Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jan;15(1):98-112.
doi: 10.1080/15548627.2018.1509608. Epub 2018 Sep 11.

Transcriptional and epigenetic profiling of nutrient-deprived cells to identify novel regulators of autophagy

J G C Peeters  1   2   3   4 , L W Picavet  2   3   4 , S G J M Coenen  2   3   4 , M Mauthe  5 , S J Vervoort  1 , E Mocholi  1   4 , C de Heus  1   6 , J Klumperman  1   6 , S J Vastert  2   3 , F Reggiori  5 , P J Coffer  1   3   4 , M Mokry  3   4   7 , J van Loosdregt  1   2   3   4
Affiliations

Transcriptional and epigenetic profiling of nutrient-deprived cells to identify novel regulators of autophagy

J G C Peeters et al. Autophagy. 2019 Jan.

Abstract

Macroautophagy (hereafter autophagy) is a lysosomal degradation pathway critical for maintaining cellular homeostasis and viability, and is predominantly regarded as a rapid and dynamic cytoplasmic process. To increase our understanding of the transcriptional and epigenetic events associated with autophagy, we performed extensive genome-wide transcriptomic and epigenomic profiling after nutrient deprivation in human autophagy-proficient and autophagy-deficient cells. We observed that nutrient deprivation leads to the transcriptional induction of numerous autophagy-associated genes. These transcriptional changes are reflected at the epigenetic level (H3K4me3, H3K27ac, and H3K56ac) and are independent of autophagic flux. As a proof of principle that this resource can be used to identify novel autophagy regulators, we followed up on one identified target: EGR1 (early growth response 1), which indeed appears to be a central transcriptional regulator of autophagy by affecting autophagy-associated gene expression and autophagic flux. Taken together, these data stress the relevance of transcriptional and epigenetic regulation of autophagy and can be used as a resource to identify (novel) factors involved in autophagy regulation.

