mutant lostits repressive capacity and transcriptional induction ofCXCL9, CXCL10, and GBP1 occurred to a similar extent

as in control cells (Fig. 4B) leading to the conclusion thatthe catalytic activity of PRMT1 is required for its in-hibitory effect on STAT1-mediated transcription.

We next asked whether PRMT1 and other PRMTswould possess methyltransferase activity toward PIAS1.Therefore, we employed an in vitro methylation assayusing recombinant baculovirus-expressed His-tagged PIAS1,GST-PRMTs, or immunopreciptitated PRMT5 from cellextracts as enzyme source and radiolabeled methyl-donor SAM (Fig. 4C; data not shown). This assay revealedthat PIAS1 is in vitro methylated by PRMT1, PRMT4,and PRMT6 (Fig. 4C). The catalytic activity of thevarious enzyme preparation was verified using estab-lished substrates in an in vitro methylation assay (Fig.4D; data not shown).

To address the question whether PRMT4 and PRMT6,which methylate PIAS1 in vitro as well, influence IFN-induced STAT1 target gene expression in a similar way asPRMT1, we depleted PRMT4 and PRMT6, respectively,using transient transfection of siRNAs in HeLa cells(Supplemental Fig. S7A,C). Transcriptional induction ofthe PRMT1/PIAS1-dependent STAT1 target genes CXCL9and GBP1 was not impaired by knockdown of neitherPRMT4 (Supplemental Fig. S7B) nor PRMT6 (Supplemen-tal Fig. S7D). These data demonstrate that among thePRMTs capable of methylating PIAS1 in vitro, PRMT1 isthe only enzyme that cooperates with PIAS1-dependentrepression in a methyltransferase-dependent mannerin vivo.

Arg 303 in PIAS1 is methylated by PRMT1 in vivo

To investigate the occurrence of in vivo methylation ofPIAS1 we performed metabolical labeling of HeLa cellswith L-[methyl-3H]-methionine. Cells were transfectedwith Flag-tagged PIAS1 and additionally with controlsiRNA (siScr) or with siRNA targeting PRMT1 (siPRMT1).Subsequently, cells were stimulated with IFNg or not andsimultaneously metabolical labeling was performed for3 h in the presence of translational inhibitors. Flag-PIAS1protein was immunopurified from the cell extracts, re-solved by SDS-PAGE and methylation was detected byfluorography. PIAS1 was methylated in response to IFNg

treatment in the control cells, whereas knockdown ofPRMT1 resulted in the loss of PIAS1 methylation (Fig. 5A).The efficiency of PIAS1 overexpression, PRMT1 deple-tion, and IFNg stimulated tyrosine 701 phosphorylationof STAT1 was verified by Western blot analysis (Fig. 5A).To confirm that translational inhibition avoided incorpo-ration of the radiolabeled methionine by de novo proteinbiosynthesis (Liu and Dreyfuss 1995), cells were incu-bated with 35S-methionine and protein synthesis inhib-itors. No label was detectable in total protein lysatefrom these cells by fluorography (Supplemental Fig. S8)indicating that radiolabeling of PIAS1 was indeed dueto post-translational modification. Similar results for invivo methylation of the endogenous PIAS1 protein wereobtained (Fig. 5B), whereas methylation of STAT1, whichwas reported by Mowen et al. (2001), was not detectablein this assay (Fig. 5C). These data indicate that in vivomethylation of PIAS1 occurs in response to IFNg treatment






and that PRMT1 is the responsible enzyme for PIAS1methylation in this pathway.

In order to map the methylation site(s) in PIAS1, weexpressed several N- and C-terminal deletion constructsof PIAS1 (Fig. 5D) as His-tagged fusions in bacteria(Fig. 5E). In vitro methylation in the presence of GST-PRMT1 revealed that the PIAS1 deletion DNDC is thesmallest fragment still methylated by PRMT1 (Fig. 5E).This indicates that the methylation site(s) of PRMT1 inPIAS1 are located between amino acid 262 and 384,which encompass the RING finger domain and the acidicdomain.

To identify the exact site(s) of PRMT1 methylationin PIAS1 we methylated PIAS1 DNDC in the presenceof GST-PRMT1 in vitro. The ESI mass spectrum revealeda doubly charged peak at m/z 540.26 (calculated m/z540.29) corresponding to the dimethylated sequence ofthe tryptic PIAS1 fragment GIRNPDHSR (amino acids301–309) (Fig. 5F). Fragment ion (MS/MS) spectrumresulting from this doubly charged precursor clearly in-dicated that the first arginine within the peptide (R303 inthe full-length context of PIAS1) is dimethylated (Fig. 5G).Mutation of the R303 to alanine (R303A) confirmed thisarginine, which is localized between the PINIT motif andthe RING finger domain, as the in vitro methylation siteof PIAS1 DNDC protein by PRMT1 (Fig. 5H).

