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PRMT1-mediated arginine methylation of 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) in interferon (IFN) signaling, we investigated the expression of STAT1 (signal transducer and activator of transcription) 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 the STAT1 inhibitor PIAS1 (protein inhibitor of activated STAT1). PIAS1 is arginine methylated by PRMT1 in vitro as well as in vivo upon IFN treatment. Mutational and mass spectrometric analysis of PIAS1 identifies Arg 303 as the single methylation site. Using both methylation-deficient and methylation-mimicking mutants, we find that arginine methylation of PIAS1 is essential for the repressive function of PRMT1 in IFN-dependent transcription and 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 the decline 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 STAT1 activation 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- mammalian STAT proteins STAT1 and STAT2 are medi- crine and paracrine proteins that regulate innate and ators of the IFN signaling pathway, whereas the remain- adaptive immunity in response to viral and microbial in- ing STAT family members respond to distinct cytokines. fections and modulate proliferation, survival, and apo- Type I IFNs, such as IFNa and IFNb, activate STAT1/ ptosis of normal cells as well as tumor cells (Borden et al. STAT2 heterodimers, and type II IFNg mainly signals via 2007). IFNs exert their effects by binding to cell surface STAT1 homodimers (Borden et al. 2007). receptors, which triggers receptor dimerization/oligomer- Proper duration and magnitude of IFN-induced gene ization, allowing transphosphorylation and activation of activation is crucial, as abnormal and imbalanced JAK– intracellular receptor-associated tyrosine kinases of the STAT activation is associated with immune disorders and JAK (Janus kinase) family. Activated JAKs phosphorylate cancer (Darnell 2002; Levy and Darnell 2002). Therefore, critical tyrosine residues on the receptor, which results in cells have evolved mechanisms to allow rapid and tran- the recruitment of a familyRETRACTED of nucleo–cytoplasmic shut- sient activation and to prevent excess responses to IFNs tling transcription factors, the STAT (signal transducer and other cytokines. Restriction and inactivation of the and activator of transcription) proteins (Levy and Darnell STAT pathway is accomplished by regulators such as 2002; Schindler et al. 2007). In the following, receptor- protein tyrosine phosphatases, which inactivate JAKs and bound STATs are activated by phosphorylation of a single STATs in the cytoplasm or in the nucleus (Levy and tyrosine residue within the C terminus leading to STAT Darnell 2002). In addition, SOCS (suppressors of cytokine dimerization via a reciprocal phosphotyrosine-SH2 (src signaling) proteins switch off the activity of JAKs by acting homology 2) interaction. The STAT dimers are retained in as pseudosubstrates or by recruiting the ubiquitination the nucleus, where they bind specific DNA sequences in machinery (Alexander and Hilton 2004). The PIAS (protein promoter regions of target genes and induce gene tran- inhibitor of activated STAT) protein family operates in scription (Meyer and Vinkemeier 2004). Among the seven the nucleus and the family member PIAS1 interacts with activated STAT1 and suppresses the DNA-binding ability of the transcription factor (Liu et al. 1998; Shuai and Liu 3Corresponding author. E-MAIL [email protected]; FAX 0049-6421-2865196. 2005). PIAS1 depletion causes increased expression of a Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.489409. subset of STAT1 target genes (Liu et al. 2004). In agreement

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Arginine methylation of PIAS1 with this observation, PIAS1 knockout mice reveal en- PRMT1 in HeLa cells using transient transfection of two hanced protection against pathogenic infections (Liu et al. different siRNAs directed against the PRMT1 transcript 2004). Whether the Sumo E3 ligase activity of PIAS1 and (siPRMT1_1 and siPRMT1_2). The efficient depletion of sumoylation of STAT1 is required for its repressive activity endogenous PRMT1 by the two alternative siRNAs in on IFN-inducible gene expression is controversial in the comparison with the control siRNA (siScr) was deter- literature (Rogers et al. 2003; Ungureanu et al. 2003, 2005; mined on RNA (Fig. 1A) and protein level (Fig. 1B). The Song et al. 2006). Until now, little was known about the expression of STAT1 or PIAS1 was not altered by knock- molecular mechanism leading to activation of PIAS1, down of PRMT1 (Fig. 1A,B). To examine the effect of which then restrains STAT1 activity, and how PIAS1 PRMT1 on IFNg-inducible STAT1-dependent transcrip- interferes with the chromatin binding of STAT1. tion we performed RT-QPCR of known STAT1 target Arginine methylation is catalyzed by a family of en- genes (Ramana et al. 2002) in control (siScr) and PRMT1- zymes, called PRMTs (protein arginine methyltransferases), depleted HeLa cells either unstimulated (0 h) or stimu- consisting of 11 members (Boisvert et al. 2005; Pal and Sif lated with IFNg for 2, 4, and 6 h. As shown in Figure 1C, 2007). These enzymes share a central highly conserved the IFNg-inducible transcription of several STAT1 tar- catalytic domain and perform mono- and dimethylation gets, including CXCL9 (Cxc chemokine ligand 9), of the guanidino group of the arginine residues using S- CXCL10 (Cxc chemokine ligand 10), and GBP1 (guany- adenosylmethionine (SAM) as methyl donor (Bedford and late-binding protein 1), was enhanced threefold to 10-fold Richard 2005). PRMTs regulate various cellular processes upon knockdown of PRMT1 using either of the two such as RNA processing, nucleo–cytoplasmic transport, siRNAs against PRMT1. In contrast, the basal transcript DNA repair, transcription, and signal transduction. Sev- level of these genes was not affected by PRMT1 depletion. eral reports implicate arginine methylation in the regu- IFN induction of other STAT1 target genes, like IRF1 (IFN lation of IFN signaling: PRMT1 interacts with the response factor 1) and TAP1 (transporter associated with cytoplasmic domain of the IFNa receptor, and its de- anti-gene presentation 1), was not significantly influ- pletion alters IFNa-induced growth arrest (Abramovich enced by PRMT1 knockdown, suggesting that tran- et al. 1997; Altschuler et al. 1999). Tyrosine phosphory- scriptional activation of only a subset of STAT1 target lation, transcriptional activity, and protein stability of genes is repressed by PRMT1 (Fig. 1C). The PRMT1- STAT transcription factors were suggested to be influ- dependent STAT1 targets identified here appeared to enced by arginine methylation (Mowen et al. 2001; Zhu match the subset of IFN-induced genes selectively con- et al. 2002; Chen et al. 2004). However, arginine methyl- trolled by the inhibitor of STAT1 signaling, PIAS1 (Liu ation of STAT transcription factors itself was not confirmed et al. 2004). We asked whether siRNA-mediated depletion by other groups, and the nonspecific methyltransferase of the PIAS1 protein would affect the same subset of inhibitor MTA, which was commonly used to study the IFNg-dependent STAT1 target genes as PRMT1 depletion in vivo relevance of arginine methylation in this pathway, does. Indeed, we found that PIAS1 knockdown, which executes strong side effects and inhibits IFN-induced was verified by Western blot (Fig. 1D), enhanced the IFN- phosphorylation and activation of STATs (Meissner inducible transcription of the PRMT1-dependent STAT1 et al. 2004; Komyod et al. 2005). Consequently, how ex- targets CXCL9, CXCL10, and GBP1, whereas PRMT1- actly PRMTs and arginine methylation might regulate independent genes, like IRF1 and TAP1, were not im- IFN signaling, is so far unclear. paired by suppression of PIAS1 (Fig. 1E). We obtained Here, we investigated the influence of PRMT1 on IFN similar results for the IFN-responsive A375 melanoma signaling and found that PRMT1 represses a subset of (Supplemental Fig. S1) and HEK293 cells (data not shown). STAT1 target genes that overlaps with genes regulated by Taken together, these data identify PRMT1 and PIAS1 as PIAS1. PRMT1 methylates PIAS1 at Arg 303 (R303) in vitro negative regulators of the same STAT1 target genes. and in vivo. Methylation-deficient and methylation- mimicking PIAS1 mutants revealed that PIAS1 methylation PRMT1 acts upstream of PIAS1 in STAT1 signaling is essential for the repressive function of PRMT1 in IFN- inducible transcriptionRETRACTED and allows the recruitment of PIAS1 In the following, we studied whether PRMT1 and PIAS1 to STAT1 target gene promoters in the late phase of the IFN synergize in their repressive function on STAT1-medi- response. Our findings identify PRMT1 as a novel inhibitor ated transcription. We performed single and double of IFN signaling that is involved in turning-off STAT1 knockdown of PRMT1 and PIAS1 in HeLa cells (Supple- activation by regulating the repressive function of PIAS1. mental Fig. S2A). Using RT-QPCR we found that the single and double depletion results in a similar increase in IFN-induced transcription of the PRMT1/PIAS1- Results dependent STAT1 targets. Double depletion of PRMT1 and PIAS1 did not lead to an additive effect compared PRMT1 coregulates PIAS1-dependent STAT1 with the effects of single depletion of PRMT1 or PIAS1 target genes (Supplemental Fig. S2B). As expected, the PRMT1/PIAS1- Since the role of arginine methylation in IFN signaling is independent targets were not influenced by the double controversial (Abramovich et al. 1997; Mowen et al. 2001; depletion (Supplemental Fig. S2B). These observations Meissner et al. 2004; Komyod et al. 2005), we investigated suggest that PRMT1 and PIAS1 functionally depend on the potential role of PRMT1 in this pathway. We depleted each other in the STAT1 pathway.

