PRMT5 modulates the metabolic response to fasting signals

Abstract

Under fasting conditions, increases in circulating glucagon maintain glucose balance by promoting hepatic gluconeogenesis. Triggering of the cAMP pathway stimulates gluconeogenic gene expression through the PKA-mediated phosphorylation of the cAMP response element binding (CREB) protein and via the dephosphorylation of the latent cytoplasmic CREB regulated transcriptional coactivator 2 (CRTC2). CREB and CRTC2 activities are increased in insulin resistance, in which they promote hyperglycemia because of constitutive induction of the gluconeogenic program. The extent to which CREB and CRTC2 are coordinately up-regulated in response to glucagon, however, remains unclear. Here we show that, following its activation, CRTC2 enhances CREB phosphorylation through an association with the protein arginine methyltransferase 5 (PRMT5). In turn, PRMT5 was found to stimulate CREB phosphorylation via increases in histone H3 Arg2 methylation that enhanced chromatin accessibility at gluconeogenic promoters. Because depletion of PRMT5 lowers hepatic glucose production and gluconeogenic gene expression, these results demonstrate how a chromatin-modifying enzyme regulates a metabolic program through epigenetic changes that impact the phosphorylation of a transcription factor in response to hormonal stimuli.

Results

In mass spectroscopy studies using epitope-tagged CRTC2 to identify relevant interacting proteins, we recovered the protein arginine methyltransferase 5 (PRMT5) (Fig. S1 A and B). We confirmed the CRTC2:PRMT5 interaction in coimmunoprecipitation studies with epitope-tagged proteins (Fig. 1A). The CRTC2:PRMT5 association was evident under basal conditions and to a greater extent following exposure to cAMP agonist forskolin (FSK).

CRTC2 and its paralogs share a similar structure consisting of an N-terminal CREB-binding domain (amino acids 1–55), central regulatory domain (amino acids 55–600), and C-terminal transactivation domain (amino acids 600–692) (2). In deletion mapping studies, PRMT5 was found to associate with the central regulatory region of CRTC2; deletion of sequences within this domain (amino acids 56–144) disrupted the CRTC2:PRMT5 interaction (Fig. 1B).

The PRMT family of arginine methylases consists of three types; they are distinguished by their ability to carry out mono- or asymmetric di-methylation (type I), mono- or symmetric di-methylation (type II), or exclusively monomethylation (type III) (8, 9). PRMT5 functions as the major type II symmetric arginine methyltransferase. Consistent with a critical role during development, mice with a knockout of Prmt5 have early embryonic lethality (10). PRMT5 has also been shown to function in transcriptional regulation as well as signal transduction (11).

Based on the ability for PRMT5 to associate with CRTC2, we tested whether this enzyme is correspondingly recruited to CREB target genes in response to fasting signals. In ChIP studies, exposure of mouse primary hepatocytes to glucagon increased amounts of CRTC2 over CREB-binding sites on the gluconeogenic glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (Pck1) promoters (Fig. 1C). Consistent with its ability to associate with CRTC2, PRMT5 was also recruited to these CREB target genes following exposure to glucagon. Depletion of Crtc2 with adenovirally encoded RNAi disrupted PRMT5 recruitment in hepatocytes exposed to glucagon (Fig. 1D).

Supporting a functional role for PRMT5 in modulating CREB activity, exposure to glucagon increased cAMP response element (CRE)-luciferase (Luc) and G6pc-Luc reporter activities robustly; these effects were disrupted following RNAi-mediated depletion of Prmt5 (Fig. 1E). Conversely, overexpression of Crtc2 and Prmt5 increased G6pc-Luc reporter activity cooperatively in transient assays of HepG2 hepatoma cells (Fig. S1C). In keeping with the role of CRTC2 in promoting PRMT5 recruitment, PRMT5 had no effect on G6pc-Luc reporter activity in cells depleted of Crtc2 (Fig. S1D). Arguing against a direct effect on cAMP signaling per se, knockdown of Prmt5 did not interfere with the dephosphorylation and hence activation of CRTC2 in hepatocytes exposed to glucagon (Fig. 1F). Taken together, these results indicate that PRMT5 up-regulates CREB activity following its CRTC2-dependent recruitment to relevant genes in response to cAMP agonist.

