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. 2016 Jun 23;534(7608):553-7.
doi: 10.1038/nature18014. Epub 2016 Jun 15.

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

Hi-Jai R Shin  1 , Hyunkyung Kim  1 , Sungryong Oh  1 , Jun-Gi Lee  1 , Minjung Kee  1 , Hyun-Jeong Ko  2 , Mi-Na Kweon  3 , Kyoung-Jae Won  4 , Sung Hee Baek  1
Affiliations

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

Hi-Jai R Shin et al. Nature. .

Abstract

Autophagy is a highly conserved self-digestion process, which is essential for maintaining homeostasis and viability in response to nutrient starvation. Although the components of autophagy in the cytoplasm have been well studied, 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.

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Figures

Extended Data Figure 1
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
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
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
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
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
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
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).
Extended Data Figure 8
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
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).
Extended Data Figure 10
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.
Figure 1
Figure 1. Increased H3R17 dimethylation by CARM1 is critical for proper autophagy
a, b, Immunoblot analysis of various histone marks and CARM1 in response to glucose starvation (Glc starv.). c, Wild-type (WT), Carm1 knockout (KO) or knock-in (KI) MEFs were subject to immunoblot analysis. The LC3-II/LC3-I ratio is indicated. d, Representative confocal images of GFP–LC3 puncta formation. Graph shows quantification of LC3-positive punctate cells (right). Nuclei counterstained with DAPI. Scale bar, 10 µm. e, Representative TEM images. Scale bar, 2 µm. High magnification of boxed areas is shown on the right. Scale bar, 0.5 µm. Autophagosomes (blue arrows), autolysosomes (red arrows) and multilamellar body (yellow arrow). f, Representative confocal images of GFP–LC3 puncta formation. Ellagic acid (100 µM). Scale bar, 10 µm. Bars, mean ± s.e.m.; n = 5, with over 100 cells; **P < 0.01 (one-tailed t-test) (d, f).
Figure 2
Figure 2. CARM1 is degraded by the SKP2-containing SCF E3 ligase in the nucleus under nutrient-rich conditions
a, MEFs were deprived of glucose in the absence (left) or presence (right) of MG132 (5 µg ml−1) and subject to immunoblotting. b, In vivo ubiquitination assay of wild-type CARM1 or ubiquitination-defective K471R mutant CARM1. HA, haemagglutinin; HM, HisMax tag. c, Identification of CARM1-interacting proteins. F–CARM1 denotes Flag-tagged CARM1 construct. d, Interactions between SKP2 and protein arginine methyltransferases (PRMTs) were analysed. e, Glucose-starved cells were subjected to immunoblotting. f, g, In vivo ubiquitination assay of CARM1. h, Representative confocal images. Scale bar, 20 µm. i, Schematic of SKP2-containing SCF E3 ligase-dependent degradation of CARM1.
Figure 3
Figure 3. Decrease in SKP2 after glucose starvation is AMPK dependent
a, MEFs deprived of glucose were analysed with the indicated antibodies. b, Nuclear fractions from wild-type and Ampk double knockout (DKO) MEFs were subjected to immunoblotting. c, d, qRT–PCR of Skp2. DN, dominant negative. e, Left, schematic of Skp2 promoter. Right, luciferase activities of wild-type Skp2 or FOXO response element (FRE) mutant promoter were measured. MT, mutant; RE, response element; RLU, relative light units; TSS, transcription start site. f, ChIP assays of the Skp2 promoter. g, Skp2 mRNA levels were analysed in Foxo1/3/4 triple knockout (TKO) MEFs. HR, H212R mutant; SA, sextuple T179A/ S399A/ S413A/S555A/S588A/S626A mutant. h, Left, ChIP assays of the Skp2 promoter. Right, schematic of SKP2 regulation by the AMPK–FOXO axis (right). Bars, mean ± s.e.m.; n = 3. NS, not significant. **P < 0.01, ***P < 0.001 (one-tailed t-test) (c–h). i, Immunoblot analysis in Foxo1/3/4 TKO MEFs. j, Representative confocal images of GFP–LC3 puncta formation. shSKP2, short-hairpin RNA (shRNA) against SKP2; shNS, nonspecific shRNA. Scale bar, 20 µm. Bars, mean ± s.e.m.; n = 5, with over 80 cells; **P < 0.01, ***P < 0.001 (one-tailed t-test). k, Left, immunoblot analysis from whole-cell extracts. Right, schematic of the AMPK–SKP2–CARM1 signalling cascade in autophagy.
Figure 4
Figure 4. CARM1 exerts a transcriptional co-activator function on autophagy-related and lysosomal genes through TFEB
a, Binding between CARM1 and TFEB. b, Representative confocal images. Scale bar, 10 µm. c, 2× CLEAR (TFEB RE)-luciferase reporter assays. Bars, mean ± s.e.m.; n = 3. **P < 0.01 (one-tailed t-test). d–f, ChIP assays on TFEB-dependent, CARM1-dependent (d, e) or CARM1-independent (f) promoters after knockdown of TFEB. Bars, mean ± s.e.m.; n = 3. g, Wild-type and Carm1 knockout MEFs transfected with Flag–TFEB were subject to immunoblot analysis. h, Liver tissues from fed or fasted mice treated with vehicle or ellagic acid were subjected to immunoblot analysis (n = 3 per group). i, Expression of autophagy-related genes and lysosomal genes in wild-type mouse livers. Bars, mean ± s.e.m.; n = 3 per group. *P < 0.05, **P < 0.01 (two-tailed t-test).
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