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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 5 ng/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 GAPDH mRNA level as a reference. Transcript levels in uninduced control cells were set to 1. (B) For detection of knockdown efficiency, protein levels of PRMT1, STAT1, and b-tubulin were analyzed by Western blot from 20 mg of protein extract (not induced with IFNg). For detection of PIAS1, 80 mg of extract were used. (C) Total RNA was analyzed for transcript levels of CXCL9, CXCL10, GBP1, IRF1, and TAP1 normalized to GAPDH. TranscriptRETRACTED levels in uninduced control cells were set to 1. (D,E) siRNA-mediated control (siScr) and PIAS1 knockdown (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 were analyzed by RT-QPCR as described in C.(F,G) HEK293 cells were transfected with control siRNA (siScr) or siRNA against PIAS1, and as indicated, after 24 h cells were transfected with Myc-tagged PRMT1. After 48 h, cells were induced with 5 ng/mL IFNg for the indicated time points. Subsequently, cells were harvested for the preparation of total RNA or protein extracts. (F) Efficiency of PRMT1 overexpression 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.

To further dissect the relationship between PRMT1 PRMT1/PIAS1-dependent subgroup of STAT1 target and PIAS1 we expressed Myc-tagged PRMT1 by transient genes (Fig. 1G), reinforcing our findings in PRMT1-de- transfection in HEK293 cells (Fig. 1F) and investigated pleted cells (Fig. 1C). When we depleted PIAS1 in cells IFN-induced transcription. Overexpression of PRMT1 overexpressing PRMT1 (Fig. 1F), the repressive effect diminished transcriptional induction of specifically the of exogenous PRMT1 was abrogated and transcriptional

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Figure 2. PRMT1 interacts with PIAS1 and binds simultaneously with PIAS1 to STAT1 target gene promoters in the late phase of the IFNg response. (A) Protein lysates of Flag-EGFP- (control) or Flag- PIAS1-overexpressing HEK293 cells were subjected to GST pulldown experiments. (Left panel) Equal amounts of GST alone and GST-PRMT1 protein, as indicated in the Coomassie-stained SDS gel (full- length protein bands marked with aster- isks), coupled to glutathione beads were incubated with 250 mg of protein lysates. (Right panel) Bound PIAS1 protein was visualized by Western blot using Flag antibody. (Middle panel) One percent in- put (2.5 mg) of HEK293 cell extract is shown. (B) GST pulldown with recom- binant His-tagged PIAS1 purified from Sf9 cells were performed using GST and GST-PRMT1 protein. Bound PIAS1 protein was detected by anti-His-tag West- ern blot. Five percent input of recom- binant PIAS1 is shown. (C,D)Co-IPsof endogenous PIAS1, PRMT1, and STAT1 were performed. HeLa cells were induced with 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 and examined by Western blot using antibodies against PRMT1, STAT1, and PIAS1. (D) Input Western blots corresponding to CoIP in C were stained for PIAS1, PRMT1, P-STAT1, and STAT1. (E–G) Recruitment of STAT1, PIAS1, and PRMT1 to STAT1 target gene promoters was investigated by ChIP. HeLa cells were treated in a time course experiment with IFNg (5 ng/mL). Subsequently, cells were harvested 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) gene promoters and displayed as percentage input. Standard deviation was calculated from the triplicates, and error bars are indicated accordingly. Representative figure legend is show in E. induction of the PRMT1/PIAS1-dependent STAT1 tar- and 6 h with IFNg. Nuclear extracts of these cells were get genes was enhanced to a similar extent as with the subjected to anti-PIAS1 immunoprecipitation (IP). Immu- sole depletion of PIAS1 (Fig. 1G). These results indicate noprecipitates were probed for the presence of PRMT1 by that PRMT1 operates upstream of PIAS1 in this path- Western blot analysis. PRMT1 und PIAS1 associated very way and requires PIAS1 to inhibit IFNg-dependent tran- weakly in the uninduced state and at the 3-h induction scription. time point, whereas the interaction of the two proteins was enhanced after 6 h of IFNg treatment (Fig. 2C). When we examined the PIAS1 precipitates for the presence of Endogenous PRMT1 and PIAS1 proteins interact STAT1, PIAS1 was found to associate with STAT1 after and are recruited to STAT1 target genes in the late 6 h of IFNg treatment in agreement with other reports, phase of the IFN response which also showed an IFN-dependent interaction (Liu Next, we investigatedRETRACTED whether the functional coopera- et al. 1998). Essentially the same results were obtained, tion between PRMT1 and PIAS1 could reflect interaction when we included ethidium bromide in the IP (Lai of both proteins. We performed GST pulldown experi- and Herr 1992) indicating that the interaction between ments of Flag-EGFP- (control) or Flag-PIAS1-overexpress- PIAS1, PRMT1, and STAT1 was not DNA-mediated (data ing HEK293 cell extracts with GST alone and GST- not shown). IFNg stimulation did not affect the protein PRMT1 (Fig. 2A). Pulldowns were analyzed by anti-Flag levels of the three proteins, as shown by Western blot Western blot and revealed a specific interaction between analysis of the corresponding nuclear extracts (Fig. 2D). PRMT1 and PIAS1 in vitro (Fig. 2A). Using recombinant These data strongly suggest that PIAS1 preferentially in- His-tagged PIAS1 protein we demonstrated in further teracts with PRMT1 and STAT1 in the late phase of the pulldown assays with GST-PRMT1 that the two proteins IFN response. interact directly (Fig. 2B). Subsequently, we explored whether PRMT1 and PIAS1 We then determined whether the two proteins bind are recruited to STAT1 target gene promoters. We per- also endogenously to each other and how such an in- formed chromatin IP (ChIP) analysis in either uninduced teraction might be influenced in the course of the IFN cells or 3, 6, and 9 h after addition of IFNg using response. HeLa cells were left untreated or treated for 3 antibodies against STAT1, PIAS1, and PRMT1 and assayed