To validate R303 also as the in vivo methylation siteof PIAS1 by PRMT1, we mutated R303 in the full-length PIAS1 to lysine (R303K) or glycine (R303G). Sub-sequently, we performed overexpression and metabolical

labeling of both Flag-tagged mutants or wild-type Flag-tagged PIAS1 (WT) in unstimulated and IFNg-stimulatedHeLa cells. Immunoprecipitates of PIAS1 wild typerevealed in vivo methylation of PIAS1 upon IFNg stim-ulation, whereas both PIAS1 mutants, R303K and R303G,were not methylated in the absence or in the presenceof IFNg (Fig. 5I). These results indicate that PIAS1 ismethylated upon IFNg stimulation at a single arginine,R303, in vivo.

Methylation of R303 in PIAS1 is required for itsrepressive effect on STAT1 and for its recruitmentto STAT1 target gene promoters

To investigate the impact of PIAS1 methylation on itsrepressive activity in STAT1-activated transcription weexplored overexpression of PIAS1 wild type, a methyla-tion-deficient PIAS1 mutant (R303K), and a methyla-tion-mimicking mutant, in which the arginine wasmutated to phenylalanine (R303F) (Mostaqul Huq et al.2006). The mutations of R303 did not affect the sumoy-lation activity of PIAS1 (Supplemental Fig. S9), itsnuclear localization, or the cellular localization ofSTAT1, and its IFN-induced shuttling (SupplementalFig. S10). Subsequent to transfection of PIAS1 wild typeand the two mutants, IFNg treatment was performedand STAT1 target gene expression was analyzed byRT-QPCR. Overexpression of PIAS1 wild type and themethylation-mimicking PIAS1 R303F mutant decreasedthe IFN-induced transcription of PRMT1/PIAS1-depen-dent targets compared with control transfected cells

Figure 4. The arginine methyltransferase activity of PRMT1 is required for its repressive function in IFN-dependent transcription, andPIAS1 is arginine methylated in vitro. (A,B) HeLa cells were transfected either with PRMT1 wild type (WT) or PRMT1 catalytic inactivemutant (XGX). After 48 h, cells were induced with 5 ng/mL IFNg for the indicated time points. Subsequently cells were harvested forthe preparation of total RNA or protein extracts. (A) Overexpression of PRMT1 WT and PRMT1 XGX in uninduced cell extracts wasmonitored by Western blot using anti-Myc antibody. (B) Transcript levels of the indicated STAT1 target genes were analyzed with RT-QPCR and normalized to GAPDH. Transcript levels in uninduced control cells were set 1. (C) In vitro methylation assays of His-taggedPIAS1 purified from Sf9 cells (as indicated in the Coomassie-stained gel in the left panel) were performed with recombinant GST-PRMTs in the presence of methyl-14C-labeled SAM. Methylation products were resolved on SDS-PAGE, blotted, and visualized byautoradiography. (D) As positive control for the enzymatic activity of the GST-PRMT preparations in C, in vitro methylation assayswere perfomed using established substrates of PRMTs, like GST-GAR protein (glycine–arginine-rich domain of fibrillarin) or bulkhistones, and analyzed by autoradiography.

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(Fig. 6A). In contrast, the methylation-deficient PIAS1R303K mutant exhibited a relief of this repression andresulted in a transcriptional induction of the PRMT1/PIAS1-dependent targets comparable with control cells

(Fig. 6A). Expression of the different PIAS1 proteins didnot affect the IFN-induced transcriptional activation ofPRMT1/PIAS1-independent STAT1 target genes (Fig.6A). These findings demonstrate that PRMT1-mediated

Figure 5. PRMT1 methylates R303 inPIAS1 in vivo. (A) HeLa cells were firsttransfected with control siRNA (siScr) orsiRNA against PRMT1 (siPRMT1_1 wasused). After 24 h cells were transfectedwith Flag-tagged PIAS1, and 48 h latercells were induced with IFNg for 3 h (+) ornot (�) and simultaneously labeled withL-(methyl-3H) methionine in the presenceof translation inhibitors. (Top panel) Sub-sequently, Flag-tagged PIAS1 was immu-noprecipitated from cell lysates andanalyzed by SDS-PAGE followed by fluo-rography. (Bottom panels) Effective knock-down of PRMT1 and overexpression ofPIAS1 was checked by Western blot. (B)HeLa cells were transfected with controlsiRNA (siScr) or siRNA against PRMT1(siPRMT1_1 was used). After 72 h cellswere induced with IFNg for 3 h (+) or not(�) and simultaneously labeled with L-(methyl-3H) methionine in the presenceof translation inhibitors. (Top panel) Sub-sequently, endogenous PIAS1 was immu-noprecipitated from cell lysates andanalyzed by SDS-PAGE followed by fluo-rography. (Bottom panels) Efficient knock-down of PRMT1 and endogenous PIAS1and STAT1 levels were checked by West-ern blot. (C) HeLa cells were induced withIFNg for 3 h (+) or not (�) and simulta-neously labeled with L-(methyl-3H) me-thionine in the presence of translationinhibitors. (Top panel) Subsequently, en-dogenous STAT1 was immunoprecipitatedfrom cell lysates and analyzed by SDS-PAGE followed by fluorography. (Bottom