We tested whether PRMT5 is required for induction of the gluconeogenic program during fasting. RNAi-mediated depletion of Prmt5 in liver decreased circulating glucose concentrations and gluconeogenic gene expression in lean mice and to a greater extent in genetically obese (ob/ob) or high-fat diet fed insulin-resistant mice (Fig. 2 A and B and Fig. S2 A–C). The effect of PRMT5 on hepatic gluconeogenesis appears cell autonomous because RNAi-mediated depletion of Prmt5 also down-regulated expression of Pck1, G6pc, and Peroxisome Proliferator Activated Receptor Gamma Coactivator 1a (Ppargc1a) genes in primary hepatocytes exposed to glucagon (Fig. 2C). As a consequence, knockdown of Prmt5 reduced glucose secretion from primary hepatocytes exposed to glucagon (Fig. 2D). Pointing to a specific effect on CREB signaling, Prmt5 knockdown did not alter mRNA amounts for insulin-like growth factor binding protein 1 (Igfbp1), a Forkhead box O1 (FOXO1) target gene that is also up-regulated during fasting (Fig. S2D).

PRMT5 has been reported to exert both positive and negative effects on cellular gene expression (8), prompting us to evaluate relative effects of this methylase in primary hepatocytes by gene profiling analysis. Depletion of Prmt5 down-regulated the expression of 1.5% of cellular genes, whereas it up-regulated the expression of about 1% of genes (Fig. 2E). By contrast, Prmt5 depletion decreased the expression of 20% of glucagon-inducible genes, many of which are direct targets for CREB that function in fasting glucose metabolism [tyrosine amino transferase (Tat), G6pc, Pck1, Ppargc1a (Fig. 2F). Consistent with its proposed role in potentiating CREB activity, knockdown of Prmt5 did not up-regulate the expression of any glucagon-inducible gene in this setting.

PRMT5 has been shown to catalyze the symmetric di-methylation of histone H3 at Arg-2 (H3R2me2-s) and at Arg-8 (H3R8me2-s); these marks have been associated with transcriptional repression as well as activation (8, 12). In turn, H3R2me2-s has been reported to modulate cellular gene expression in part by promoting the recruitment of WD repeat protein 5 (WDR5) (12), a core subunit of lysine methyl transferase complexes (13) and a component of the transcriptional adaptor (Ada) 2A containing complex (ATAC) (14). We confirmed that PRMT5 catalyzes increases in H3R2me2-s in vitro and that H3R2me2-s in turn associates with WDR5 by GST pull-down assays (Fig. 3 A and B).

Consistent with these observations, exposure of primary hepatocytes to glucagon increased H3R2me2-s amounts alongside the active mark histone H3 trimethyl-lysine 4 at CREB-binding sites over gluconeogenic genes (Fig. 3C and Fig. S3A). In keeping with these increases, exposure to glucagon stimulated WDR5 recruitment to gluconeogenic promoters (Fig. 3C). Conversely, Prmt5 depletion decreased both H3R2me2-s and WDR5 amounts over these genes in primary hepatocytes following glucagon treatment (Fig. 3D). Depletion of Crtc2 also decreased H3R2me2-s amounts at Pck1 and G6pc promoters, demonstrating the importance of CRTC2 in mediating PRMT5 recruitment (Fig. S3B).

Based on the ability for PRMT5 to increase H3R2me2-s amounts over CREB target genes, we considered whether this methylase could open chromatin structure at relevant promoters in response to glucagon. Supporting this idea, exposure to glucagon decreased nucleosome occupancy over CREB-binding sites in primary hepatocytes as revealed by decreases in histone H3 occupancy; these effects were blocked following RNAi-mediated knockdown of Prmt5 or Crtc2 (Fig. 4 A and B and Fig. S4A). Taken together, these results indicate that PRMT5 enhances CREB activation in part by increasing H3R2me2-s amounts over CREB binding sites in response to cAMP agonist.