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

for the enrichment of the PRMT1/PIAS1-dependent detachment of STAT1 and with the gradual decline of STAT1 target genes CXCL9 and CXCL10 and the PRMT1/ transcriptional activation. PIAS1-independent target IRF1. STAT1 binding to the CXCL9 and CXCL10 promoter peaked at 3 h IFNg treat- PRMT1 is required for promoter recruitment of PIAS1 ment, gradually declined betweenRETRACTED 6 and 9 h and reached and promoter detachment of STAT1 the basal (uninduced) level of recruitment at the 9 h time point (Fig. 2E,F). Consistent with this detachment of To test whether PRMT1 influences the promoter recruit- STAT1 from the CXCL9 and CXCL10 promoter and the ment of PIAS1 we conducted ChIP analysis in PRMT1- decrease in transcriptional induction in the late phase of depleted cells and control (siScr) cells, which were either IFN treatment (Supplemental Fig. S3), PIAS1 and PRMT1 untreated or IFN-treated. PRMT1 depletion in the corre- bound to both promoters after 6 and 9 h of IFN induction sponding chromatin samples was verified by Western blot (Fig. 2E,F). In contrast, the promoter of the PRMT1/ (Fig. 3A). In control cells STAT1, PIAS1, and PRMT1 PIAS1-independent STAT1 target gene IRF1 was never binding to the CXCL9 and CXCL10 promoter overlapped occupied by PIAS1 or PRMT1 throughout the whole after 6 h of IFNg stimulation (Fig. 3B, top and middle IFN time course, but STAT1 was strongly enriched after panels), in agreement with Figure 2, E and F. Depletion 3 and 6 h of IFNg stimulation (Fig. 2G). These results of PRMT1 resulted in a loss of PIAS1 recruitment and in indicate that both PRMT1 and PIAS1 are simultaneously an enhanced and prolonged recruitment of STAT1 to the recruited to the PRMT1/PIAS1-dependent genes in the CXCL9 and CXCL10 promoter during the IFN response late phase of IFNg signaling, which coincides with the (Fig. 3B, top and middle panels). In contrast association of

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STAT1 with the PRMT1/PIAS1-independent IRF1 pro- as in control cells (Fig. 4B) leading to the conclusion that moter was not influenced by PRMT1 suppression (Fig. 3B, the catalytic activity of PRMT1 is required for its in- bottom panel). A similar effect on STAT1 binding was hibitory effect on STAT1-mediated transcription. also observed in PIAS1-depleted cells, in which IFN- We next asked whether PRMT1 and other PRMTs induced STAT1 binding of the CXCL9 and CXCL10 would possess methyltransferase activity toward PIAS1. promoter was increased (Fig. 3A,B, top and middle pan- Therefore, we employed an in vitro methylation assay els), but binding of the IRF1 promoter was unchanged using recombinant baculovirus-expressed His-tagged PIAS1, (Fig. 3B, bottom panel). PIAS1 knockdown did not affect GST-PRMTs, or immunopreciptitated PRMT5 from cell the recruitment of PRMT1 to the CXCL9 and CXCL10 extracts as enzyme source and radiolabeled methyl- gene promoter (Fig. 3B, top and middle panels) corrobo- donor SAM (Fig. 4C; data not shown). This assay revealed rating our observation that PRMT1 acts upstream of that PIAS1 is in vitro methylated by PRMT1, PRMT4, PIAS1 in this pathway (Fig. 1G). However, PRMT1 pro- and PRMT6 (Fig. 4C). The catalytic activity of the moter recruitment requires IFNg-induced binding of various enzyme preparation was verified using estab- STAT1 to its target genes, as ChIP analysis in STAT1- lished substrates in an in vitro methylation assay (Fig. deficient U3A cells revealed (Supplemental Fig. S4). To 4D; data not shown). strengthen our observation that PRMT1 and PIAS1 reg- To address the question whether PRMT4 and PRMT6, ulate the magnitude and kinetic of STAT1 promoter which methylate PIAS1 in vitro as well, influence IFN- binding we performed ChIP analysis for STAT1 in a more induced STAT1 target gene expression in a similar way as detailed IFN induction time course including additional PRMT1, we depleted PRMT4 and PRMT6, respectively, time points. As shown in Figure 3C, depletion of PRMT1 using transient transfection of siRNAs in HeLa cells or PIAS1 resulted in an increased and prolonged STAT1 (Supplemental Fig. S7A,C). Transcriptional induction of binding with a clearly delayed peak of STAT1 recruitment the PRMT1/PIAS1-dependent STAT1 target genes CXCL9 to a subset of target genes. These results indicate that and GBP1 was not impaired by knockdown of neither PRMT1 regulates PIAS1-mediated repression of STAT1 PRMT4 (Supplemental Fig. S7B) nor PRMT6 (Supplemen- target genes at the chromatin level by allowing promoter tal Fig. S7D). These data demonstrate that among the recruitment of PIAS1, which subsequently diminishes PRMTs capable of methylating PIAS1 in vitro, PRMT1 is promoter association of STAT1. the only enzyme that cooperates with PIAS1-dependent Recent studies suggested a role of PIAS1-mediated repression in a methyltransferase-dependent manner sumoylation of STAT1 in the repression of STAT1 signal- in vivo. ing (Ungureanu et al. 2003, 2005). We asked whether Arg 303 in PIAS1 is methylated by PRMT1 in vivo PRMT1 has an impact on the sumoylation of STAT1. PRMT1 depletion did not change the levels of sumoylated To investigate the occurrence of in vivo methylation of STAT1 (Supplemental Fig. S5) indicating that PRMT1 does PIAS1 we performed metabolical labeling of HeLa cells not control the sumoylation activity of PIAS1. Moreover, with L-[methyl-3H]-methionine. Cells were transfected overexpression of the sumoylation-deficient PIAS1 mu- with Flag-tagged PIAS1 and additionally with control tant W372A (Liu et al. 2007) revealed a similar repressive siRNA (siScr) or with siRNA targeting PRMT1 (siPRMT1). effect on the IFN induction of the same STAT1 target Subsequently, cells were stimulated with IFNg or not and genes as wild-type PIAS1 (Supplemental Fig. S6). These simultaneously metabolical labeling was performed for results imply that sumoylation activity by PIAS1 is not 3 h in the presence of translational inhibitors. Flag-PIAS1 essential for transcriptional inhibition of the STAT1 protein was immunopurified from the cell extracts, re- target genes investigated here. solved by SDS-PAGE and methylation was detected by fluorography. PIAS1 was methylated in response to IFNg treatment in the control cells, whereas knockdown of The methyltransferase activity of PRMT1 is required PRMT1 resulted in the loss of PIAS1 methylation (Fig. 5A). for its repressive function in STAT1 signaling The efficiency of PIAS1 overexpression, PRMT1 deple- As PRMT1 possesses arginineRETRACTED methyltransferase activity tion, and IFNg stimulated tyrosine 701 phosphorylation toward histones and other nuclear proteins (Wang et al. of STAT1 was verified by Western blot analysis (Fig. 5A). 2001; Kwak et al. 2003; Barrero and Malik 2006) we To confirm that translational inhibition avoided incorpo- studied in the following whether the catalytic activity ration of the radiolabeled methionine by de novo protein of PRMT1 is required for its repressive function in STAT1- biosynthesis (Liu and Dreyfuss 1995), cells were incu- dependent transcription. Therefore, we established tran- bated with 35S-methionine and protein synthesis inhib- sient overexpression of wild-type PRMT1 (WT) and the itors. No label was detectable in total protein lysate catalytically inactive PRMT1 mutant XGX (Balint et al. from these cells by fluorography (Supplemental Fig. S8) 2005), which were equally well expressed (Fig. 4A). As indicating that radiolabeling of PIAS1 was indeed due expected IFNg-induced expression of CXCL9, CXCL10, to post-translational modification. Similar results for in and GBP1 was diminished in the presence of exogenous vivo methylation of the endogenous PIAS1 protein were wild-type PRMT1 compared with control cells (Fig. 4B). In obtained (Fig. 5B), whereas methylation of STAT1, which contrast, the catalytically inactive PRMT1 mutant lost was reported by Mowen et al. (2001), was not detectable its repressive capacity and transcriptional induction of in this assay (Fig. 5C). These data indicate that in vivo CXCL9, CXCL10, and GBP1 occurred to a similar extent methylation of PIAS1 occurs in response to IFNg treatment

