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PRMT1-Mediated Arginine Methylation of PIAS1 Regulates STAT1 Signaling

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PRMT1-Mediated Arginine Methylation of PIAS1 Regulates STAT1 Signaling Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press 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 118 GENES & DEVELOPMENT 23:118–132 Ó 2009 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/09; www.genesdev.org Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press 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
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