panels) The endogenous STAT1 and PRMT1levels were checked by Western blot.(D) Schematic representation of PIAS1deletion constructs used in E. Conserveddomains are indicated: The N-terminalSAP box (Saf-A/B, Acinus, PIAS) is re-quired for DNA-binding, PINIT motif isinvolved in nuclear retention of certain

family members, RING finger-like domain is essential for Sumo ligase activity and the acidic domain is important for protein–proteininteractions (Duval et al. 2003; Shuai and Liu 2005). (E) His-tagged PIAS1 deletion constructs were purified from E. coli, and equalamounts were used for in vitro methylation with recombinant GST-PRMT1 in the presence of methyl-14C-labeled SAM. Coomassiestaining illustrates the protein amounts used for the methylation assay (left panel). Methylation products were resolved on SDS-PAGE,blotted, and visualized by autoradiography (right panel). PIAS1-specific methylation products are marked with asterisks and nonspecificmethylation products with a circle. (F,G) PIAS1 DNDC was in vitro methylated by recombinant PRMT1 and subsequently analyzedby mass spectrometry. (F) The NanoESI-MS spectrum of the tryptic fragment 301GIRNPDHSR309 revealed a peak with m/z 540.26(calculated m/z 540.29) corresponding to the doubly charged ion of the dimethylated sequence. (G) Fragment ion (MS/MS) spectrumresulting from the doubly charged precursor with m/z 540.26 produced y ions and the b ions by consecutive fragmentation reactions.Relevant ions were labeled according to the accepted nomenclature. Dimethylation of Arg 303 was detected in the mass of the b3, b4,b6, and b7 ions. (H, right panel) In vitro methylation of PIAS1 DNDC or the mutant PIAS1 DNDC R303A by GST-PRMT1 was monitoredby autoradiography. (Left panel) The Coomassie staining reveals that equal amounts of both PIAS1 deletion proteins were used for themethylation assay. (I) HeLa cells were transiently transfected with Flag-tagged EGFP (control), Flag-tagged PIAS1 wild type (WT), PIAS1R303K mutant, and PIAS1 R303G mutant, respectively. Metabolic labeling with L-(methyl-3H) methionine, anti-Flag IP, fluorography(top panel), and corresponding Western blot (bottom panels) were performed as in A.






methylation of R303 in PIAS1 is crucial for repression ofSTAT1-activated transcription.

To determine the impact of R303 methylation on thepromoter recruitment of STAT1 and PIAS1, and to avoidat the same time an interference of the endogenousPIAS1 protein in this assay, we first depleted the endog-enous PIAS1 expression in HeLa cells by using siRNAstargeting the 39 untranslated region (UTR) of PIAS1

(Supplemental Fig. S11A). Subsequently, we transientlytransfected the PIAS1-depleted cells with empty vector(control), Flag-PIAS1 wild type, methylation-deficientFlag-PIAS1 R303K mutant, and methylation-mimickingFlag-PIAS1 R303F mutant. The exogenous PIAS1 expres-sion was not affected by the siPIAS1 targeting the 39UTR,and the expression levels of the different PIAS1 con-structs were equal (Supplemental Fig. S11A). When we

Figure 6. Methylation of R303 in PIAS1 is crucial for PIAS1-dependent repression of STAT1 signaling and for PIAS1 promoterrecruitment. (A) HeLa cells transiently overexpressing either Flag-EGFP, Flag-PIAS1 wild type (WT), methylation-deficient Flag-PIAS1R303K mutant or methylation-mimicking Flag-PIAS1 R303F mutant were induced with IFNg (5 ng/mL) for the indicated time points.Subsequently, RNA was prepared and analyzed by RT-QPCR for transcript levels of STAT1 target genes. Results were normalized toGAPDH and transcript levels in uninduced control cells were set to 1. (B,C) HeLa cells were transfected with control siRNA (siScr) orsiRNA against the 39UTR of PIAS1 (siPIAS1 UTR = siPIAS1 UTR1 + 2). After 24 h, the siPIAS1 UTR-treated cells were transfected withempty vector (control), Flag-tagged PIAS1 wild-type (WT), R303K mutant, or R303F mutant constructs. After 48 h of this secondtransfection, cells were treated with IFN for 6 h and subjected to ChIP analysis. Recruitment of STAT1 (B) and Flag-PIAS1 (C) to theindicated gene promoters was determined by QPCR. Results were displayed as percentage input. (D) HeLa cells were transfected withFlag-tagged PIAS1 wild-type (WT), R303K mutant, or R303F mutant constructs. After 48 h, cells were treated with IFN for 6 h andsubjected to co-IP analysis. IP of Flag-PIAS1 (anti-Flag) or control IP (IgG) was carried out from 500-mg nuclear extract and examined byWestern blot using antibodies against STAT1 and Flag-PIAS1. Input Western blots of the nuclear extracts corresponding to the co-IPwere stained for P-STAT1, STAT1, Flag-PIAS1, PRMT1, and HDAC1 (the latter served as loading control).