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Figure 4. The arginine methyltransferase activity of PRMT1 is required for its repressive function in IFN-dependent transcription, and PIAS1 is arginine methylated in vitro. (A,B) HeLa cells were transfected either with PRMT1 wild type (WT) or PRMT1 catalytic inactive mutant (XGX). After 48 h, cells were induced with 5 ng/mL IFNg for the indicated time points. Subsequently cells were harvested for the preparation of total RNA or protein extracts. (A) Overexpression of PRMT1 WT and PRMT1 XGX in uninduced cell extracts was monitored 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-tagged PIAS1 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 by autoradiography. (D) As positive control for the enzymatic activity of the GST-PRMT preparations in C, in vitro methylation assays were perfomed using established substrates of PRMTs, like GST-GAR protein (glycine–arginine-rich domain of fibrillarin) or bulk histones, and analyzed by autoradiography.

and that PRMT1 is the responsible enzyme for PIAS1 labeling of both Flag-tagged mutants or wild-type Flag- methylation in this pathway. tagged PIAS1 (WT) in unstimulated and IFNg-stimulated In order to map the methylation site(s) in PIAS1, we HeLa cells. Immunoprecipitates of PIAS1 wild type expressed several N- and C-terminal deletion constructs revealed in vivo methylation of PIAS1 upon IFNg stim- of PIAS1 (Fig. 5D) as His-tagged fusions in bacteria ulation, whereas both PIAS1 mutants, R303K and R303G, (Fig. 5E). In vitro methylation in the presence of GST- were not methylated in the absence or in the presence PRMT1 revealed that the PIAS1 deletion DNDC is the of IFNg (Fig. 5I). These results indicate that PIAS1 is smallest fragment still methylated by PRMT1 (Fig. 5E). methylated upon IFNg stimulation at a single arginine, This indicates that the methylation site(s) of PRMT1 in R303, in vivo. PIAS1 are located between amino acid 262 and 384, Methylation of R303 in PIAS1 is required for its which encompass the RING finger domain and the acidic repressive effect on STAT1 and for its recruitment domain. to STAT1 target gene promoters To identify the exact site(s) of PRMT1 methylation in PIAS1 we methylated PIAS1 DNDC in the presence To investigate the impact of PIAS1 methylation on its of GST-PRMT1 in vitro. The ESI mass spectrum revealed repressive activity in STAT1-activated transcription we a doubly charged peak atRETRACTED m/z 540.26 (calculated m/z explored overexpression of PIAS1 wild type, a methyla- 540.29) corresponding to the dimethylated sequence of tion-deficient PIAS1 mutant (R303K), and a methyla- the tryptic PIAS1 fragment GIRNPDHSR (amino acids tion-mimicking mutant, in which the arginine was 301–309) (Fig. 5F). Fragment ion (MS/MS) spectrum mutated to phenylalanine (R303F) (Mostaqul Huq et al. resulting from this doubly charged precursor clearly in- 2006). The mutations of R303 did not affect the sumoy- dicated that the first arginine within the peptide (R303 in lation activity of PIAS1 (Supplemental Fig. S9), its the full-length context of PIAS1) is dimethylated (Fig. 5G). nuclear localization, or the cellular localization of Mutation of the R303 to alanine (R303A) confirmed this STAT1, and its IFN-induced shuttling (Supplemental arginine, which is localized between the PINIT motif and Fig. S10). Subsequent to transfection of PIAS1 wild type the RING finger domain, as the in vitro methylation site andthetwomutants,IFNg treatment was performed of PIAS1 DNDC protein by PRMT1 (Fig. 5H). and STAT1 target gene expression was analyzed by To validate R303 also as the in vivo methylation site RT-QPCR. Overexpression of PIAS1 wild type and the of PIAS1 by PRMT1, we mutated R303 in the full- methylation-mimicking PIAS1 R303F mutant decreased length PIAS1 to lysine (R303K) or glycine (R303G). Sub- the IFN-induced transcription of PRMT1/PIAS1-depen- sequently, we performed overexpression and metabolical dent targets compared with control transfected cells

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Figure 5. PRMT1 methylates R303 in PIAS1 in vivo. (A) HeLa cells were first transfected with control siRNA (siScr) or siRNA against PRMT1 (siPRMT1_1 was used). After 24 h cells were transfected with Flag-tagged PIAS1, and 48 h later cells were induced with IFNg for 3 h (+)or not (À) and simultaneously labeled with L-(methyl-3H) methionine in the presence of translation inhibitors. (Top panel) Sub- sequently, Flag-tagged PIAS1 was immu- noprecipitated from cell lysates and analyzed by SDS-PAGE followed by fluo- rography. (Bottom panels) Effective knock- down of PRMT1 and overexpression of PIAS1 was checked by Western blot. (B) HeLa cells were transfected with control siRNA (siScr) or siRNA against PRMT1 (siPRMT1_1 was used). After 72 h cells were induced with IFNg for 3 h (+)ornot (À) and simultaneously labeled with L- (methyl-3H) methionine in the presence of translation inhibitors. (Top panel) Sub- sequently, endogenous PIAS1 was immu- noprecipitated from cell lysates and analyzed by SDS-PAGE followed by fluo- rography. (Bottom panels) Efficient knock- down of PRMT1 and endogenous PIAS1 and STAT1 levels were checked by West- ern blot. (C) HeLa cells were induced with IFNg for 3 h (+)ornot(À) and simulta- neously labeled with L-(methyl-3H) me- thionine in the presence of translation inhibitors. (Top panel) Subsequently, en- dogenous STAT1 was immunoprecipitated from cell lysates and analyzed by SDS- PAGE followed by fluorography. (Bottom panels) The endogenous STAT1 and PRMT1 levels were checked by Western blot. (D) Schematic representation of PIAS1 deletion constructs used in E. Conserved domains are indicated: The N-terminal SAP box (Saf-A/B, Acinus, PIAS) is re- quired for DNA-binding, PINIT motif is involved 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–protein interactions (Duval et al. 2003; Shuai and Liu 2005). (E) His-tagged PIAS1 deletion constructs were purified from E. coli, and equal amounts were used for in vitro methylation with recombinant GST-PRMT1 in the presence of methyl-14C-labeled SAM. Coomassie staining 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 nonspecific methylation products withRETRACTED a circle. (F,G) PIAS1 DNDC was in vitro methylated by recombinant PRMT1 and subsequently analyzed by 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) spectrum resulting 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 monitored by autoradiography. (Left panel) The Coomassie staining reveals that equal amounts of both PIAS1 deletion proteins were used for the methylation assay. (I) HeLa cells were transiently transfected with Flag-tagged EGFP (control), Flag-tagged PIAS1 wild type (WT), PIAS1 R303K 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.

