Autophagy is a highly conserved self-digestion process, which is essential for maintaining homeostasis and viability in response to nutrient starvation1,2,3,4. Although the components of autophagy in the cytoplasm have been well studied5,6, the molecular basis for the transcriptional and epigenetic regulation of autophagy is poorly understood. Here we identify co-activator-associated arginine methyltransferase 1 (CARM1) as a crucial component of autophagy in mammals. Notably, CARM1 stability is regulated by the SKP2-containing SCF (SKP1-cullin1-F-box protein) E3 ubiquitin ligase in the nucleus, but not in the cytoplasm, under nutrient-rich conditions. Furthermore, we show that nutrient starvation results in AMP-activated protein kinase (AMPK)-dependent phosphorylation of FOXO3a in the nucleus, which in turn transcriptionally represses SKP2. This repression leads to increased levels of CARM1 protein and subsequent increases in histone H3 Arg17 dimethylation. Genome-wide analyses reveal that CARM1 exerts transcriptional co-activator function on autophagy-related and lysosomal genes through transcription factor EB (TFEB). Our findings demonstrate that CARM1-dependent histone arginine methylation is a crucial nuclear event in autophagy, and identify a new signalling axis of AMPK–SKP2–CARM1 in the regulation of autophagy induction after nutrient starvation.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Yang, Z. & Klionsky, D. J. Eaten alive: a history of macroautophagy. Nat. Cell Biol. 12, 814–822 (2010)
Mizushima, N., Levine, B., Cuervo, A. M. & Klionsky, D. J. Autophagy fights disease through cellular self-digestion. Nature 451, 1069–1075 (2008)
Rabinowitz, J. D. & White, E. Autophagy and metabolism. Science 330, 1344–1348 (2010)
Choi, A. M., Ryter, S. W. & Levine, B. Autophagy in human health and disease. N. Engl. J. Med. 368, 651–662 (2013)
Mizushima, N. Autophagy: process and function. Genes Dev. 21, 2861–2873 (2007)
Klionsky, D. J. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat. Rev. Mol. Cell Biol. 8, 931–937 (2007)
Mizushima, N. & Yoshimori, T. How to interpret LC3 immunoblotting. Autophagy 3, 542–545 (2007)
Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010)
Bjørkøy, G. et al. Monitoring autophagic degradation of p62/SQSTM1. Methods Enzymol. 452, 181–197 (2009)
Selvi, B. R. et al. Identification of a novel inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1)-mediated methylation of histone H3 Arg-17. J. Biol. Chem. 285, 7143–7152 (2010)
Carrano, A. C., Eytan, E., Hershko, A. & Pagano, M. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat. Cell Biol. 1, 193–199 (1999)
Hardie, D. G. AMPK and autophagy get connected. EMBO J. 30, 634–635 (2011)
Mihaylova, M. M. & Shaw, R. J. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat. Cell Biol. 13, 1016–1023 (2011)
Inoki, K., Kim, J. & Guan, K.-L. AMPK and mTOR in cellular energy homeostasis and drug targets. Annu. Rev. Pharmacol. Toxicol. 52, 381–400 (2012)
Salt, I. et al. AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the α2 isoform. Biochem. J. 334, 177–187 (1998)
Eijkelenboom, A. & Burgering, B. M. FOXOs: signalling integrators for homeostasis maintenance. Nat. Rev. Mol. Cell Biol. 14, 83–97 (2013)
Greer, E. L. et al. The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor. J. Biol. Chem. 282, 30107–30119 (2007)
Potente, M. et al. Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization. J. Clin. Invest. 115, 2382–2392 (2005)
Wang, K. & Li, P.-F. Foxo3a regulates apoptosis by negatively targeting miR-21. J. Biol. Chem. 285, 16958–16966 (2010)
Yang, Y.-C. et al. DNMT3B overexpression by deregulation of FOXO3a-mediated transcription repression and MDM2 overexpression in lung cancer. J. Thorac. Oncol. 9, 1305–1315 (2014)