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subjected these cells to IFNg treatment, we found thatsimilar to the siRNA targeting the PIAS1 ORF (Fig. 1E),the siPIAS1 UTR resulted in an increased transcriptionalactivation of the specific subset of STAT1 targets incomparison with siScr transfected cells (SupplementalFig. S11B). Overexpression of PIAS1 wild type and R303Fmutant rescued this effect, whereas PIAS1 R303K wasnot able to perform the rescue (Supplemental Fig. S11B).ChIP analysis of these cells revealed that IFN-inducedpromoter recruitment of STAT1 was enhanced in thesiPIAS1 UTR-transfected cells compared with siScr-transfected cells, in agreement with Figure 3B. Further,overexpression of PIAS1 wild type and methylation-mimicking R303F mutant rescued this effect; i.e., bothPIAS1 proteins decreased the STAT1 recruitment back tothe level detected in siScr-transfected cells (Fig. 6B;Supplemental Fig. S12A). In contrast, the methylation-deficient PIAS1 R303K mutant was unable to substitutefor the function of the endogenous PIAS1 protein inthe PIAS1-depleted cells and the STAT1 recruitmentremained increased (Fig. 6B; Supplemental Fig. S12A).These observations apply to the PRMT1/PIAS1-depen-dent STAT1 targets, and are consistent with the tran-scriptional effects of PIAS1 wild type and the mutants onthis particular subset of genes (Fig. 6A; Supplemental Fig.11B). In agreement with the PRMT1/PIAS1-independenttranscriptional regulation of IRF1, PIAS1 depletion aswell as overexpression of the different PIAS1 constructsdid not influence STAT1 recruitment to this promoter(Fig. 6B). When we analyzed the promoter recruitment ofPIAS1 in these cells, we noticed that the binding ofPIAS1 R303K to the PRMT1/PIAS1-dependent STAT1targets was completely lost, whereas the binding ofmethylation-mimicking PIAS1 R303F mutant was sim-ilar to PIAS1 wild type (Fig. 6C; Supplemental Fig. S12B).In agreement with the PRMT1/PIAS1-independenttranscriptional regulation of IRF1, the different PIAS1constructs did not associate with this promoter (Supple-mental Fig. S12B). These results show that PRMT1-mediated methylation of R303 in PIAS1 is essential forthe recruitment of PIAS1, for the release of STAT1 fromtarget gene promoters, and for turning off of STAT1-activated transcription.

To test whether the methylation of R303 itself contrib-utes to the interaction between PIAS1 and STAT1, weperformed co-IP experiments from cells overexpressingeither Flag-PIAS1 wild type, Flag-PIAS1 R303F, or Flag-PIAS1 R303K in the absence or presence of IFNg stimu-lation. Nuclear extracts were subjected to anti-Flag IPand, subsequently, immunoprecipitates were stained forthe presence of endogenous STAT1. As expected, theinteraction between PIAS1 wild type and STAT1 was en-hanced upon addition of IFNg (Fig. 6D). The methylation-mimicking PIAS1 R303F mutant revealed already as-sociation with STAT1 without IFNg stimulation, whichwas not further increased upon treatment with IFNg. Incontrast, the methylation-deficient PIAS1 R303K mutantexhibited reduced binding to STAT1, under uninduced aswell as induced conditions (Fig. 6D). These data suggestthat the PRMT1-mediated methylation of R303 in PIAS1

facilitates its interaction with STAT1, and in this waymight allow promoter recruitment of PIAS1.

Knockdown of PRMT1 or PIAS1 enhancesthe anti-proliferative effect of IFNg

Finally, we elucidated the impact of PRMT1-regulatedrepression by PIAS1 on the biological outcome of IFNsignaling and STAT1 function. Therefore we measuredthe growth/proliferation of HeLa cells in the presence ofdifferent concentrations of IFNg. A concentration of 15ng IFNg/mL resulted in a growth arrest, whereas at lowerconcentration of 7.5 ng IFNg/mL the anti-proliferativeeffect of IFN was not detectable (Supplemental Fig. S13)(Bromberg et al. 1996). In the following, HeLa cells weretransfected with control siRNA (siScr), siPRMT1, orsiPIAS1 and subjected to growth curve analysis in thepresence of IFNg. Depletion of endogenous PRMT1 andPIAS1, respectively, was monitored and verified by West-ern blot analysis (Supplemental Fig. S14). Relative to cellstransfected with siScr, depletion of PRMT1 or PIAS1resulted in the occurrence of anti-proliferative activityat low doses of IFNg (Fig. 7A–C) and enhanced anti-proliferative activity at high doses of IFNg (data notshown). These results demonstrate that PRMT1/PIAS1-mediated repression of STAT1 activity is important forthe biological effects of IFN.