(Fig. 6A). In contrast, the methylation-deficient PIAS1 (Fig. 6A). Expression of the different PIAS1 proteins did R303K mutant exhibited a relief of this repression and not affect the IFN-induced transcriptional activation of resulted in a transcriptional induction of the PRMT1/ PRMT1/PIAS1-independent STAT1 target genes (Fig. PIAS1-dependent targets comparable with control cells 6A). These findings demonstrate that PRMT1-mediated

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Figure 6. Methylation of R303 in PIAS1 is crucial for PIAS1-dependent repression of STAT1 signaling and for PIAS1 promoter recruitment. (A) HeLa cells transiently overexpressing either Flag-EGFP, Flag-PIAS1 wild type (WT), methylation-deficient Flag-PIAS1 R303K 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 to GAPDH and transcript levels in uninduced control cells were set to 1. (B,C) HeLa cells were transfected with control siRNA (siScr) or siRNA against the 39UTR of PIAS1 (siPIAS1 UTR = siPIAS1 UTR1 + 2). After 24 h, the siPIAS1 UTR-treated cells were transfected with empty vector (control), Flag-tagged PIAS1 wild-type (WT), R303K mutant, or R303F mutant constructs. After 48 h of this second transfection, cells were treated with IFN for 6 h and subjected to ChIP analysis. Recruitment of STAT1 (B) and Flag-PIAS1 (C) to the indicated gene promoters was determined by QPCR. Results were displayed as percentage input. (D) HeLa cells were transfected with Flag-tagged PIAS1 wild-type (WT),RETRACTED R303K mutant, or R303F mutant constructs. After 48 h, cells were treated with IFN for 6 h and subjected to co-IP analysis. IP of Flag-PIAS1 (anti-Flag) or control IP (IgG) was carried out from 500-mg nuclear extract and examined by Western blot using antibodies against STAT1 and Flag-PIAS1. Input Western blots of the nuclear extracts corresponding to the co-IP were stained for P-STAT1, STAT1, Flag-PIAS1, PRMT1, and HDAC1 (the latter served as loading control).

methylation of R303 in PIAS1 is crucial for repression of (Supplemental Fig. S11A). Subsequently, we transiently STAT1-activated transcription. transfected the PIAS1-depleted cells with empty vector To determine the impact of R303 methylation on the (control), Flag-PIAS1 wild type, methylation-deficient promoter recruitment of STAT1 and PIAS1, and to avoid Flag-PIAS1 R303K mutant, and methylation-mimicking at the same time an interference of the endogenous Flag-PIAS1 R303F mutant. The exogenous PIAS1 expres- PIAS1 protein in this assay, we first depleted the endog- sion was not affected by the siPIAS1 targeting the 39UTR, enous PIAS1 expression in HeLa cells by using siRNAs and the expression levels of the different PIAS1 con- targeting the 39 untranslated region (UTR) of PIAS1 structs were equal (Supplemental Fig. S11A). When we

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Arginine methylation of PIAS1 subjected these cells to IFNg treatment, we found that similar to the siRNA targeting the PIAS1 ORF (Fig. 1E), the siPIAS1 UTR resulted in an increased transcriptional activation of the specific subset of STAT1 targets in comparison with siScr transfected cells (Supplemental Fig. S11B). Overexpression of PIAS1 wild type and R303F mutant rescued this effect, whereas PIAS1 R303K was not able to perform the rescue (Supplemental Fig. S11B). ChIP analysis of these cells revealed that IFN-induced promoter recruitment of STAT1 was enhanced in the siPIAS1 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., both PIAS1 proteins decreased the STAT1 recruitment back to the level detected in siScr-transfected cells (Fig. 6B; Supplemental Fig. S12A). In contrast, the methylation- deficient PIAS1 R303K mutant was unable to substitute for the function of the endogenous PIAS1 protein in the PIAS1-depleted cells and the STAT1 recruitment remained 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 on Figure 7. Knockdown of PRMT1 or PIAS1 enhances the anti- this particular subset of genes (Fig. 6A; Supplemental Fig. proliferative effect of IFNg.(A–C) HeLa cells were transfected 11B). In agreement with the PRMT1/PIAS1-independent with control siRNA, siRNA against PRMT1 (siPRMT1_1 was used) or against PIAS1. After 48 h, cells were trypsinized and transcriptional regulation of IRF1, PIAS1 depletion as 10,000 cells per well were seeded and after 4 h induced with 7.5 well as overexpression of the different PIAS1 constructs ng/mL IFNg. At the indicated time points, cells were harvested did not influence STAT1 recruitment to this promoter and cell number was determined. For each condition and time (Fig. 6B). When we analyzed the promoter recruitment of point triplicates were counted. Error bars are depicted accord- PIAS1 in these cells, we noticed that the binding of ingly. (D) Model for the regulation of PIAS1-mediated repression PIAS1 R303K to the PRMT1/PIAS1-dependent STAT1 of STAT1 signaling by PRMT1. targets was completely lost, whereas the binding of methylation-mimicking PIAS1 R303F mutant was sim- facilitates its interaction with STAT1, and in this way ilar to PIAS1 wild type (Fig. 6C; Supplemental Fig. S12B). might allow promoter recruitment of PIAS1. In agreement with the PRMT1/PIAS1-independent transcriptional regulation of IRF1, the different PIAS1 Knockdown of PRMT1 or PIAS1 enhances constructs did not associate with this promoter (Supple- the anti-proliferative effect of IFNg mental Fig. S12B). These results show that PRMT1- mediated methylation of R303 in PIAS1 is essential for Finally, we elucidated the impact of PRMT1-regulated the recruitment of PIAS1, for the release of STAT1 from repression by PIAS1 on the biological outcome of IFN target gene promoters, and for turning off of STAT1- signaling and STAT1 function. Therefore we measured activated transcription. the growth/proliferation of HeLa cells in the presence of To test whether the methylation of R303 itself contrib- different concentrations of IFNg. A concentration of 15 utes to the interaction between PIAS1 and STAT1, we ng IFNg/mL resulted in a growth arrest, whereas at lower performed co-IP experimentsRETRACTED from cells overexpressing concentration of 7.5 ng IFNg/mL the anti-proliferative either Flag-PIAS1 wild type, Flag-PIAS1 R303F, or Flag- effect of IFN was not detectable (Supplemental Fig. S13) PIAS1 R303K in the absence or presence of IFNg stimu- (Bromberg et al. 1996). In the following, HeLa cells were lation. Nuclear extracts were subjected to anti-Flag IP transfected with control siRNA (siScr), siPRMT1, or and, subsequently, immunoprecipitates were stained for siPIAS1 and subjected to growth curve analysis in the the presence of endogenous STAT1. As expected, the presence of IFNg. Depletion of endogenous PRMT1 and interaction between PIAS1 wild type and STAT1 was en- PIAS1, respectively, was monitored and verified by West- hanced upon addition of IFNg (Fig. 6D). The methylation- ern blot analysis (Supplemental Fig. S14). Relative to cells mimicking PIAS1 R303F mutant revealed already as- transfected with siScr, depletion of PRMT1 or PIAS1 sociation with STAT1 without IFNg stimulation, which resulted in the occurrence of anti-proliferative activity was not further increased upon treatment with IFNg.In at low doses of IFNg (Fig. 7A–C) and enhanced anti- contrast, the methylation-deficient PIAS1 R303K mutant proliferative activity at high doses of IFNg (data not exhibited reduced binding to STAT1, under uninduced as shown). These results demonstrate that PRMT1/PIAS1- well as induced conditions (Fig. 6D). These data suggest mediated repression of STAT1 activity is important for that the PRMT1-mediated methylation of R303 in PIAS1 the biological effects of IFN.

