The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy

Abstract

Autophagy is an evolutionarily conserved catabolic process involved in several physiological and pathological processes. Although primarily cytoprotective, autophagy can also contribute to cell death; it is thus important to understand what distinguishes the life or death decision in autophagic cells. Here we report that induction of autophagy is coupled to reduction of histone H4 lysine 16 acetylation (H4K16ac) through downregulation of the histone acetyltransferase hMOF (also called KAT8 or MYST1), and demonstrate that this histone modification regulates the outcome of autophagy. At a genome-wide level, we find that H4K16 deacetylation is associated predominantly with the downregulation of autophagy-related genes. Antagonizing H4K16ac downregulation upon autophagy induction results in the promotion of cell death. Our findings establish that alteration in a specific histone post-translational modification during autophagy affects the transcriptional regulation of autophagy-related genes and initiates a regulatory feedback loop, which serves as a key determinant of survival versus death responses upon autophagy induction.

Figure 1. Autophagy is associated with reduced acetylation of histone H4 lysine 16

(a) Starvation (3 h)-induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells. (b) Upon rapamycin treatment (300 nM) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy-deficient Atg5^−/− and Atg7^−/− MEF cells. (c) Rapamycin treatment increased the LC3-II/LC3-I ratio and promoted H4K16ac decrease in Sirt1^−/− and WT MEF cells. (d) Rapamycin-induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells, and after 6 h in U1810 cells. (e) Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin-treated cells. Data are expressed as mean ± SEM (n=3–5); *Pvalue<0.05; **Pvalue<0.01.

Figure 2. Deacetylation of H4K16 by rapamycin treatment is associated with transcriptional regulation of autophagy-related genes

(a) Heat map of H4K16Ac ChIP-Seq performed in U1810 cells without or with 8 h rapamycin treatment. Data are shown as log2 values of tag counts in the 3422 regions defined as peaks in the control sample. (b) De novo detection of transcripts using GRO-Seq analysis was performed in 8 h rapamycin-treated U1810 cells and compared to untreated U1810 cells. GRO-seq data visualized as ‘MA’ plots (log ratio versus abundance). The plot shows GRO-Seq gene expression for pair-wise comparison between rapamycin-treated vs control cells. The red points denote the differentially expressed genes. (c) Autophagy-related genes identified as regulated by rapamycin in the GRO-Seq data analysis and in the ChIP-Seq data analysis.

Figure 3. Rapamycin-induced hMOF downregulation promotes deacetylation of H4K16

Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a) and transfected HeLa cells (b). VPA (1mM) treatment counteracted rapamycin-induced H4K16ac downregulation (c) and decreased the LC3 ratio (d). (e) Cotreatment with chloroquine (CQ, 10 μM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux. (f) Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux. (g) The yeast homolog of hMOF, Sas2, tagged with 3xHA showed a complete disappearance of HA signal upon autophagy induction after 3 h. (h) Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment.

Figure 4. Inhibition of H4K16ac downregulation upon autophagy induction results in cell death

(a) VPA increases the autophagic flux in rapamycin-treated HeLa cells transfected with the mRFP-GFP-LC3 tandem reporter construct which allows distinction between autophagosomes (GFP+/RFP+ yellow puncta) and autolysosomes (GFP−/RFP+ red puncta). (b) Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux. (c) Co-treatment with VPA and rapamycin led to increased cell death. (d) Co-treatment with CQ abrogated VPA+rapamycin-induced cell death. (e–f) Increasing H4K16ac levels by either overexpression of hMOF, inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment. Co-treatment with the autophagy inhibitor Bafilomycin A (BafA, 40nM) (g) or 3-methyladenine (3MA, 5 mM) (h) abrogated VPA+rapamycin-induced cell death. Treatment of HeLa cells upon amino-acid starvation with VPA induced cell death (i), which was rescued when cells were co-treated with CQ (j). Data are expressed as mean ± SEM (n=3).