Figure 7. Knockdown of PRMT1 or PIAS1 enhances the anti-proliferative effect of IFNg. (A–C) HeLa cells were transfectedwith control siRNA, siRNA against PRMT1 (siPRMT1_1 wasused) or against PIAS1. After 48 h, cells were trypsinized and10,000 cells per well were seeded and after 4 h induced with 7.5ng/mL IFNg. At the indicated time points, cells were harvestedand cell number was determined. For each condition and timepoint triplicates were counted. Error bars are depicted accord-ingly. (D) Model for the regulation of PIAS1-mediated repressionof STAT1 signaling by PRMT1.






Discussion

Previous reports suggested that arginine methylationinfluences IFN signaling, since PRMT1 was shown tointeract with the cytoplasmic domain of the IFNa re-ceptor (Abramovich et al. 1997) and its depletion in-terfered with IFNa-induced growth arrest (Altschuleret al. 1999). PRMT1 was reported to methylate thetranscription factor STAT1, and this was shown to inhibitthe association of STAT1 and PIAS1 and consequently toenhance transcriptional activation of IFN responsivegenes (Mowen et al. 2001; Nien et al. 2007). However,arginine methylation of STAT1 was not confirmed byothers (Meissner et al. 2004) and also not in the presentwork (Fig. 5C). Furthermore, most studies investigatingthe impact of arginine methylation in IFN signaling usednonspecific methyltransferase inhibitors, like MTA,which execute strong side effects and inhibit variouscellular kinase cascades (p38 and ERK) and the IFN-induced phosphorylation of STAT1 and STAT3 (Meissneret al. 2004; Komyod et al. 2005). Therefore, we set out inthe present study to elucidate the role of arginine meth-ylation in STAT1 signaling and identified the PIAS1protein as an important target of PRMT1 in this pathway.

Our findings reveal a novel mechanism how thenuclear inhibitor of STAT1 signaling, PIAS1, is activatedin the course of the IFN response to restrain STAT1function and thus how duration and magnitude of IFNsignaling is balanced. We demonstrate that argininemethylation by PRMT1 influences the transcriptionalactivity of STAT1 negatively by regulating the interactionbetween STAT1 and it repressor protein PIAS1. Interest-ingly, in the literature PRMT1 has been reported to act astranscriptional coactivator (Wang et al. 2001; An et al.2004; Wagner et al. 2006) as well as corepressor (Kwaket al. 2003; Yu et al. 2006). Our model for the cooperationbetween PRMT1 and PIAS1 in STAT1 signaling is sum-marized in Figure 7D. We find that PRMT1 repressesa subset of IFNg-activated STAT1 target genes, whichoverlaps with genes regulated by PIAS1 (Liu et al. 2004).PRMT1 mediates its function in STAT1 signalingthrough PIAS1, as depletion of PIAS1 abrogates the in-hibitory activity of PRMT1. In search of the molecularmechanism of this cooperation, we discover that PRMT1dimethylates PIAS1 at R303. Association between PRMT1and PIAS1 and methylation of R303 is induced in thelate phase of the IFNg response and enables gene-specificrecruitment of PIAS1. Therefore, depletion of PRMT1results in the loss of PIAS1 promoter binding. Consistently,transcriptional repression and promoter recruitment ofa methylation-deficient PIAS1 mutant is abrogated, rein-forcing that PRMT1-dependent methylation of PIAS1 isimportant for its repressive function. The promoter-associ-ated methylation of PIAS1 then triggers the interactionbetween STAT1 and PIAS1 and enforces the release ofSTAT1 from its target genes, which subsequently results inthe decline of transcriptional activation. This interpreta-tion is supported by our findings that the methylation-deficient PIAS1 mutant exhibits a decreased interactionwith STAT1 in co-IP, and that depletion of PRMT1 or

PIAS1 results in an enhanced and prolonged recruitment ofSTAT1 to a subset of its target genes.

Regulation of the repressive activity of PIAS1 is neces-sary, as the protein is predominantly localized in thenucleus. A constitutively active form of PIAS1 wouldstraight away inhibit activated STAT1 dimers, whichenter the nucleus upon stimulus. But initially, STAT1dimers need to perform their transactivation function andto enable a proinflammatory and anti-proliferative geneexpression pattern. Therefore, mechanisms must existto allow a targeted interaction between STAT1 and itsnuclear repressor protein in the late phase of the immuneresponse. Here, we describe such a mechanism: R303methylation catalyzed by PRMT1 facilitates the STAT1–PIAS1 interaction, which was reported to be a directinteraction (Liao et al. 2000). R303 is localized in a con-served but functionally not assigned part between thePINIT motif and the RING finger domain of PIAS1.Although the C-terminal portion of PIAS1 (not encom-passing the R303) is responsible for the direct interactionbetween PIAS1 and STAT1, the N-terminal part of PIAS1including R303 modulates the association between thetwo proteins. This N-terminal domain restricts the in-teraction of PIAS1 to STAT1 dimers by inhibiting itsinteraction with STAT1 monomers (Liao et al. 2000)supporting our findings that methylation of R303 isimportant for the PIAS1–STAT1 interaction. Interest-ingly, the methylation site is not embedded in a consensusmethylation sequence of PRMT1, which typically har-bors the methylated arginine in a RGG or RXR context(Smith et al. 1999). In general, methylation of the arginineresidue is thought to regulate protein–protein and pro-tein–nucleic acid interactions potentially by increasingits ‘‘hydrophobic’’ character (Boisvert et al. 2005). Meth-ylation of arginine residues occurs also in other chroma-tin proteins involved in cytokine signaling, like thePRMT1-driven methylation of the arginine–glycine richN terminus of the coactivator NIP45, which promotesthe association between NIP45 and the transcriptionfactor NFAT and results in an augmented activity of theIL-4 and IFNg gene promoter (Mowen et al. 2004).