Lam, E. W.-F., Brosens, J. J., Gomes, A. R. & Koo, C.-Y. Forkhead box proteins: tuning forks for transcriptional harmony. Nat. Rev. Cancer 13, 482–495 (2013)
Tsai, K.-L. et al. Crystal structure of the human FOXO3a-DBD/DNA complex suggests the effects of post-translational modification. Nucleic Acids Res. 35, 6984–6994 (2007)
Sardiello, M. et al. A gene network regulating lysosomal biogenesis and function. Science 325, 473–477 (2009)
Settembre, C. et al. TFEB links autophagy to lysosomal biogenesis. Science 332, 1429–1433 (2011)
Settembre, C. & Medina, D. L. TFEB and the CLEAR network. Methods Cell Biol. 126, 45–62 (2015)
Kim, I. S. et al. Roles of Mis18α in epigenetic regulation of centromeric chromatin and CENP-A loading. Mol. Cell 46, 260–273 (2012)
Kim, H. et al. DNA damage-induced RORα is crucial for p53 stabilization and increased apoptosis. Mol. Cell 44, 797–810 (2011)
Chen, Z., Zhou, Y., Song, J. & Zhang, Z. hCKSAAP_UbSite: improved prediction of human ubiquitination sites by exploiting amino acid pattern and properties. Biochim. Biophys. Acta 1834, 1461–1467 (2013)
Kim, J. et al. Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy. Cell 152, 290–303 (2013)
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)
Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010)
Boo, K. et al. Pontin functions as an essential coactivator for Oct4-dependent lincRNA expression in mouse embryonic stem cells. Nat. Commun. 6, 6810 (2015)
We thank members of the Chromatin Dynamics Research Center for technical assistance and discussions and J. Kim and J. Chung for valuable reagents and discussions. We thank Y. S. Yu for illustrations. The TEM data were analysed in the Korean Basic Science Institute. Carm1 knockout and knock-in MEFs were provided by M. T. Bedford. Ampk DKO MEFs was a gift from B. Viollet, and Foxo1.3.4f/f MEFs were a gift from R. DePinho and J.-H. Paik. This work was supported by Creative Research Initiatives Program (Research Center for Chromatin Dynamics, 2009-0081563) to S.H.B.; the National Junior Research Fellowship (NRF-2011-A01496-0001806) to H.-J.R.S.; the Basic Science Research Program (NRF-2014R1A6A3A0405 7910) to H.K. from the National Research Foundation (NRF) grant funded by the South Korean government (MSIP); NIH grant (R01DK106027) to K.-J.W.
The authors declare no competing financial interests.
Extended data figures and tables
a, b, Immunoblot analysis of various histone marks in response to amino acid (AA) starvation or rapamycin (100 nM). c, Immunoblot analysis of CARM1 and LC3 conversion (LC3-II). d, Amino acid-starved wild-type, Carm1 knockout or knock-in MEFs were analysed by immunoblot. e, Representative confocal images of GFP–LC3 puncta formation. GFP–LC3 (green); DAPI (blue). Scale bar, 20 μm. The graph shows quantification of LC3-positive punctate cells (right). Bars, mean ± s.e.m.; n = 5, with over 100 cells. **P < 0.01 (one-tailed t-test).
a, LC3 flux was analysed in MEFs infected with nonspecific shRNA (shNS) or CARM1 shRNAs (shCARM1-1 and -2). Bafilomycin A1 (BafA1; 200 nM, 2 h). The LC3-II/LC3-I ratio is indicated. b, LC3 flux was analysed in wild-type and Carm1 knockout MEFs in the absence or presence of Bafilomycin A1. The LC3-II/LC3-I ratio is indicated. c, mCherry-GFP–LC3 was transfected in wild-type and Carm1 knockout MEFs and the formation of autophagosome (mCherry-positive; GFP-positive) and autolysosome (mCherry-positive; GFP-negative) was examined. Scale bar, 20 μm. d, Immunoblot analysis in MEFs. e, Representative confocal images of GFP–LC3 puncta formation. Scale bar, 10 μm. Bars, mean ± s.e.m.; n = 5, over 150 cells. *P < 0.05 (one-tailed t-test). f, Immunoblot analysis in MEFs.