Keywords: Autophagy; ChIP-seq; EGR1; RNA-seq; nutrient-deprivation.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Increased expression of autophagy-associated genes after nutrient deprivation. (a) Western Blot of HAP1 cells in control and starved (6 h EBSS) condition, with and without bafilomycin A1 (40 nM). Representative blot is shown (n = 4). (b) Representative EM images of HAP1 cells in control and starved (6 h EBSS) condition, treated with bafilomycin A1. Autolysosomal structures are indicated by arrows. (c) Representative images of HAP1 cells transfected with a plasmid encoding mCherry-EGFP-LC3B in control and starved (6 h EBSS) condition. mCherry+ EGFP+ dots (yellow) are autophagosomes and mCherry+ dots (red) are autolysosomes. (d) MA plot of HAP1 cells upon 6 h starvation with EBSS, displaying all expressed genes. Red dots indicate genes with a FDR < 0.05. (e) Heatmap of genes differentially expressed in HAP1 cells after 6 h starvation with EBSS. (f) Gene set enrichment analysis for autophagy-associated genes in HAP1 cells upon starvation (6 h EBSS). (g) Heatmap depicting expression of key autophagy proteins upon starvation (6 h EBSS) of HAP1 cells. See also Figure S1.
Figure 2.
Figure 2.
Increased expression of autophagy-associated genes upon nutrient deprivation in ATG7 KO and RB1CC1 KO cells. (a) Western Blot of WT, ATG7 KO, and RB1CC1 KO HAP1 cells in control and starved (3 h EBSS) condition, treated with bafilomycin A1 (40 nM). Representative blot is shown (n = 4). (b) Representative EM images of WT, ATG7 KO, and RB1CC1 KO HAP1 cells in starved (6 h EBSS) condition, treated with bafilomycin A1. Autolysosomes are indicated by arrows. (c) Representative images of HAP1 WT, ATG7 KO, and RB1CC1 KO cells transfected with a plasmid encoding mCherry-EGFP-LC3B in starved (6 h EBSS) condition. mCherry+ EGFP+ dots (yellow) are autophagosomes and mCherry+ dots (red) are autolysosomes. (d) Fold change of significantly differentially expressed genes in either WT, ATG7 KO and/or RB1CC1 KO HAP1 cells. Blue dots represent autophagy-associated genes. (e) Heatmap of WT, ATG7 KO, and RB1CC1 KO HAP1 cells upon starvation (6 h EBSS) displaying genes significantly different in one of the cell lines. (f) MA plot of genes differentially expressed between WT and ATG7 KO or RB1CC1 KO HAP1 cells upon starvation (6 h EBSS). Red dots indicate genes with a FDR < 0.05. (g) Gene set enrichment analysis of autophagy-associated genes in ATG7 KO and RB1CC1 KO HAP1 cells upon starvation (6 h EBSS). (h) Heatmap depicting expression of key autophagy proteins upon starvation (6 h EBSS) of ATG7 KO and RB1CC1 KO cells. See also Figure S2.
Figure 3.
Figure 3.
Increased transcription of autophagy-associated genes contributes to increased expression of autophagy-associated genes. Rank analysis of gene body POLR2 occupancy and RNA-sequencing signal in control (a) condition and after starvation (b). Genes are ranked according to RNA-sequencing data. (c) Boxplots with 5%-95% whiskers displaying log2 fold change of body POLR2 signal for genes unchanged, increased, and decreased ≥ log1 based on RNA-sequencing. (d) Gene set enrichment analysis of autophagy-associated genes for genes associated with an alteration of body POLR2 signal upon starvation (3 h EBSS). (e) Heatmap depicting body POLR2 signal for key autophagy genes upon starvation (3 h EBSS). (f) Gene track for MAP1LC3B displaying ChIP-seq signals for POLR2. See also Figure S3.
Figure 4.
Figure 4.
Increased transcription of autophagy-associated genes is reflected at the epigenetic level. (a) MA plots of H3K4me3, H3K27ac and H3K56ac signal upon starvation (3 h EBSS). Red dots indicate genes with a FDR < 0.05. (b) Boxplots with 5%-95% whiskers displaying log2 fold change in H3K4me3, H3K27ac or H3K56ac signal for genes unchanged, increased, and decreased ≥ log1 based on RNA-sequencing. (c) Gene set enrichment analysis of autophagy-associated genes for genes associated with an alteration of H3K4me3, H3K27ac or H3K56ac signal upon starvation (3 h EBSS). (d) Gene tracks for MAP1LC3B and ATG4D displaying ChIP-seq signals for H3K4me3, H3K27ac, and H3K56ac with and without starvation (3 h EBSS). See also Figure S4.
Figure 5.
Figure 5.
Epigenetic and transcriptomic analysis identifies EGR1 as a candidate transcriptional regulator of autophagy. (a) Top 10 transcription factor binding motifs enriched in autophagy-associated genes. Log2 FPKM (b) and fold change (c) after starvation (6 h EBSS) of transcription factors with binding motifs enriched in autophagy-associated genes. (d) Gene track for EGR1 displaying ChIP-seq signal for POLR2 with and without starvation (3 h EBSS). (e) EGR1 and TUBA4A/tubulin expression in HAP1 and U2OS cells upon starvation (6 h EBSS). Representative blots are shown (n = 3). (f) Gene track for MAP1LC3B displaying ChIP-seq signals for EGR1 (obtained from GEO GSE32465, samples GSM803414 and GSM803434), H3K4me3, H3K27ac, H3K56ac, DNAse I hypersensitivity sites, and EGR1 motif.
Figure 6.
Figure 6.
EGR1 acts as a transcriptional regulator of autophagy. (a) Expression of autophagy-associated genes in HAP1 cells with and without EGR1 knockdown starved for 6 h with EBSS. Fold change relative to cells transfected with scrambled siRNA is shown. Data are represented as mean ± SEM (n = 6–9). (b) Expression of autophagy-associated genes in HAP1 cells with and without EGR1 overexpression starved for 6 h with EBSS. Fold change relative to empty vector (EV)-transfected cells is shown. Data are represented as mean ± SEM (n = 3–6). (c) Expression of autophagy-associated genes in HAP1 EGR1 KO cells starved for 6 h with EBSS. Fold change relative to WT HAP1 starved for 6 h is shown. Data are represented as mean ± SEM (n = 6). (d) LC3 and TUBA4A expression in WT and EGR1 KO HAP1 cells in control and starved (3 h and 6 h EBSS) condition, treated with bafilomycin A1 (40 nM). Representative blot is shown (3 h: n = 4; 6 h: n = 6). (e) EGR1, LC3, and TUBA4A expression in WT HAP1 cells with and without EGR1 overexpression in control and starved (3 h and 6 h EBSS) condition, treated with bafilomycin A1 (40 nM). Representative blot is shown (3 h: n = 3; 6 h: n = 3). (f) LC3, EGR1, and TUBA4A expression in HEK293 cells with and without EGR1 overexpression in control and starved (3 h and 6 h EBSS) condition, treated with bafilomycin A1 (40 nM). Representative blot is shown (3 h: n = 3; 6 h: n = 6). (g) Representative images of HAP1 WT and EGR1 KO cells transfected with a plasmid encoding mCherry-EGFP-LC3B in starved (6 h EBSS) condition. mCherry+ EGFP+ dots (yellow) are autophagosomes and mCherry+ dots (red) are autolysosomes. (h) Boxplots with 5%-95% whiskers displaying the ratio red vs. yellow dots per cell and the total amount of dots per cell (75 cells were counted within 2 independent experiments). * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Klionsky DJ, Emr SD.. Autophagy as a regulated pathway of cellular degradation. Science. 2000;290:1717–1721. - PMC - PubMed
    1. Yang Z, Klionsky DJ. Eaten alive: a history of macroautophagy. Nat Cell Biol. 2010;12:814–822. - PMC - PubMed
    1. Mizushima N. Autophagy in Protein and Organelle Turnover. Cold Spring Harb Symp Quant Biol. 2011;76:397–402. - PubMed
    1. Feng Y, Yao Z, Klionsky DJ. How to control self-digestion: transcriptional, post-transcriptional, and post-translational regulation of autophagy. Trends Cell Biol. 2015;25:354–363. - PMC - PubMed
    1. Tasdemir E, Maiuri MC, Galluzzi L, et al. Regulation of autophagy by cytoplasmic p53. Nat Cell Biol. 2008;10:676–687. - PMC - PubMed

Publication types

Substances

Grants and funding

This work was supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek [022.004.018];Nederlandse Organisatie voor Wetenschappelijk Onderzoek [91615113];Wilhelmina Children’s Hospital;Marie Skłodowska-Curie Cofund [713660];Marie Skłodowska-Curie ITN [765912];Reumafonds [14-3-201];ZonMw [016.130.606].