Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

AMPK–SKP2–CARM1 signalling cascade in transcriptional regulation of autophagy

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Increased H3R17 dimethylation by CARM1 is critical for proper autophagy.
Figure 2: CARM1 is degraded by the SKP2-containing SCF E3 ligase in the nucleus under nutrient-rich conditions.
Figure 3: Decrease in SKP2 after glucose starvation is AMPK dependent.
Figure 4: CARM1 exerts a transcriptional co-activator function on autophagy-related and lysosomal genes through TFEB.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The RNA-seq and H3R17me2 ChIP–seq data sets have been deposited in the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE72901.

References

  1. Yang, Z. & Klionsky, D. J. Eaten alive: a history of macroautophagy. Nat. Cell Biol. 12, 814–822 (2010)

    Article  CAS  Google Scholar 

  2. Mizushima, N., Levine, B., Cuervo, A. M. & Klionsky, D. J. Autophagy fights disease through cellular self-digestion. Nature 451, 1069–1075 (2008)

    Article  ADS  CAS  Google Scholar 

  3. Rabinowitz, J. D. & White, E. Autophagy and metabolism. Science 330, 1344–1348 (2010)

    Article  ADS  CAS  Google Scholar 

  4. Choi, A. M., Ryter, S. W. & Levine, B. Autophagy in human health and disease. N. Engl. J. Med. 368, 651–662 (2013)

    Article  CAS  Google Scholar 

  5. Mizushima, N. Autophagy: process and function. Genes Dev. 21, 2861–2873 (2007)

    Article  CAS  Google Scholar 

  6. Klionsky, D. J. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat. Rev. Mol. Cell Biol. 8, 931–937 (2007)

    Article  CAS  Google Scholar 

  7. Mizushima, N. & Yoshimori, T. How to interpret LC3 immunoblotting. Autophagy 3, 542–545 (2007)

    Article  CAS  Google Scholar 

  8. Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010)

    Article  CAS  Google Scholar 

  9. Bjørkøy, G. et al. Monitoring autophagic degradation of p62/SQSTM1. Methods Enzymol. 452, 181–197 (2009)

    Article  Google Scholar 

  10. 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)

    Article  CAS  Google Scholar 

  11. 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)

    Article  CAS  Google Scholar 

  12. Hardie, D. G. AMPK and autophagy get connected. EMBO J. 30, 634–635 (2011)

    Article  CAS  Google Scholar 

  13. Mihaylova, M. M. & Shaw, R. J. The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat. Cell Biol. 13, 1016–1023 (2011)

    Article  CAS  Google Scholar 

  14. 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)

    Article  CAS  Google Scholar 

  15. 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)

    Article  CAS  Google Scholar 

  16. Eijkelenboom, A. & Burgering, B. M. FOXOs: signalling integrators for homeostasis maintenance. Nat. Rev. Mol. Cell Biol. 14, 83–97 (2013)

    Article  CAS  Google Scholar 

  17. 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)

    Article  CAS  Google Scholar 

  18. Potente, M. et al. Involvement of Foxo transcription factors in angiogenesis and postnatal neovascularization. J. Clin. Invest. 115, 2382–2392 (2005)

    Article  CAS  Google Scholar 

  19. Wang, K. & Li, P.-F. Foxo3a regulates apoptosis by negatively targeting miR-21. J. Biol. Chem. 285, 16958–16966 (2010)

    Article  CAS  Google Scholar 

  20. 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)

    Article  CAS  Google Scholar 

  21. 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)

    Article  CAS  Google Scholar 

  22. 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)

    Article  CAS  Google Scholar 

  23. Sardiello, M. et al. A gene network regulating lysosomal biogenesis and function. Science 325, 473–477 (2009)

    Article  ADS  CAS  Google Scholar 

  24. Settembre, C. et al. TFEB links autophagy to lysosomal biogenesis. Science 332, 1429–1433 (2011)

    Article  ADS  CAS  Google Scholar 

  25. Settembre, C. & Medina, D. L. TFEB and the CLEAR network. Methods Cell Biol. 126, 45–62 (2015)

    Article  CAS  Google Scholar 

  26. Kim, I. S. et al. Roles of Mis18α in epigenetic regulation of centromeric chromatin and CENP-A loading. Mol. Cell 46, 260–273 (2012)

    Article  CAS  Google Scholar 

  27. Kim, H. et al. DNA damage-induced RORα is crucial for p53 stabilization and increased apoptosis. Mol. Cell 44, 797–810 (2011)

    Article  CAS  Google Scholar 

  28. 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)

    Article  CAS  Google Scholar 

  29. Kim, J. et al. Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy. Cell 152, 290–303 (2013)

    Article  CAS  Google Scholar 

  30. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

    Article  Google Scholar 

  31. 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)

    Article  CAS  Google Scholar 

  32. 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)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

H.-J.R.S., H.K., S.O., J.-G.L. and M.K. performed the cell biology and biochemistry experiments; H.-J.K. and M.-N.K. provided TEM analysis and critical comments; H.-J.R.S. and K.J.W. performed RNA and ChIP–seq preparation and systemic analysis; H.-J.R.S., H.K., K.J.W. and S.H.B. organized and analysed the data; H.-J.R.S., K.J.W. and S.H.B. wrote the manuscript.

Corresponding author

Correspondence to Sung Hee Baek.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Increased H3R17me2 by CARM1 in amino acid starvation-induced autophagy.

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).

Extended Data Figure 2 Loss of CARM1 and inhibition of H3R17me2 impair autophagy.

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.

Extended Data Figure 3 CARM1 is degraded by SKP2-containing SCF E3 ligase in the nucleus.

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.

Extended Data Figure 6 Identification of CARM1 target genes by RNA-seq and ChIP–seq analyses.

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) (fi).

Extended Data Figure 8 CARM1 functions as a co-activator of TFEB.

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) (af).

Extended Data Figure 10 Graphical summary of the AMPK–SKP2–CARM1 signalling cascade.

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.

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1 showing the original immunoblot images and Supplementary Tables 1-3. (PDF 2461 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shin, HJ., 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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature18014

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing