Epigenetic regulation of autophagy by the methyltransferase G9a

Abstract

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the cytoplasmic machinery that orchestrates autophagy induction during starvation, hypoxia, or receptor stimulation has been widely studied, the key epigenetic events that initiate and maintain the autophagy process remain unknown. Here we show that the methyltransferase G9a coordinates the transcriptional activation of key regulators of autophagosome formation by remodeling the chromatin landscape. Pharmacological inhibition or RNA interference (RNAi)-mediated suppression of G9a induces LC3B expression and lipidation that is dependent on RNA synthesis, protein translation, and the methyltransferase activity of G9a. Under normal conditions, G9a associates with the LC3B, WIPI1, and DOR gene promoters, epigenetically repressing them. However, G9a and G9a-repressive histone marks are removed during starvation and receptor-stimulated activation of naive T cells, two physiological inducers of macroautophagy. Moreover, we show that the c-Jun N-terminal kinase (JNK) pathway is involved in the regulation of autophagy gene expression during naive-T-cell activation. Together, these findings reveal that G9a directly represses genes known to participate in the autophagic process and that inhibition of G9a-mediated epigenetic repression represents an important regulatory mechanism during autophagy.

Fig 1

Inhibition or depletion of G9a promotes the formation of vacuole-like structures and increases LC3B-II. (A) Bright-field images of SU86.86 cells depleted of G9a expression show the distinct vacuolar phenotype of enlarged autophagosomes. (B and C) Immunoblot analysis for LC3B expression and lipidation in SU86.86 cells treated with 3 mM YT-2-6 for the indicated times (B) or with the indicated concentrations of YT-2-6 or UNC0638 for 12 h (C). (D) Bright-field analysis of SU86.86 cells treated with diluent, 3 μM YT-2-6, 5 μM UNC0638, and/or 2 μg/ml actinomycin D (ACT-D) for 36 h. (E) Immunoblot analysis for LC3B in MEFs treated with diluent, 3 μM YT-2-6, 20 μg/ml cycloheximide, 2 μg/ml actinomycin D, or the indicated combinations for 24 h. (F) Immunoblot analysis for LC3B in HeLa cells transiently depleted of G9a expression along with reexpression of the wt HA-YFP-tagged G9a or G9a ΔSet domain.

Fig 2

G9a inhibition results in formation of autophagosomes but not autophagic flux. (A) Panc04.03 cells were treated over time with 3 μM YT-2-6, 5 μM UNC0638, or 250 nM torin for 12 h, and lysates were immunoblotted with the indicated antibodies. (B) Immunoblot analysis for LC3B lipidation in ATG5^−/− mouse fibroblasts treated with diluent or 3 μM YT-2-6 for 12 h. (C) Pancreatic SU86.86 cells were analyzed by immunofluorescence for LAMP1 (red) along with p62 (green) after treatment with 3 μM YT-2-6 for 12 h. The nucleus was visualized by Hoechst staining (blue). (D) Immunoblot analysis of SU86.86 cells treated for 12 h with a 3 μM concentration of the G9a inhibitor YT-2-6, the TORC inhibitors torin (250 nM) and rapamycin (100 nM), or combinations as indicated shows that although p62 is upregulated upon G9a inhibition, it does not get degraded within autolysosomes unless G9a inhibition is combined with TORC inhibition. (E) Analysis of dually tagged GFP-mCherry-p62 to study autophagic flux in HeLa cells upon treatment with 3 μM YT-2-6 and/or 250 nM torin for 12 h. (F) Colocalization coefficients were determined from the results in panel E for mCherry overlap with GFP (lower colocalization indicates mCherry fluorescence independent of GFP and exposure of the tagged p62 to an acidic compartment).

Fig 3

G9a inhibition leads to increased expression of genes involved in autophagosome formation. (A) Quantitative RT-PCR analysis of identified target genes in Panc04.03 cells transiently depleted of G9a expression. (B to D) Quantitative RT-PCR analysis in SU86.86 cells treated with diluent or 3 μM or 5 μM YT-2-6 for 24 h (B), 3 μM YT-2-6 for the indicated times (C), or 3 μM YT-2-6 or 5 μM UNC0638 for 24 h (D). Results were normalized to RPLP0 and are displayed as x-fold over the value for controls (mean plus standard deviation [SD]).

Fig 4

Inhibition or genomic depletion of G9a alters G9a promoter binding and chromatin modifications of genes involved in autophagosome formation. (A) Cartoon of the LC3B promoter showing the localization of the primers (P1 to P3) used for panels B to D, relative to the transcription start (arrow). (B) Quantitative PCR of ChIP analysis shows G9a association with the LC3B, WIPI1, and DOR promoters relative to input and displayed as fold over IgG (mean plus SD) in untreated pancreatic SU86.86 cells. (C and D) Quantitative analysis following ChIP in SU86.86 cells treated with 3 μM YT-2-6 for 24 h or stably depleted of G9a shows H3K9me2 and binding of RNA polymerase II to the LC3B, WIPI1, and DOR promoter relative to input and displayed as fold over the value for control cells (mean plus SD). (E to G) Quantitative ChIP PCR analysis shows H3K9me2 (E), H3K9ac (F), and RNA polymerase II (G) binding to the LC3B promoter. Results are relative to input and displayed as fold over the value for control cells (mean plus SD). Primer P1 (for H3K9me2 and RNA polymerase II [Pol II]) and primer P2 (for H3K9ac) was used to analyze the promoter (see the cartoon in Fig. 2A). (H) Quantitative RT-PCR for LC3B and WIPI1 expression in HeLa cells transfected with the indicated suppression and reexpression constructs for G9a. Results are normalized to RPLP0 and displayed as fold over the value for controls (mean plus SD).

Fig 5

Nutrient starvation, a physiologic stressor which triggers autophagy induction, leads to G9a-dependent, epigenetic changes at autophagy-related target gene promoters. (A) Immunoblot analysis of Panc04.03 cells upon glucose starvation for 18 h shows expression and lipidation of LC3B. (B) Quantitative RT-PCR analysis of selected genes in Panc04.03 cells upon glucose starvation for 18 h. Results were normalized to RPLP0 and are displayed as x-fold over the value for controls (mean plus SD). (C to E) Quantitative PCR of ChIP analysis in glucose-starved Panc04.03 cells shows association of G9a (C), H3K9me2 (D), and RNA polymerase II binding (E) to the LC3B, WIPI1, and DOR promoters. Results are relative to input and displayed as fold over the value for control cells (mean plus SD). Primer P1 (see the cartoon in Fig. 4A) was used in ChIP for analyzing all three promoters.

Fig 6

Nutrient deprivation leads to G9a-specific loss from autophagy gene promoters. (A) Quantitative ChIP PCR analysis for G9a association, H3K9me2, and RNA Pol II association with the WIPI1 promoter upon treatment with indicated concentrations of the G9a inhibitors YT-2-6 or UNC0638 for 24 h using primer P1. (B) Quantitative RT-PCR shows mRNA expression of LC3B, WIPI1, and MAGE A1 upon treatment with 3 μM YT-2-6 for the indicated times. Results were normalized to RPLP0 and are relative to diluent-treated cells (mean ± SD). (C) Quantitative RT-PCR shows mRNA expression of LC3B, WIPI1, and MAGE A1 upon glucose starvation for the indicated times. (D) Quantitative ChIP PCR analysis for G9a association, H3K9me2, and RNA Pol II association with the WIPI1 and MAGE A1 promoters upon treatment with 3 μM YT-2-6 for the indicated times using primer P1. (E) Quantitative ChIP PCR analysis for G9a association, H3K9me2, and RNA Pol II association with the WIPI1 and MAGE A1 promoters upon glucose starvation for indicated times using primer P1. Results in panels A, D, and E were normalized to input and are relative to diluent-treated cells (mean ± SD).

Fig 7

Naive-T-cell activation leads to G9a-dependent epigenetic changes at autophagy-related target gene promoters. (A and B) Immunoblot analysis shows expression and lipidation of LC3B in naive human CD4 T cells treated with increasing amounts of YT-2-6 for 24 h (A) or stimulated with anti-CD3/CD28 for 24 h and 48 h or treated with 3 μM YT-2-6 for 24 h (B). (C) Quantitative RT-PCR analysis of selected genes in naive human CD4 T cells stimulated with anti-CD3/CD28 for 24 h and 48 h or treated with 3 μM YT-2-6 for 36 h. (D) Quantitative PCR of ChIP analysis shows G9a association in naive human T cells with the LC3B, WIPI1, or DOR promoter relative to input and displayed as x-fold over the value for IgG (mean plus SD). (E to H) Quantitative PCR of ChIP analysis in naive human T cells stimulated with anti-CD3/CD28 for 24 h and 48 h or treated with 3 μM YT-2-6 for 36 h shows association of G9a (E), H3K9me2 (F), H3K9ac (G), and RNA polymerase II binding (H) to the LC3B, WIPI1, and DOR promoters. Results are relative to input and displayed as x-fold over the value for control cells (mean plus SD). Primer P1 for G9a, H3K9me2, and RNA Pol II and primer P2 for H3K9ac (see the cartoon in Fig. 4A) were used. (I) Immunoblot showing the levels of G9a after activation of naive T cells.

Fig 8

JNK activity is required for autophagy gene expression following the stimulation of naive CD4⁺ T cells. (A) Quantitative RT-PCR for LC3B and p62 expression in naive human CD4⁺ T cells stimulated with anti-CD3/CD28 for 36 h and simultaneously treated with diluent (DMSO), PP2 (2 μM), SP600125 (25 μM), or YT-2-6 (3 μM). Results are displayed as relative to DMSO control and normalized to RPLP0 (mean plus SD). (B) Naive CD4⁺ T cells were stimulated with anti-CD3/CD28 for 48 h and simultaneously treated with diluent (DMSO), PP2 (2 μM), SP600125 (25 μM), or YT-2-6 (3 μM). Cellular proteins were isolated and immunoblotted for the expression of LC3B and p62. (C) Quantitative PCR analysis for G9a and H3K9me2 ChIP at the LC3B and p62 promoters. Naive CD4⁺ T cells were stimulated with anti-CD3/CD28 for 36 h and simultaneously treated with diluent (DMSO), PP2 (2 μM), or SP600125 (25 μM). (D) Quantitative PCR analysis for c-Jun ChIP at the LC3B and p62 promoters. Naive CD4⁺ T cells were stimulated with anti-CD3/CD28 for 36 h and simultaneously treated with diluent (DMSO), PP2 (2 μM), or SP600125 (25 μM). Results in panels C and D were normalized to input and are relative to unstimulated control cells (mean plus SD). (E) Under basal conditions, G9a epigenetically represses the expression of LC3B, DOR, and WIPI1 through H3K9me2. However, following physiologic stimuli that induce macroautophagy (glucose starvation or T cell activation), G9a is removed from these gene promoters and silencing marks are lost from the indicated macroautophagy-related target genes. Acetylases, transcription factors, and RNA Pol II can then access these target gene promoters, resulting in increased transcription of these genes and sustained autophagosome formation.