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Discussion PIAS1 results in an enhanced and prolonged recruitment of STAT1 to a subset of its target genes. Previous reports suggested that arginine methylation Regulation of the repressive activity of PIAS1 is neces- influences IFN signaling, since PRMT1 was shown to sary, as the protein is predominantly localized in the interact with the cytoplasmic domain of the IFNa re- nucleus. A constitutively active form of PIAS1 would ceptor (Abramovich et al. 1997) and its depletion in- straight away inhibit activated STAT1 dimers, which terfered with IFNa-induced growth arrest (Altschuler enter the nucleus upon stimulus. But initially, STAT1 et al. 1999). PRMT1 was reported to methylate the dimers need to perform their transactivation function and transcription factor STAT1, and this was shown to inhibit to enable a proinflammatory and anti-proliferative gene the association of STAT1 and PIAS1 and consequently to expression pattern. Therefore, mechanisms must exist enhance transcriptional activation of IFN responsive to allow a targeted interaction between STAT1 and its genes (Mowen et al. 2001; Nien et al. 2007). However, nuclear repressor protein in the late phase of the immune arginine methylation of STAT1 was not confirmed by response. Here, we describe such a mechanism: R303 others (Meissner et al. 2004) and also not in the present methylation catalyzed by PRMT1 facilitates the STAT1– work (Fig. 5C). Furthermore, most studies investigating PIAS1 interaction, which was reported to be a direct the impact of arginine methylation in IFN signaling used interaction (Liao et al. 2000). R303 is localized in a con- nonspecific methyltransferase inhibitors, like MTA, served but functionally not assigned part between the which execute strong side effects and inhibit various PINIT motif and the RING finger domain of PIAS1. cellular kinase cascades (p38 and ERK) and the IFN- Although the C-terminal portion of PIAS1 (not encom- induced phosphorylation of STAT1 and STAT3 (Meissner passing the R303) is responsible for the direct interaction et al. 2004; Komyod et al. 2005). Therefore, we set out in between PIAS1 and STAT1, the N-terminal part of PIAS1 the present study to elucidate the role of arginine meth- including R303 modulates the association between the ylation in STAT1 signaling and identified the PIAS1 two proteins. This N-terminal domain restricts the in- protein as an important target of PRMT1 in this pathway. teraction of PIAS1 to STAT1 dimers by inhibiting its Our findings reveal a novel mechanism how the interaction with STAT1 monomers (Liao et al. 2000) nuclear inhibitor of STAT1 signaling, PIAS1, is activated supporting our findings that methylation of R303 is in the course of the IFN response to restrain STAT1 important for the PIAS1–STAT1 interaction. Interest- function and thus how duration and magnitude of IFN ingly, the methylation site is not embedded in a consensus signaling is balanced. We demonstrate that arginine methylation sequence of PRMT1, which typically har- methylation by PRMT1 influences the transcriptional bors the methylated arginine in a RGG or RXR context activity of STAT1 negatively by regulating the interaction (Smith et al. 1999). In general, methylation of the arginine between STAT1 and it repressor protein PIAS1. Interest- residue is thought to regulate protein–protein and pro- ingly, in the literature PRMT1 has been reported to act as tein–nucleic acid interactions potentially by increasing transcriptional coactivator (Wang et al. 2001; An et al. its ‘‘hydrophobic’’ character (Boisvert et al. 2005). Meth- 2004; Wagner et al. 2006) as well as corepressor (Kwak ylation of arginine residues occurs also in other chroma- et al. 2003; Yu et al. 2006). Our model for the cooperation tin proteins involved in cytokine signaling, like the between PRMT1 and PIAS1 in STAT1 signaling is sum- PRMT1-driven methylation of the arginine–glycine rich marized in Figure 7D. We find that PRMT1 represses N terminus of the coactivator NIP45, which promotes a subset of IFNg-activated STAT1 target genes, which the association between NIP45 and the transcription overlaps with genes regulated by PIAS1 (Liu et al. 2004). factor NFAT and results in an augmented activity of the PRMT1 mediates its function in STAT1 signaling IL-4 and IFNg gene promoter (Mowen et al. 2004). through PIAS1, as depletion of PIAS1 abrogates the in- Mechanistically, it was suggested that the interaction hibitory activity of PRMT1. In search of the molecular between PIAS1 and STAT1 inhibits the DNA-binding mechanism of this cooperation, we discover that PRMT1 ability of STAT1 and causes the reduction of transcrip- dimethylates PIAS1 at R303. Association between PRMT1 tional activity (Liu et al. 1998; Shuai and Liu 2005). and PIAS1 and methylationRETRACTED of R303 is induced in the Similar data were also reported for other PIAS family late phase of the IFNg response and enables gene-specific members; for example, PIAS3 interacts with STAT3 and recruitment of PIAS1. Therefore, depletion of PRMT1 suppresses the interaction of STAT3 with the DNA results in the loss of PIAS1 promoter binding. Consistently, (Chung et al. 1997). Given that R303 is conserved also transcriptional repression and promoter recruitment of in other PIAS family members, arginine methylation of a methylation-deficient PIAS1 mutant is abrogated, rein- PIAS proteins might play a more general role in cytokine- forcing that PRMT1-dependent methylation of PIAS1 is dependent gene regulation. PIAS1 knockout mice reveal important for its repressive function. The promoter-associ- enhanced protection against pathogenic infections, al- ated methylation of PIAS1 then triggers the interaction though PIAS1 represses only a subset of 9% of the IFN- between STAT1 and PIAS1 and enforces the release of induced STAT target genes (Liu et al. 2004), indicating STAT1 from its target genes, which subsequently results in that PIAS1 is a physiologically important negative regu- the decline of transcriptional activation. This interpreta- lator. A common characteristic of genes regulated by tion is supported by our findings that the methylation- PIAS1 and also PRMT1, like the GBP1, CXCL9, and deficient PIAS1 mutant exhibits a decreased interaction CXCL10 gene, is that they contain low-affinity binding with STAT1 in co-IP, and that depletion of PRMT1 or sites of STAT1, whereas the high-affinity binding site of

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STAT1 in the IRF1 gene promoter does not depend on Antibodies PIAS1 as well as PRMT1 (Liu et al. 2004). The following antibodies were employed: anti-Flag (F3165) from Methylation by PRMT1 seems to be not the only Sigma, anti-HA (sc-805) from Santa Cruz Biotechnologies, anti-5- mechanism for how PIAS1 function is regulated. It was His (34,660) from Qiagen, anti-STAT1 (sc-346) from Santa Cruz reported recently that PIAS1 is phosphorylated at Ser 90 by Biotechologies, anti-STAT1pY701 (9171) from Cell Signaling, the IKKa kinase particularly in response to TNFa and LPS anti-b-tubulin (MAB 3408) from Chemicon, anti-HDAC1 (sc- stimulation, and that this modification is required to 7872) from Santa Cruz Biotechnologies, anti-PRMT6 (Wagner repress the NF-kB/STAT1 activity (Liu et al. 2007). Similar et al. 2006), anti-PRMT4 (Kleinschmidt et al. 2008), anti-PRMT1 to the regulation of PIAS1 by PRMT1, S90 phosphoryla- (07-404) from Upstate Biotechnologies for Western blot, anti- tion was needed to allow promoter recruitment of PIAS1 PRMT1 (Kleinschmidt et al. 2008) for ChIP, and anti-PIAS1 (sc- 8152) from Santa Cruz Biotechnologies for Western blot/IP. For k in NF- B-activated transcription and resulted in the de- ChIP analysis we generated a polyclonal rabbit antibody against tachment of NF-kB from its targets. In contrast to the the C terminus of human PIAS1 (amino acids 384–651). The Myc regulation by PRMT1, the Sumo E3-ligase activity was (9E10) antibody was a kind gift from Martin Eilers. As control necessary for the repressive function of PIAS1, since antibodies in ChIP and IP we used anti-p15 (sc-1429) from Santa a sumoylation-deficient PIAS1 mutant was defective in Cruz Biotechnologies or rabbit IgG from Pierce (31,235) and from S90 phosphorylation (Liu et al. 2007). Interestingly, we did Sigma (I5006), respectively. not find phosphorylated PIAS1 species in response to IFNg treatment (data not shown). Thus, the mechanism of Cell culture and transfections PIAS1 regulation might differ depending on the stimulus, transcription factor, target genes, and cell type. In the HeLa and HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine future it will be important to also study the role of arginine serum (FBS). A375 cells were cultured in RPMI supplemented methylation of PIAS1 in its STAT1-independent functions. with 10% FBS. IFNg (Strathmann Biotec) was used at concen- In summary, our results identify PIAS1 as the crucial trations between 5 and 15 ng/mL for the indicated time points. target of arginine methylation and PRMT1 in IFN signal- Transfection of HeLa and HEK293 cells was carried out using the ing. PRMT1 is involved in turning off transcriptional Ca-Phosphat method. activity of STAT1 and represses the transcription factor siRNA oligonucleotide duplexes were purchased from Dhar- by regulating the interaction between STAT1 and it re- macon for targeting the human PRMT1, PIAS1, PRMT4, and pressor protein PIAS1 in a methylation-dependent manner. PRMT6, respectively. The transfections were carried out with Hence, PRMT1 is a very interesting target in therapies of Oligofectamine (Invitrogen). siRNA duplexes were transfected at viral infections and also deregulated STAT signal trans- a final concentration of 100 nM in HeLa, HEK293, or A375 cells. Afer 2 d cells were collected for protein or RNA preparation. The duction. How PRMT1 itself is regulated in the course of siRNA sequences are listed in the Supplemental Material. IFN stimulation, needs to be addressed in the future.

RT-QPCR Materials and methods Total RNA was isolated using PeqGold total RNA Kit (PeqLab). Two micrograms of RNA were applied to reverse transcription by Plasmids incubation with 0.5 mg of oligo(dT)17 primer and 100 U of M- MLV reverse transcriptase (Invitrogen). cDNA was analyzed by The following plasmids were used: pcDNA3.1-PRMT1 wild-type QPCR, which was performed using ABsolute QPCR SybrGreen and pcDNA3.1-PRMT1-Myc XGX mutant were published re- Mix (Abgene) and the Mx3000P real-time detection system cently (Balint et al. 2005). pGex2T-GAR and pGex2T-PRMT3 (Agilent). The primers used in RT-QPCR are indicated in the were described previously (Tang et al. 1998). pGex4T-1-PRMT1 Supplemental Material. All amplifications were performed in and pGex4T-1-PRMT2 were published (Scott et al. 1998). Murine triplicate using 0.5 mL of cDNA per reaction. The triplicate mean pGex4T-1-PRMT4 (Chen et al. 1999) and human pGex2T- values were calculated according to the DDCt quantification PRMT6 (Hyllus et al. 2007) were described. method (Pfaffl 2001) using the GAPDH gene transcription as PIAS1 full-length and deletion constructs were generated by reference for normalization. Relative mRNA levels in uninduced PCR amplification from the pBK-CMV-PIAS1 plasmid (Sapetsch- control cells were equated to 1 and the other values were nig et al. 2002) introducingRETRACTED at the 59-end of a XhoI site and at the expressed relative to this. The RT-QPCR results were reproduced 39-end of a Hind III site and cloned via these sites in the pFastBac at least three times in independent experiments and representa- HT vector or the pRSETA vector (Invitrogen). The following con- tive data sets are shown. structs were generated: pFastBac HT-Pias1 full-length (amino acids 1–651), pRSETA-PIAS1 DN45 (amino acids 46–651), DN261 (amino acids 262–651), DN383 (amino acids 384–651), DC402 ChIP (amino acids 1–253), and DNDC (amino acids 262–474). Further- more, pRSETA-PIAS1 DNDC was used for site-directed muta- HeLa cells were cross-linked in the presence of 1% formal- genesis (Stratagene QuickChange Kit) of Arg 303 to alanine. The dehyde for 10 min at room temperature. Subsequent ChIP pN3-3xFlag PIAS1 (full-length) construct was generated by PCR analysis was performed as described previously (Wagner et al. amplification introducing at the 59-end of an EcoRI site and at the 2006). Immunoprecipitated and eluted DNA was purified with 39-end of a SalI site and cloned into a modified pEGFP-N3 vector QIAquick columns (Qiagen) and amplified by QPCR. The ChIP- (Clontech; EGFP tag replaced by triple Flag tag). pN3-3xFlag QPCR primers are indicated in the Supplemental Material. PIAS1 R303 mutants were generated by site-directed mutagen- All amplifications were performed in triplicate using 0.6 mLof esis (Stratagene Quickchange Kit). R303 was exchanged to K, G, DNA per reaction. The triplicate mean values were displayed and F, respectively. as percentage input and standard deviation was calculated. The

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Weber et al. presented results were reproduced several times in independent bicarbonate, shrunk by dehydration in acetonitrile, and dried in experiments. a vacuum centrifuge. Reduction/alkylation of disulfide bonds and enzymatic in-gel digestion was performed as described (Bauer and Krause 2005). MS and MS/MS experiments were Preparation of protein extracts and IP carried out on a quadrupole orthogonal acceleration time-of- Whole-cell extract were prepared from HeLa or HEK293, re- flight mass spectrometer Q-Tof Ultima equipped with a CapLC spectively. Cells were rinsed in PBS and lysed in IPH buffer (50 liquid chromatography system (Micromass). Peptides were sep- ˚ mM Tris at pH 8, 150 mM NaCl, 5 mM EDTA, 0.5% NP40, arated using a capillary column (Atlantis dC18,3mm, 100 A, 150 mm 3 75 mm i.d.; Waters GmbH) and an eluent flow rate of 200 1 mM DTT, 150 mM NaF, 2 mM Na3VO4). After lysis the extracts were cleared by centrifugation and supernatant was used for nL/min. Mobile phase A was 0.1% formic acid (v/v) in acetoni- Western blot analysis. trile-water (3:97, v/v) and B was 0.1% formic acid in acetonitrile- Nuclear extract were prepared from HeLa cells. Cells were water (8:2, v/v). Runs were performed using a gradient of 3%– washed with PBS and subsequently lysed in buffer A1 (10 mM 64% B in 100 min. Data-dependent aquisition was used, whereas peptides, which covered the methylation sites, were preferen- HEPES KOH at pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM tially selected for MS/MS. The processed MS/MS spectra (Mas- DTT, 0.04% NP40, 2 mM Na3VO4) for 5 min on ice. After centrifugation cytoplasmic supernatant was removed and nuclei sLynx version 4.0 software) were compared with the theoretical were washed in buffer A2 (10 mM HEPES KOH at pH 7.9, 10 mM fragment ions of peptides of human PIAS1 protein.