Mechanistically, it was suggested that the interactionbetween PIAS1 and STAT1 inhibits the DNA-bindingability of STAT1 and causes the reduction of transcrip-tional activity (Liu et al. 1998; Shuai and Liu 2005).Similar data were also reported for other PIAS familymembers; for example, PIAS3 interacts with STAT3 andsuppresses the interaction of STAT3 with the DNA(Chung et al. 1997). Given that R303 is conserved alsoin other PIAS family members, arginine methylation ofPIAS proteins might play a more general role in cytokine-dependent gene regulation. PIAS1 knockout mice revealenhanced protection against pathogenic infections, al-though PIAS1 represses only a subset of 9% of the IFN-induced STAT target genes (Liu et al. 2004), indicatingthat PIAS1 is a physiologically important negative regu-lator. A common characteristic of genes regulated byPIAS1 and also PRMT1, like the GBP1, CXCL9, andCXCL10 gene, is that they contain low-affinity bindingsites of STAT1, whereas the high-affinity binding site of

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STAT1 in the IRF1 gene promoter does not depend onPIAS1 as well as PRMT1 (Liu et al. 2004).

Methylation by PRMT1 seems to be not the onlymechanism for how PIAS1 function is regulated. It wasreported recently that PIAS1 is phosphorylated at Ser 90 bythe IKKa kinase particularly in response to TNFa and LPSstimulation, and that this modification is required torepress the NF-kB/STAT1 activity (Liu et al. 2007). Similarto the regulation of PIAS1 by PRMT1, S90 phosphoryla-tion was needed to allow promoter recruitment of PIAS1in NF-kB-activated transcription and resulted in the de-tachment of NF-kB from its targets. In contrast to theregulation by PRMT1, the Sumo E3-ligase activity wasnecessary for the repressive function of PIAS1, sincea sumoylation-deficient PIAS1 mutant was defective inS90 phosphorylation (Liu et al. 2007). Interestingly, we didnot find phosphorylated PIAS1 species in response to IFNg

treatment (data not shown). Thus, the mechanism ofPIAS1 regulation might differ depending on the stimulus,transcription factor, target genes, and cell type. In thefuture it will be important to also study the role of argininemethylation of PIAS1 in its STAT1-independent functions.

In summary, our results identify PIAS1 as the crucialtarget of arginine methylation and PRMT1 in IFN signal-ing. PRMT1 is involved in turning off transcriptionalactivity of STAT1 and represses the transcription factorby regulating the interaction between STAT1 and it re-pressor protein PIAS1 in a methylation-dependent manner.Hence, PRMT1 is a very interesting target in therapies ofviral infections and also deregulated STAT signal trans-duction. How PRMT1 itself is regulated in the course ofIFN stimulation, needs to be addressed in the future.

Materials and methods

Plasmids

The following plasmids were used: pcDNA3.1-PRMT1 wild-typeand pcDNA3.1-PRMT1-Myc XGX mutant were published re-cently (Balint et al. 2005). pGex2T-GAR and pGex2T-PRMT3were described previously (Tang et al. 1998). pGex4T-1-PRMT1and pGex4T-1-PRMT2 were published (Scott et al. 1998). MurinepGex4T-1-PRMT4 (Chen et al. 1999) and human pGex2T-PRMT6 (Hyllus et al. 2007) were described.

PIAS1 full-length and deletion constructs were generated byPCR amplification from the pBK-CMV-PIAS1 plasmid (Sapetsch-nig et al. 2002) introducing at the 59-end of a XhoI site and at the39-end of a Hind III site and cloned via these sites in the pFastBacHT vector or the pRSETA vector (Invitrogen). The following con-structs were generated: pFastBac HT-Pias1 full-length (aminoacids 1–651), pRSETA-PIAS1 DN45 (amino acids 46–651), DN261(amino acids 262–651), DN383 (amino acids 384–651), DC402(amino acids 1–253), and DNDC (amino acids 262–474). Further-more, pRSETA-PIAS1 DNDC was used for site-directed muta-genesis (Stratagene QuickChange Kit) of Arg 303 to alanine. ThepN3-3xFlag PIAS1 (full-length) construct was generated by PCRamplification introducing at the 59-end of an EcoRI site and at the39-end of a SalI site and cloned into a modified pEGFP-N3 vector(Clontech; EGFP tag replaced by triple Flag tag). pN3-3xFlagPIAS1 R303 mutants were generated by site-directed mutagen-esis (Stratagene Quickchange Kit). R303 was exchanged to K, G,and F, respectively.