We reasoned that PRMT5 may enhance chromatin accessibility over CREB-regulated promoters. To test this idea, we evaluated effects of PRMT5 on micrococcal nuclease (MNase) sensitivity in control and Prmt5-depleted cells in response to glucagon. Although it did not alter MNase sensitivity globally, Prmt5 depletion blocked glucagon-dependent increases in MNase sensitivity over G6pc and Pck1 promoters (Fig. 4 C and D). As a result, Ser133-phosphorylated CREB (p-CREB) amounts over gluconeogenic genes were substantially reduced in Prmt5-depleted compared with control cells exposed to glucagon (Fig. 4E and Fig. S4B). Similarly, depletion of Crtc2 reduced p-CREB amounts over gluconeogenic promoters in cells exposed to glucagon, reflecting in part the importance of this coactivator in mediating PRMT5 recruitment (Fig. S4C). Although Prmt5 depletion decreased p-CREB amounts in cells exposed to glucagon, it did not interfere with the phosphorylation of cytosolic PKA substrates such as liver kinase B1 (LKB1) (Fig. 4F). Collectively, these results demonstrate that PRMT5 modulates the expression of signal-dependent genes through increases in chromatin accessibility that are permissive for the phosphorylation of nuclear factors such as CREB by PKA and perhaps other regulatory enzymes (Fig. 4G).

Discussion

Under fasting conditions, increases in circulating glucagon promote hepatic glucose production via the cAMP-dependent phosphorylation of CREB and dephosphorylation of CRTC2 (2). CREB and CRTC2 activities are increased in diabetes, where they contribute to the attendant hyperglycemia via up-regulation of the gluconeogenic program.

We found that CRTC2 stimulates gluconeogenic gene expression via an association with the protein arginine methyltransferase PRMT5 in response to glucagon stimulation. Following its recruitment to CREB-binding sites, PRMT5 promotes increases in H3R2me2-s that in turn trigger the expression of relevant CREB target genes. PRMT5-dependent increases in H3R2me2-s appeared to stimulate cellular gene expression in part by enhancing the recruitment of WDR5. The CRTC2-dependent recruitment of this chromatin-modifying enzyme to CREB-binding sites appears critical for the subsequent phosphorylation of CREB.

In addition to effects of PRMT5, the type I PRMT family member PRMT4/coactivator-associated arginine methyltransferase 1 (CARM1) has also been found to promote gluconeogenic gene expression via an interaction with CREB and through subsequent increases in H3 methylation (15). Indeed, we have also detected increases in asymmetric H3R8me2 amounts at CREB-binding sites, potentially catalyzed by the type I enzyme PRMT2, in response to glucagon simulation. Collectively, these studies indicate that histone arginine methylation plays an important role in hepatic gluconeogenesis.

Phosphorylation has been shown to modulate transcription factors and coactivators in the cytoplasm by regulating their nuclear translocation, and in the nucleus by increasing their transactivation potential (16). Consistent with its proximity to the signaling machinery in the cytoplasm, CRTC2 undergoes rapid and quantitative dephosphorylation in response to glucagon. Although the two events are closely spaced, CRTC2 dephosphorylation and promoter recruitment appear to precede cAMP-dependent increases in phospho-CREB levels, suggesting a potential role for CRTC2 in contributing to CREB activation (6).

In reconstitution studies, CREB phosphorylation by PKA was strongly inhibited on chromatin compared with naked DNA templates, whereas CREB occupancy was more resistant (7). We found that CRTC2 enhances CREB phosphorylation in response to glucagon via its association with PRMT5. In turn, PRMT5 potentiated CREB phosphorylation through increases in H3R2me2-s that stimulate nucleosome clearance and thereby increase accessibility to PKA. Taken together, these results indicate that local changes in chromatin structure may be required for signaling to certain nuclear factors. Future studies should reveal whether such changes are predictive for the selective activation of target gene subsets by CREB and other phosphorylation-dependent factors in response to extracellular stimuli.

Materials and Methods

Supplementary Material

Acknowledgments

We thank M. Bedford for PRMT plasmids and K. Ravnskjaer and J. Butler for advice and reagents. Funding for this work was provided by National Institutes of Health Grants F32 DK096778 (to W.-W.T.), R01 DK049777, R01 DK083834, and R01 DK091618; the Clayton Foundation for Medical Research; the Leona M. and Harry B. Helmsley Charitable Trust; and the Kieckefer Foundation.

Footnotes

References

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