KCl, 1.5 mM MgCl2, 0.5 mM DTT, 2 mM Na3VO4) to exclude cytoplasmatic contamination. The remaining nuclear pellet was lysed in IPH buffer, and thereafter a benzonase digestion Growth curves was performed. For IP, 1–2 mg of the indicated antibodies were For analysis of IFNg-induced growth arrest, 10,000 cells per well incubated with 500 mg of nuclear extract as described previously were seeded and subsequently stimulated with 7.5 or 15 ng/mL (Kleinschmidt et al. 2008). IFNg. Cells were harvested at the indicated time points and cell numbers were determined in triplicates for each condition and time point by using the Neubauer counting chamber. For growth Recombinant proteins and GST pulldown experiments arrest analysis in PRMT1- or PIAS1-depleted cells, we performed GST fusion proteins and His tag proteins were prepared from transient transfection of the corresponding siRNAs in HeLa cells Escherichia coli BL21 according to standard procedures. His- for 48 h. Subsequently, cells were trypsinized, seeded (10,000 tagged PIAS1 (full length) was expressed and purified from Sf9 cells per well) and treated with IFNg. The knockdown was cells according to the manufacturer’s instructions for the Bac-to- monitored in parallel by Western blot analysis. Bac Baculovirus Expression System (Invitrogen). GST and GST- PRMT1 immobilized on glutathione beads were blocked with 10% goat serum in HEGN buffer (30 mM HEPES-KOH at pH 7.6, Acknowledgments 125 mM KCl, 0.16 mM EDTA, 16% glycerol, 0.16% NP-40, 1 mM DTT) for 10 min at room temperature. Precleared whole-cell We thank Hervey Herschman for providing the pGEX2T-GAR extract (250 mg) (Kleinschmidt et al. 2008) or recombinant His- plasmid. We thank Alexander Brehm, Martin Eilers, Jean-Fran- tagged PIAS1 (0.5 mg) was incubated with the blocked beads for cois Naud, and Uwe Vinkemeier for critical reading of the 1 h at room temperature. Subsequently, beads were washed five manuscript and valuable suggestions. We thank all members of times with IPH buffer and examined by Western blot analysis. the U.M.B. laboratory and Alexandra Sapetschnig for their help during work progress, and Inge Pelz for technical assistance. This work was supported by the DFG (Deutsche Forschungsgemein- In vitro methyltransferase assay schaft), by funding from Land Hessen, and from BMBF (Bundes- Eluted and dialyzed GST-PRMTs (0.5 mg) were incubated with ministerium fu¨ r Bildung und Forschung) to U.M.B. approximatly 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 buffer References and analyzed by SDS-PAGE, blotting and autoradiography. Abramovich, C., Yakobson, B., Chebath, J., and Revel, M. 1997. A protein–arginine methyltransferase binds to the intracyto- In vivo methylation assay plasmic domain of the IFNAR1 chain in the type I interferon RETRACTEDreceptor. EMBO J. 16: 260–266. Transfected Hela cells were pretreated with cycloheximid and Alexander, W.S., and Hilton, D.J. 2004. The role of suppressors chloramphenicol in the presences or absence of IFNg (5 ng/mL). of cytokine signaling (SOCS) proteins in regulation of the After a 30-min pretreatment, L-[methyl-3H]-methionine (70 Ci/ immune response. Annu. Rev. Immunol. 22: 503–529. mM; GE Healthcare Life Science) was added and cells were Altschuler, L., Wook, J.O., Gurari, D., Chebath, J., and Revel, M. cultured for another 3 h (Liu and Dreyfuss 1995). Whole-cell 1999. Involvement of receptor-bound protein methyltrans- extracts were prepared and employed for IP of Flag-tagged PIAS1 ferase PRMT1 in antiviral and antiproliferative effects of or endogenous PIAS1 as well as STAT1. Immunoprecipitates type I interferons. J. Interferon Cytokine Res. 19: 189–195. were separated by SDS-PAGE. The gel was incubated in Enlight An, W., Kim, J., and Roeder, R.G. 2004. Ordered cooperative solution (MoBiTec) according to manufacturer instructions and functions of PRMT1, p300, and CARM1 in transcriptional 3 H-labeled proteins were visualized by fluorography. activation by p53. Cell 117: 735–748. Balint, B.L., Szanto, A., Madi, A., Bauer, U.M., Gabor, P., Benko, S., Puskas, L.G., Davies, P.J., and Nagy, L. 2005. Arginine Mass spectrometric analysis methylation provides epigenetic transcription memory for Gel-excised methylated PIAS1 protein (DNDC) bands were retinoid-induced differentiation in myeloid cells. Mol. Cell. washed with 50% (v/v) acetonitrile in 25 mM ammonium Biol. 25: 5648–5663.

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RETRACTED

132 GENES & DEVELOPMENT Retraction Genes & Development 23: 118–132 (2009)

PRMT1-mediated arginine methylation of PIAS1 regulates STAT1 signaling Susanne Weber, Florian Maaß, Michael Schuemann, Eberhard Krause, Guntram Suske, and Uta-Maria Bauer

‘‘In agreement with all authors, we herewith retract our above-mentioned article. In this article, we identified PRMT1 as a novel transcriptional corepressor of IFN-induced STAT1 target genes that cooperates with PIAS1. In the process of following up these findings, researchers in the group of the corresponding author recently uncovered that the original experimental data points of a subset of quantitative PCR (qPCR) results were intentionally falsified by the first author. Therefore, the reported results of Figures 3 (B and C) and 6 (B and C) and Supplemental Figures S7, S11B, and S12 are not supported by the raw qPCR data. As a consequence, we cannot maintain the central conclusions of our paper that PIAS1 is recruited to STAT1 target gene promoters in a PRMT1- and methylation-dependent manner; that PIAS1 methylation leads to promoter release of STAT1 from a subset of target genes and, consequently, to transcriptional repression; and that PRMT4 and PRMT6 do not influence PIAS1-dependent STAT1 target genes. Other results of the paper (Figs. 1, 2, 4, 5, 7) and their conclusions remain unaffected.

‘‘We deeply apologize for any inconvenience this scientific misconduct might have caused.’’

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PRMT1-mediated arginine methylation of PIAS1 regulates STAT1 signaling

Susanne Weber, Florian Maaß, Michael Schuemann, et al.

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

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Related Content PRMT1-mediated arginine methylation of PIAS1 regulates STAT1 signaling Susanne Weber, Florian Maaß, Michael Schuemann, et al. Genes Dev. July , 2011 25: 1451

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