Antibodies

The following antibodies were employed: anti-Flag (F3165) fromSigma, anti-HA (sc-805) from Santa Cruz Biotechnologies, anti-5-His (34,660) from Qiagen, anti-STAT1 (sc-346) from Santa CruzBiotechologies, anti-STAT1pY701 (9171) from Cell Signaling,anti-b-tubulin (MAB 3408) from Chemicon, anti-HDAC1 (sc-7872) from Santa Cruz Biotechnologies, anti-PRMT6 (Wagneret al. 2006), anti-PRMT4 (Kleinschmidt et al. 2008), anti-PRMT1(07-404) from Upstate Biotechnologies for Western blot, anti-PRMT1 (Kleinschmidt et al. 2008) for ChIP, and anti-PIAS1 (sc-8152) from Santa Cruz Biotechnologies for Western blot/IP. ForChIP analysis we generated a polyclonal rabbit antibody againstthe C terminus of human PIAS1 (amino acids 384–651). The Myc(9E10) antibody was a kind gift from Martin Eilers. As controlantibodies in ChIP and IP we used anti-p15 (sc-1429) from SantaCruz Biotechnologies or rabbit IgG from Pierce (31,235) and fromSigma (I5006), respectively.

Cell culture and transfections

HeLa and HEK293 cells were maintained in Dulbecco’s modifiedEagle’s medium (DMEM) supplemented with 10% fetal bovineserum (FBS). A375 cells were cultured in RPMI supplementedwith 10% FBS. IFNg (Strathmann Biotec) was used at concen-trations between 5 and 15 ng/mL for the indicated time points.Transfection of HeLa and HEK293 cells was carried out using theCa-Phosphat method.

siRNA oligonucleotide duplexes were purchased from Dhar-macon for targeting the human PRMT1, PIAS1, PRMT4, andPRMT6, respectively. The transfections were carried out withOligofectamine (Invitrogen). siRNA duplexes were transfected ata final concentration of 100 nM in HeLa, HEK293, or A375 cells.Afer 2 d cells were collected for protein or RNA preparation. ThesiRNA sequences are listed in the Supplemental Material.

RT-QPCR

Total RNA was isolated using PeqGold total RNA Kit (PeqLab).Two micrograms of RNA were applied to reverse transcription byincubation with 0.5 mg of oligo(dT)17 primer and 100 U of M-MLV reverse transcriptase (Invitrogen). cDNA was analyzed byQPCR, which was performed using ABsolute QPCR SybrGreenMix (Abgene) and the Mx3000P real-time detection system(Agilent). The primers used in RT-QPCR are indicated in theSupplemental Material. All amplifications were performed intriplicate using 0.5 mL of cDNA per reaction. The triplicate meanvalues were calculated according to the DDCt quantificationmethod (Pfaffl 2001) using the GAPDH gene transcription asreference for normalization. Relative mRNA levels in uninducedcontrol cells were equated to 1 and the other values wereexpressed relative to this. The RT-QPCR results were reproducedat least three times in independent experiments and representa-tive data sets are shown.

ChIP

HeLa cells were cross-linked in the presence of 1% formal-dehyde for 10 min at room temperature. Subsequent ChIPanalysis was performed as described previously (Wagner et al.2006). Immunoprecipitated and eluted DNA was purified withQIAquick columns (Qiagen) and amplified by QPCR. The ChIP-QPCR primers are indicated in the Supplemental Material.All amplifications were performed in triplicate using 0.6 mL ofDNA per reaction. The triplicate mean values were displayedas percentage input and standard deviation was calculated. The






presented results were reproduced several times in independentexperiments.

Preparation of protein extracts and IP

Whole-cell extract were prepared from HeLa or HEK293, re-spectively. Cells were rinsed in PBS and lysed in IPH buffer (50mM Tris at pH 8, 150 mM NaCl, 5 mM EDTA, 0.5% NP40,1 mM DTT, 150 mM NaF, 2 mM Na3VO4). After lysis the extractswere cleared by centrifugation and supernatant was used forWestern blot analysis.

