Transcriptional regulation of autophagy by RNA polymerase II

ABSTRACT

Macroautophagy/autophagy is a catabolic recycling pathway and is tightly regulated by upstream signals. Autophagy genes are quickly upregulated upon stimuli such as nutrition limitation in response to the external environment. However, how the transcriptional activation of autophagy genes occurs is not well understood. We recently found that in yeast, the RNA polymerase II subunit Rpb9 specifically and efficiently upregulates the transcription of the autophagy gene ATG1 with the mediation of Gcn4. Such regulation was shown to be essential for autophagic activities induced by starvation. Furthermore, the function of Rpb9 in autophagy and the activation of ATG1 transcription is conserved in mammalian cells. In conclusion, Rpb9 specifically and positively regulates ATG1 transcription as a key regulator of autophagy.

KEYWORDS:

• ATG1 gene
• autophagy
• Gcn4
• Rpb9
• starvation
• transcription
• yeast

Macroautophagy/autophagy is an evolutionarily conserved process in eukaryotes. Autophagy involves processes such as initiation, expansion, completion, and fusion. Intracellular materials, including damaged organelles and misfolded proteins, are transported into vacuoles (in yeast and plants) or lysosomes (in mammals) for degradation. The degraded substances are recycled to sustain cell life. As a dynamic and multistep process, autophagy is subject to multiple regulatory mechanisms, including transcriptional regulation.

The importance of transcriptional regulation in autophagy has been highlighted in recent years, mainly in characterizing the release of transcriptional suppression of autophagy genes (by Dr. Daniel Klionsky’s laboratory). For example, the autophagy gene ATG8 is negatively regulated by the transcriptional repressor Ume6, whereas ATG9 is inhibited by the transcriptional repressor Pho23-Rpd3 complex. The histone demethylase Rph1 functions in the transcriptional repression of several ATG genes (especially ATG7). Upon autophagy induction, such repression is released by the upstream kinase Rim15. Compared to these mechanisms of transcriptional repression, the transcriptional activation of autophagy ATG genes is less known.

In our recent study, we demonstrated the importance and mechanism of transcriptional activation of the essential autophagy gene ATG1 [1]. Autophagy induced by rapamycin treatment or nitrogen starvation is blocked by cycloheximide, an inhibitor of protein synthesis. Treatment with thiolutin, an RNA polymerase inhibitor, can also cause autophagy blockage. Then, mRNA sequencing assays demonstrated that the transcriptional level of ATG1 is significantly upregulated during nutrient deprivation, and the protein levels of Atg1 are also dramatically induced.

To analyze the transcription of autophagy genes (especially ATG1), we focused on transcriptional activators. We identified RNA polymerase 2 subunit Rpb9 as an essential autophagic regulator from a high-throughput screen using a yeast gene knockout library. Therefore, we speculated that Rpb9 could transcriptionally upregulate autophagy genes. Using autophagy substrates such as GFP-50Q and GFP-Atg8, we found that Rpb9 deficiency blocks autophagy. In addition, the fluorescence analysis of Cherry-Atg8 demonstrated that the deletion of RPB9 causes diffuse cytoplasmic distribution of Atg8 rather than punctate localization, which indicates that Rpb9 participates in regulating the formation of autophagosomes. The transcriptomic analysis of wild-type (WT) and Rpb9-deficient yeast cells demonstrated that Rpb9 positively regulates the mRNA levels of ATG1. Through the overexpression of multiple Atg proteins in Rpb9-deficient cells, we confirmed that Rpb9 deficiency blocks autophagy by downregulating ATG1 transcription.

Rpb9 contains two zinc finger domains with a linker between zinc finger 1 (Zn1) and zinc finger 2 (Zn2). In Rpb9-deficient cells, the Zn1 and linker domains (Rpb9 1–52 aa) together are sufficient to restore autophagy, whereas the absence of Zn2 has no effect on the autophagic function of Rpb9. Overexpression of Zn1 and the linker domain (Rpb9 1–52 aa) can restore starvation-induced transcription of ATG1, suggesting that the transcriptional regulation of ATG1 by Rpb9 is dependent on its Zn1 and linker domains.

Through chromosomal immunoprecipitation/ChIP experiments and an electrophoretic mobility shift assay/EMSA, we found that although Rpb9 activates the transcription of ATG1, there is no direct interaction between Rpb9 and the promoter regions of ATG1. In previous studies, Gcn4 has been found to bind to the promoter regions of ATG1. Our results confirmed that Gcn4 and Rpb9 bind to the same regions of the ATG1 promoter and that Rpb9 can efficiently bind to the promoter of ATG1 in the presence of Gcn4. A direct interaction between Gcn4 and Rpb9 is detected. Furthermore, the overexpression of Gcn4 partially rescues the autophagy blockade caused by rpb9 deletion, whereas Rpb9 cannot restore the autophagy blockade caused by gcn4 deletion. According to these results, we speculated that Gcn4 binds to the ATG1 promoter and interacts with Rpb9, which recruits the RNA polymerase II complex to the ATG1 promoter. In the absence of Rpb9, Gcn4 may weakly bind to RNA polymerase II directly or through other factors, thereby recruiting RNA polymerase II to the ATG1 promoter.

Through domain and sequence similarity analyses of Rpb9 and its homologs in other eukaryotes, we speculated that Rpb9, like most autophagy factors, is evolutionarily conserved. When expressed in yeast cells, Rpb9 homologs restore the degradation of autophagic substrates, cell survival upon starvation and ATG1 transcription in Rpb9-deficient yeast cells. In addition, the Zn1 and linker domains in the Rpb9 homologs are also confirmed to support autophagy. Knockout of POLR2I, the human homolog of yeast Rpb9, specifically reduces the mRNA levels of ULK1, the homologous gene of yeast ATG1. Reduced degradation of the autophagy receptor SQSTM1/p62 and reduced autophagosome formation demonstrate decreased autophagic activities upon POLR2I deficiency, suggesting the conserved function of POLR2I in autophagy in mammalian cells. In conclusion, the transcriptional regulation of ATG1 and the autophagy function of Rpb9 are highly conserved in eukaryotes.

Based on this evidence, we confirmed that the transcriptional regulatory effect of Rpb9 on ATG1 is specific. As the core factor at the upstream initial stage of the autophagy pathway, Atg1 guards the normal progress of the pathway, indicating that its expression induction is crucial to autophagy and the circulation of substances in cells upon nutrient limitation. Many studies have uncovered the relationships between autophagy dysfunction and neurodegenerative diseases, immune disorders, and cancer. However, scientific research and clinical treatments have not been well combined, and the search for specific targets in the autophagy pathway is an urgent problem to be solved. Considering the essential functions of Atg1 at the very early stage of the autophagy process, the specific regulation of ATG1 by Rpb9 not only reveals the importance of transcriptional regulation of autophagy genes and provides clues for discovering the transcriptional regulation mechanisms of other essential ATG genes but also has clinical potential to provide targets for the treatment of diseases related to dysregulated autophagy.

Disclosure statement

All authors have no conflicts of interest to declare.

Additional information

Funding

This work was supported by the National Key R&D Program of China under grant [2017YFA0506300] (to K.L.), the National Natural Science Foundation under grants [32022020] (to K.L.) and [31970693] (to K.L.) and the Disciplinary Excellence Development 135 program of West China Hospital under grant [ZYYC20015] (to K.L.)