a, Wild-type CARM1 and ubiquitination-defective mutant K471R were analysed for their expression in MEFs after MG132 treatment. b, Interaction between CARM1 and CUL proteins was analysed. c, Lysates were analysed by immunoblot. d, Left, HepG2 cells infected with two different SKP2 shRNAs were subject to cycloheximide (CHX) experiment. Right, protein half-life of CARM1 was quantitatively defined (right). e, Left, CHX experiment in HepG2 expressing wild-type SKP2 or ΔF mutant. Right, protein half-life of CARM1 was quantitatively defined. Data are mean ± s.e.m.; n = 3. **P < 0.01 (one-tailed t-test) (d, e).
Extended Data Figure 4 CARM1 is degraded by CUL1-containing SCF E3 ligase in the nucleus under nutrient-rich condition.
a, HepG2 cells transfected with Flag–CUL1 were deprived of glucose for 18 h and treated with MG132 before collecting. Interaction between CARM1 and CUL1 was analysed. b, c, In vivo ubiquitination assay of CARM1 after knockdown of CUL1 (b) or overexpression of wild-type or K720R mutant (MT) CUL1 (c). d, e, Left, HepG2 cells infected with two different CUL1 shRNAs (d) or overexpressing wild-type or mutant CUL1 (e) were subject to cycloheximide treatment. Right, protein half-life of CARM1 was quantitatively defined. Data are mean ± s.e.m.; n = 3. *P < 0.05, **P < 0.01 (one-tailed t-test) (d, e).
Extended Data Figure 5 AMPKα2 accumulates in the nucleus leading to repression of SKP2 and stabilization of CARM1 under nutrient-starved conditions.
a, b, qRT–PCR of Ampka1 and Ampka2 in MEFs (a) and HepG2 cells (b) upon glucose starvation. c, The nuclear AMPKα2 expression level was analysed in the absence or presence of MG132. d, Binding between CARM1 and AMPK was assessed. e, In vitro kinase assay with AMPK. f, MEFs were treated with AICAR (1 mM) or phenformin (2 mM) for 4 h. The nuclear fraction was analysed by immunoblot. g, MEFs were deprived of glucose in the absence or presence of 10 μM compound C and the nuclear fraction was analysed by immunoblot. h, Left, cycloheximide treatment in wild-type and Ampk DKO MEFs. Right, protein half-life of CARM1 was quantitatively defined. i, j, Ampk DKO MEF lysates were analysed by immunoblot. k, CARM1–CUL1 interaction was analysed after SKP2 knockdown in wild-type and Ampk DKO MEFs. l, SKP2 expression levels were analysed in the absence or presence of MG132. m, Foxo1/3/4f/f MEFs infected with Cre virus were analysed for Skp2 mRNA. n, SKP2 and phosphorylated FOXO3a were analysed by immunoblot. o, ChIP assay of the Skp2 promoter. Data are mean ± s.e.m.; n = 3. *P < 0.05, **P < 0.01 (one-tailed t-test) (a, b, h, m, o). p, Representative confocal images. Scale bar, 20 μm.
a, Flow chart showing the strategy of RNA-seq analysis. b, Hierarchical clustering results applied to 4,998 differentially expressed genes (DEGs). c, Autophagy-related and lysosomal genes significantly observed in cluster 1. Hyper-geometric P values were calculated. d, Genes from cluster 1 were analysed for transcription factor (TF) motif enrichment at their promoter region (−500–100). Hypergeometric P values were calculated. e, qRT–PCR analysis of CARM1-dependent autophagy-related and lysosomal genes. Data are mean ± s.e.m.; n = 3. *P < 0.05, **P < 0.01 (one-tailed t-test). f, Enrichment of H3R17me2 at promoters (left) and enhancers (right). The data on H3R17me2, H3K4me1, H3K4me3 and H3K27ac were obtained from MEFs under normal condition. g, Increase in H3R17me2 at promoters of genes from cluster 1 after glucose starvation. h, Increased H3R17me2 levels in response to 18 h of glucose starvation at the autophagy-related gene Map1lc3b. The direction of transcription is indicated by the arrow and the beginning of the arrow indicates the TSS.