Nuclear extract were prepared from HeLa cells. Cells werewashed with PBS and subsequently lysed in buffer A1 (10 mMHEPES KOH at pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mMDTT, 0.04% NP40, 2 mM Na3VO4) for 5 min on ice. Aftercentrifugation cytoplasmic supernatant was removed and nucleiwere washed in buffer A2 (10 mM HEPES KOH at pH 7.9, 10 mMKCl, 1.5 mM MgCl2, 0.5 mM DTT, 2 mM Na3VO4) to excludecytoplasmatic contamination. The remaining nuclear pelletwas lysed in IPH buffer, and thereafter a benzonase digestionwas performed. For IP, 1–2 mg of the indicated antibodies wereincubated with 500 mg of nuclear extract as described previously(Kleinschmidt et al. 2008).

Recombinant proteins and GST pulldown experiments

GST fusion proteins and His tag proteins were prepared fromEscherichia coli BL21 according to standard procedures. His-tagged PIAS1 (full length) was expressed and purified from Sf9cells according to the manufacturer’s instructions for the Bac-to-Bac Baculovirus Expression System (Invitrogen). GST and GST-PRMT1 immobilized on glutathione beads were blocked with10% goat serum in HEGN buffer (30 mM HEPES-KOH at pH 7.6,125 mM KCl, 0.16 mM EDTA, 16% glycerol, 0.16% NP-40, 1mM DTT) for 10 min at room temperature. Precleared whole-cellextract (250 mg) (Kleinschmidt et al. 2008) or recombinant His-tagged PIAS1 (0.5 mg) was incubated with the blocked beads for1 h at room temperature. Subsequently, beads were washed fivetimes with IPH buffer and examined by Western blot analysis.

In vitro methyltransferase assay

Eluted and dialyzed GST-PRMTs (0.5 mg) were incubated withapproximatly 2–4 mg of substrate in the presence of [14C-methyl]-SAM (58.3 mCi/mM; GE Healthcare Life Science) for 2 h at 37°C.Reaction was stopped by addition of SDS-PAGE sample bufferand analyzed by SDS-PAGE, blotting and autoradiography.

In vivo methylation assay

Transfected Hela cells were pretreated with cycloheximid andchloramphenicol in the presences or absence of IFNg (5 ng/mL).After a 30-min pretreatment, L-[methyl-3H]-methionine (70 Ci/mM; GE Healthcare Life Science) was added and cells werecultured for another 3 h (Liu and Dreyfuss 1995). Whole-cellextracts were prepared and employed for IP of Flag-tagged PIAS1or endogenous PIAS1 as well as STAT1. Immunoprecipitateswere separated by SDS-PAGE. The gel was incubated in Enlightsolution (MoBiTec) according to manufacturer instructions and3H-labeled proteins were visualized by fluorography.

Mass spectrometric analysis

Gel-excised methylated PIAS1 protein (DNDC) bands werewashed with 50% (v/v) acetonitrile in 25 mM ammonium

bicarbonate, shrunk by dehydration in acetonitrile, and dried ina vacuum centrifuge. Reduction/alkylation of disulfide bondsand enzymatic in-gel digestion was performed as described(Bauer and Krause 2005). MS and MS/MS experiments werecarried out on a quadrupole orthogonal acceleration time-of-flight mass spectrometer Q-Tof Ultima equipped with a CapLCliquid chromatography system (Micromass). Peptides were sep-arated using a capillary column (Atlantis dC18, 3 mm, 100 A, 150mm 3 75 mm i.d.; Waters GmbH) and an eluent flow rate of 200nL/min. Mobile phase A was 0.1% formic acid (v/v) in acetoni-trile-water (3:97, v/v) and B was 0.1% formic acid in acetonitrile-water (8:2, v/v). Runs were performed using a gradient of 3%–64% B in 100 min. Data-dependent aquisition was used, whereaspeptides, which covered the methylation sites, were preferen-tially selected for MS/MS. The processed MS/MS spectra (Mas-sLynx version 4.0 software) were compared with the theoreticalfragment ions of peptides of human PIAS1 protein.

Growth curves

For analysis of IFNg-induced growth arrest, 10,000 cells per wellwere seeded and subsequently stimulated with 7.5 or 15 ng/mLIFNg. Cells were harvested at the indicated time points and cellnumbers were determined in triplicates for each condition andtime point by using the Neubauer counting chamber. For growtharrest analysis in PRMT1- or PIAS1-depleted cells, we performedtransient transfection of the corresponding siRNAs in HeLa cellsfor 48 h. Subsequently, cells were trypsinized, seeded (10,000cells per well) and treated with IFNg. The knockdown wasmonitored in parallel by Western blot analysis.

Acknowledgments

We thank Hervey Herschman for providing the pGEX2T-GARplasmid. We thank Alexander Brehm, Martin Eilers, Jean-Fran-cois Naud, and Uwe Vinkemeier for critical reading of themanuscript and valuable suggestions. We thank all members ofthe U.M.B. laboratory and Alexandra Sapetschnig for their helpduring work progress, and Inge Pelz for technical assistance. Thiswork was supported by the DFG (Deutsche Forschungsgemein-schaft), by funding from Land Hessen, and from BMBF (Bundes-ministerium fur Bildung und Forschung) to U.M.B.

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prmt1-mediated arginine methylation of pias1 regulates stat1

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