PRMT5 modulates the metabolic response to fasting signals Wen-Wei Tsaia, Sherry Niessenb, Naomi Goebela, John R. Yates IIIb, Ernesto Guccionec,d, and Marc Montminya,1 aThe Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, CA 92037; bDepartment of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037; cInstitute of Molecular and Cell Biology, Proteos, Singapore 138673; dDepartment of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119074 Contributed by Marc Montminy, March 11, 2013 (sent for review February 20, 2013) Under fasting conditions, increases in circulating glucagon maintain sites. The results provide a mechanism to explain how latent cy- glucose balance by promoting hepatic gluconeogenesis. Triggering toplasmic regulators such as CRTC2 may contribute to signaling of the cAMP pathway stimulates gluconeogenic gene expression in the nucleus through their association with chromatin mod- through the PKA-mediated phosphorylation of the cAMP response ifying enzymes. element binding (CREB) protein and via the dephosphorylation of the latent cytoplasmic CREB regulated transcriptional coactivator 2 Results (CRTC2). CREB and CRTC2 activities are increased in insulin resis- In mass spectroscopy studies using epitope-tagged CRTC2 to tance, in which they promote hyperglycemia because of constitu- identify relevant interacting proteins, we recovered the protein tive induction of the gluconeogenic program. The extent to which arginine methyltransferase 5 (PRMT5) (Fig. S1 A and B). We CREB and CRTC2 are coordinately up-regulated in response to glu- confirmed the CRTC2:PRMT5 interaction in coimmunopreci- cagon, however, remains unclear. Here we show that, following its pitation studies with epitope-tagged proteins (Fig. 1A). The activation, CRTC2 enhances CREB phosphorylation through an as- CRTC2:PRMT5 association was evident under basal conditions sociation with the protein arginine methyltransferase 5 (PRMT5). In and to a greater extent following exposure to cAMP agonist turn, PRMT5 was found to stimulate CREB phosphorylation via forskolin (FSK). increases in histone H3 Arg2 methylation that enhanced chromatin CRTC2 and its paralogs share a similar structure consisting of accessibility at gluconeogenic promoters. Because depletion of an N-terminal CREB-binding domain (amino acids 1–55), central BIOCHEMISTRY PRMT5 lowers hepatic glucose production and gluconeogenic gene regulatory domain (amino acids 55–600), and C-terminal trans- expression, these results demonstrate how a chromatin-modifying activation domain (amino acids 600–692) (2). In deletion map- enzyme regulates a metabolic program through epigenetic ping studies, PRMT5 was found to associate with the central changes that impact the phosphorylation of a transcription factor regulatory region of CRTC2; deletion of sequences within this in response to hormonal stimuli. domain (amino acids 56–144) disrupted the CRTC2:PRMT5 interaction (Fig. 1B). hronic elevations in circulating blood glucose contribute to the The PRMT family of arginine methylases consists of three types; Cchronic complications of type II diabetes, which include retinal they are distinguished by their ability to carry out mono- or asym- blindness, renal failure, peripheral neuropathy, and cardiovascular metric di-methylation (type I), mono- or symmetric di-methylation diseases (1). Fasting hyperglycemia represents an early sign of (type II), or exclusively monomethylation (type III) (8, 9). PRMT5 insulin resistance, which is characterized by an inability for insulin functions as the major type II symmetric arginine methyltransfer- to promote glucose uptake into muscle and to inhibit glucose ase. Consistent with a critical role during development, mice with production by the liver. aknockoutofPrmt5 have early embryonic lethality (10). PRMT5 During fasting, increases in circulating pancreatic glucagon has also been shown to function in transcriptional regulation as well stimulate the gluconeogenic program via the protein kinase A as signal transduction (11). (PKA)-mediated phosphorylation and activation of the tran- Based on the ability for PRMT5 to associate with CRTC2, we scription factor cAMP response element binding (CREB) pro- tested whether this enzyme is correspondingly recruited to CREB tein, a modification that promotes recruitment of the coactivators target genes in response to fasting signals. In ChIP studies, expo- P300 and CREB binding protein (CBP) (2). In parallel, increases sure of mouse primary hepatocytes to glucagon increased amounts in cAMP signaling also promote the dephosphorylation of the of CRTC2 over CREB-binding sites on the gluconeogenic glucose- CREB