Extended Data Figure 7 Binding mapping of CARM1 and TFEB and their target gene regulation in glucose starvation.
a, Bimolecular fluorescence complementation (BiFC) analysis of the CARM1–TFEB interaction. Scale bar, 20 μm. b, Interaction between CARM1 and TFEB was analysed in wild-type and Ampk DKO MEFs after glucose starvation. c, d, In vitro GST pull-down assays for domain mapping of CARM1–TFEB interaction. BHLH, basic helix–loop–helix; LZ: leucine zipper. MD, methyltransferase domain; TA, transcription activation domain. e, Endogenous co-immunoprecipitation from nuclear fraction of wild-type MEFs. f, g, qRT–PCR analysis in MEFs after knockdown of TFEB or TFE3. h, i, qRT–PCR analysis showing mRNA levels of TFEB-dependent and CARM1-dependent genes after knockdown of TFEB (h) or CARM1 (i). Bars, mean ± s.e.m.; n = 3. *P < 0.05, **P < 0.01 (one-tailed t-test) (f–i).
a, ChIP assays on TFEB-dependent, CARM1-dependent promoters after knockdown of CARM1. b, ChIP assays of the Hspa5 promoter, a TFEB-dependent, CARM1-independent target promoter. c, MEFs were analysed with indicated antibodies. d, Two-step ChIP assays were performed on promoters of TFEB-dependent, CARM1-dependent target genes or TFEB-dependent, CARM1-independent target genes in MEFs after 18 h of glucose starvation. The chromatin fractions were first subject to pull-down with anti-TFEB antibody, eluted from immunocomplexes and applied for the second pull-down with control IgG or anti-CARM1 antibody. Bars, mean ± s.e.m.; n = 3 (a, b, d). e, Representative confocal images. Scale bar, 10 μm.
Extended Data Figure 9 A subset of autophagy-related and lysosomal genes regulated by TFEB requires CARM1.
a, qRT–PCR analysis showing mRNA levels of TFEB-dependent and CARM1-dependent autophagy-related and lysosomal genes in wild-type and Ampk DKO MEFs in response to glucose starvation. b, ChIP assays on TFEB-dependent, CARM1-dependent target genes in wild-type and Ampk DKO MEFs. c, qRT–PCR analysis of CARM1-dependent genes after knockdown of SKP2 in Ampk DKO MEFs. d, qRT–PCR analysis was performed in MEFs deprived of glucose in the absence or presence of H3R17me2-specific inhibitor, ellagic acid. e, f, ChIP assays on TFEB-dependent, CARM1-dependent promoters. Hspa5 promoter was also analysed as a CARM1-independent promoter. Bars, mean ± s.e.m.; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 (one-tailed t-test) (a–f).
Proposed model depicting the AMPK–SKP2–CARM1 signalling axis in the transcriptional and epigenetic regulation of autophagy. The SKP2-containing SCF E3 ubiquitin ligase complex degrades CARM1 under nutrient-rich conditions, but in nutrient-deprived conditions, AMPK-dependent phosphorylation of FOXO3a downregulates SKP2 and stabilizes CARM1, which in turn functions as a co-activator of TFEB in regulation of autophagy.
About this article
Cite this article
Shin, H., Kim, H., Oh, S. et al. AMPK–SKP2–CARM1 signalling cascade in transcriptional regulation of autophagy. Nature 534, 553–557 (2016). https://doi.org/10.1038/nature18014
Modulating the modulators: regulation of protein arginine methyltransferases by post-translational modifications
Drug Discovery Today (2020)
Applied and Environmental Microbiology (2020)
Cancer Cell (2020)
Cell Metabolism (2020)
CD74 knockout protects against LPS‐induced myocardial contractile dysfunction through AMPK‐Skp2‐SUV39H1 ‐mediated demethylation of BCLB
British Journal of Pharmacology (2020)