pubmed.ncbi.nlm.nih.gov

PRMT5 modulates the metabolic response to fasting signals - PubMed

  • ️Tue Jan 01 2013

PRMT5 modulates the metabolic response to fasting signals

Wen-Wei Tsai et al. Proc Natl Acad Sci U S A. 2013.

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.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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) Coimmunoprecipitation 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.

Fig. 2.
Fig. 2.

PRMT5 stimulates hepatic gluconeogenesis during fasting and in diabetes. (A) Effect of hepatic Prmt5 depletion with adenovirally encoded RNAi on fasting blood glucose concentrations in genetically obese (ob/ob) mice compared with lean controls (ob/+). Each bar represents averaged results, n = 5; error bars, SD. **P < 0.01; ***P < 0.001. (B) Effect of hepatic PRMT5 knockdown on gluconeogenic gene expression in livers of fasted obese (ob/ob) mice. Each bar represents averaged results, n = 5; error bars, SD. **P < 0.01; ***P < 0.001. (C) Effect of PRMT5 knockdown on gluconeogenic gene expression in primary hepatocytes exposed to glucagon. Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Effect of PRMT5 depletion on glucose secretion from primary hepatocytes exposed to glucagon. Each bar represents averaged results, n = 3; error bars, SD. **P < 0.01. (E) Gene profiling analysis of primary mouse hepatocytes following RNAi-mediated knockdown of Prmt5. Pie charts show proportion of total cellular genes up or down-regulated 1.5-fold or better under basal conditions and following exposure to glucagon. (F) Heat map showing effects of Prmt5 knockdown on top 40 scoring glucagon-inducible genes in primary hepatocytes.

Fig. 3.
Fig. 3.

PRMT5 promotes H3R2 symmetric dimethylation and recruitment of WDR5 to gluconeogenic promoters in response to glucagon. (A) Western blot showing effect of PRMTs (–7) on H3R2me2 and H3R2me2-s of histone H3 Arg-2 in vitro. (B) Western blot showing effect of PRMT5 and PRMT6-catalyzed histone H3 Arg-2 di-methylation on WDR5 binding by pull-down assay with GST-WDR5. (C) ChIP assay of primary hepatocytes, showing effect of glucagon (1 h) on H3R2me2-s methylation and WDR5 recruitment at CREB-binding sites over gluconeogenic promoters. Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SD. *P < 0.05; **P < 0.01. (D) ChIP assay showing effect of Prmt5 knockdown on H3R2me2-s and WDR5 amounts over gluconeogenic promoters in hepatocytes exposed to glucagon (1 h). Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SD. *P < 0.05; **P < 0.01.

Fig. 4.
Fig. 4.

PRMT5 promotes CREB phosphorylation at gluconeogenic promoters by increasing chromatin accessibility at CREB-binding sites. (A) Effect of glucagon (1 h) exposure on total histone H3 amounts over CREB-binding sites on gluconeogenic genes in primary hepatocytes. Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SD. *P < 0.05; **P < 0.01. (B) Effect of Prmt5 knockdown on H3 occupancy over CREB-binding sites at gluconeogenic promoters in hepatocytes exposed to glucagon for 1 h. Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SD. *P < 0.05. (C) Effect of PRMT5 depletion on MNase digestion of chromatin from primary hepatocytes exposed to glucagon (1 h). Increasing amounts of MNase indicated. (D) Effect of Prmt5 RNAi on MNase sensitivity over CREB-binding sites on Pck1 and G6pc promoters in cells exposed to glucagon (1 h). Representative experiment from three independent experiments shown. Each bar represents averaged results, assayed three times each; error bars, SD. *P < 0.05; **P < 0.01. ***P < 0.001. (E) ChIP assay showing effect of Prmt5 RNAi on CREB and p-CREB amounts over gluconeogenic promoters in primary hepatocytes exposed to glucagon (1 h). Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SD. *P < 0.05; **P < 0.01. ***P < 0.001. (F) Western blot showing effect of PRMT5 depletion on amounts of p-CREB and total CREB in primary hepatocytes under basal conditions and following exposure to glucagon (30 min). Relative phosphorylation of LKB in response to glucagon shown for comparison. (G) Model for activation of gluconeogenic genes during fasting. Exposure to glucagon leads to CRTC2 activation and association with PRMT5. Following its CRTC2-mediated recruitment to relevant target genes, PRMT5 increases nucleosome clearance over CREB-binding sites, in part through increases in histone H3 Arg2 di-methylation that promote WDR5 occupancy. As a consequence of the increase in chromatin accessibility, CREB phosphorylation by PKA is up-regulated over PRMT5 occupied promoters, leading to increases in target gene expression. NSi, nonspecific RNAi; P5i, PRMT5 RNAi; p-LKB1, phospho-liver kinase B1.

Similar articles

Cited by

References

    1. Saltiel AR. New perspectives into the molecular pathogenesis and treatment of type 2 diabetes. Cell. 2001;104(4):517–529. - PubMed
    1. Altarejos JY, Montminy M. CREB and the CRTC co-activators: Sensors for hormonal and metabolic signals. Nat Rev Mol Cell Biol. 2011;12(3):141–151. - PMC - PubMed
    1. Koo SH, et al. The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism. Nature. 2005;437(7062):1109–1111. - PubMed
    1. Screaton RA, et al. The CREB coactivator TORC2 functions as a calcium- and cAMP-sensitive coincidence detector. Cell. 2004;119(1):61–74. - PubMed
    1. Bittinger MA, et al. Activation of cAMP response element-mediated gene expression by regulated nuclear transport of TORC proteins. Curr Biol. 2004;14(23):2156–2161. - PubMed

Publication types

MeSH terms

Substances