regulated transcriptional coactivator 2 (CRTC2) (3). Se- 6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase questered in the cytoplasm through phosphorylation-dependent (Pck1) promoters (Fig. 1C). Consistent with its ability to associate interactions with 14-3-3 proteins, CRTC2 undergoes de- with CRTC2, PRMT5 was also recruited to these CREB target Crtc2 phosphorylation in response to cAMP, where it shuttles to the genes following exposure to glucagon. Depletion of with adenovirally encoded RNAi disrupted PRMT5 recruitment in nucleus and mediates induction of cellular genes via an interaction hepatocytes exposed to glucagon (Fig. 1D). with CREB over relevant binding sites (4, 5). Supporting a functional role for PRMT5 in modulating CREB CRTC2 dephosphorylation represents an early event in the activity, exposure to glucagon increased cAMP response element cAMP response relative to CREB phosphorylation, although the (CRE)-luciferase (Luc) and G6pc-Luc reporter activities ro- extent to which the two processes are linked remains unclear (6). bustly; these effects were disrupted following RNAi-mediated The PKA-mediated phosphorylation of CREB on chromatin depletion of Prmt5 (Fig. 1E). Conversely, overexpression of Crtc2 templates appears to be generally inefficient compared with its phosphorylation on naked DNA templates; these inhibitory effects can be circumvented through the acetylation of resident nucleo- Author contributions: W.-W.T. and M.M. designed research; W.-W.T. and N.G. performed somes and subsequent increases in chromatin accessibility (7). The research; S.N., J.R.Y., and E.G. contributed new reagents/analytic tools; W.-W.T., S.N., J.R.Y., extent to which these or other chromatin modifications are per- E.G., and M.M. analyzed data; and W.-W.T. and M.M. wrote the paper. missive for CREB activation in vivo has not be addressed, however. The authors declare no conflict of interest. Here we characterize a CRTC2-associated arginine methylase 1To whom correspondence should be addressed. E-mail: [email protected]. that promotes CREB phosphorylation and target gene activation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in part by enhancing chromatin accessibility over CREB binding 1073/pnas.1304602110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1304602110 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 A B C input input IP: myc CRTC2 CRTC2 - + - + - + FSK - + + + + + myc-PRMT5 - + + + + + CRTC2 myc PRMT5 myc-PRMT5 PRMT5 PRMT5 CREB Flag- Flag- CRTC2s CRTC2s IP: HA PRMT5 D E F CRTC2 CRTC2 CRE-Luc NSi Prmt5i glucagon - + - + PRMT5 p-CRTC2 PRMT5 PRMT5 G6pc-Luc CRTC2 CREB Hsp90 Fig. 1. PRMT5 associates with CRTC2 and is recruited to gluconeogenic genes in response to glucagon. (A) Coimmunoprecipitation assay of HEK293T cells expressing epitope-tagged PRMT5 and CRTC2. No interaction with epitope-tagged CREB as shown. Exposure to FSK (30 min) indicated. (B) Coimmunopre- cipitation assay of WT and mutant CRTC2 polypeptides with PRMT5 in cells exposed to FSK for 30 min. Amino acid endpoints for each construct indicated. (C) ChIP assay of primary hepatocytes showing effect of glucagon (1 h) on recruitment of PRMT5 and CRTC2 to CREB-binding sites over gluconeogenic (Pck1, G6pc) promoters. Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SD. **P < 0.01; ***P < 0.001. (D) Effect of CRTC2 depletion by RNAi-mediated knockdown on PRMT5 recruitment to gluconeogenic promoters in primary hepatocytes exposed to glucagon (1 h). Each bar represents averaged results, n = 2 biological replicates, assayed three times each; error bars, SD. *P < 0.05; **P < 0.01. (E) Effect of PRMT5 depletion on CRE-Luc and G6pc-Luc reporter activities in primary hepatocytes exposed to glucagon (4–6 h). Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SD. ***P < 0.001. (F) Western blot showing effect of Prmt5 RNAi on PRMT5 protein amounts and on CRTC2 dephosphorylation in primary hepatocytes exposed to glucagon (30 min). Hsp, heat shock protein 90. and Prmt5 increased G6pc-Luc reporter activity cooperatively in exposed to glucagon (Fig. 2C). As a consequence, knockdown of transient assays of HepG2 hepatoma cells (Fig. S1C). In keeping Prmt5 reduced glucose secretion from primary hepatocytes exposed with the role of CRTC2 in promoting PRMT5 recruitment, to glucagon (Fig. 2D). Pointing to a specific effect on CREB sig- PRMT5 had no effect on G6pc-Luc reporter activity in cells naling, Prmt5 knockdown did not alter mRNA amounts for in- depleted of Crtc2 (Fig. S1D). Arguing against a direct effect on sulin-like growth factor binding protein 1 (Igfbp1), a Forkhead box cAMP signaling per se, knockdown of Prmt5