A gene toolbox for monitoring autophagy transcription

Abstract

Autophagy is a highly dynamic and multi-step process, regulated by many functional protein units. Here, we have built up a comprehensive and up-to-date annotated gene list for the autophagy pathway, by combining previously published gene lists and the most recent publications in the field. We identified 604 genes and created main categories: MTOR and upstream pathways, autophagy core, autophagy transcription factors, mitophagy, docking and fusion, lysosome and lysosome-related genes. We then classified such genes in sub-groups, based on their functions or on their sub-cellular localization. Moreover, we have curated two shorter sub-lists to predict the extent of autophagy activation and/or lysosomal biogenesis; we next validated the "induction list" by Real-time PCR in cell lines during fasting or MTOR inhibition, identifying ATG14, ATG7, NBR1, ULK1, ULK2, and WDR45, as minimal transcriptional targets. We also demonstrated that our list of autophagy genes can be particularly useful during an effective RNA-sequencing analysis. Thus, we propose our lists as a useful toolbox for performing an informative and functionally-prognostic gene scan of autophagy steps.

Fig. 1. Graphic representation of the 604 genes that compose the entire autophagic list.

Genes were classified in 6 main groups: mTOR and upstream pathways (135 genes), autophagy core (197 genes), autophagy regulators (68 genes), mitophagy (80 genes), docking and fusion (22 genes), lysosome (162 genes) and lysosome-related genes (34 genes). For each group, also subgroups are reported.

Fig. 2. The “induction list” and the “lysosomal biogenesis list”.

The two shorter lists were generated by identifying the gene targets related to autophagy activation (Induction list) and lysosomal biogenesis.

Fig. 3. Validation of the “induction list” by Real-time PCR in cell lines during fasting or MTOR inhibition.

HEK-293 (A) and SH-SY5Y (B) cells were cultured in EBSS starvation medium for 4 h or treated with AZD8055 (100 nM) for 8 h. ATG10, ATG14, ATG16L1, ATG3, ATG4, ATG7, ATG9A, BCL2, GABARAP, GABARAPL1, MAP1LC3B, NBR1, OPTN, PINK1, SQSTM1, ULK1, ULK2, UVRAG, WDR45, and WIPI1 mRNA expression were assessed by qPCR and were normalized to HPRT1 mRNA levels, used as internal control. Data display the fold-changes relative to control cells (n = 3-4-5-6, based on the variation of each gene expression) and are expressed as the mean value ± SEM. C Summary of those genes that were significantly upregulated in both cell lines upon starvation (STV), upon AZD8055 treatment (AZD) or upon both conditions (both). D List of genes for the evaluation of lysosomal biogenesis. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig. 4. Experimental validation of the “autophagy core” list.

GSEA analysis obtained by using the “autophagy core” list, cleaned of “Negative Regulator of Autophagy” and “Its mutation leads to Autophagy defects” sub-categories, in starved HAP1 cells (A) or in DS fibroblasts compared to control fibroblast (B).