Old factors, new players: transcriptional regulation of autophagy

ABSTRACT

Macroautophagy/autophagy is a catabolic process that allows cells to adapt to environmental changes and maintain energy homeostasis. This multistep process is regulated at several levels, including transcriptionally regulating autophagy-related (ATG) gene expression through the action of transcription regulators. Very recently, Wen et al. and we have provided more evidence that two well-known transcription factors regulate different ATG genes to control either nonselective or selective forms of autophagy, respectively. Under nitrogen-starvation conditions, the Spt4-Spt5 complex derepresses ATG8 and ATG41 expression and upregulates bulk autophagy activity. By contrast, under glucose-starvation conditions, the Paf1 complex (the polymerase-associated factor 1 complex, Paf1C) specifically modulates expression of ATG11 and ATG32 to regulate mitophagy. These studies suggest the potential existence of other transcription regulators yet to be discovered that function in the regulation of diverse autophagy pathways.

Abbreviations: AMPK: AMP-activated protein kinase; ATG: autophagy-related; NELF: negative elongation factor; Paf1C/PAF1C: polymerase-associated factor 1 complex; RNAP II: RNA polymerase II; Rpd3L: Rpd3 large complex

KEYWORDS:

• Autophagy
• transcriptional regulation
• the Spt4-Spt5 complex
• the Paf1 complex
• mitophagy

Autophagy is a major intracellular degradation process in eukaryotes that is essential for the removal of cellular components and organelles in response to different types of stress. Nonselective autophagy involves the sequestration of cytoplasm within double-membrane autophagosomes that subsequently deliver their cargo to vacuoles (in yeast and plants) or lysosomes (in mammals) for degradation and recycling. In addition, it has also been extensively demonstrated that selective autophagy is responsible for specifically removing superfluous or damaged components and superfluous organelles through particular cargo-receptor recognition under certain types of nutrient or environmental stress.

Autophagy is a dynamic process that requires tight control to maintain the appropriate timing of induction and magnitude in the cell. Dysregulation of autophagy is associated with a wide range of pathophysiologies including cancer, neurodegeneration, and lysosomal storage diseases. Therefore, eukaryotic cells have evolved mechanisms to integrate signals at various levels to fine-tune autophagy. For example, at transcriptional levels, the expression of many ATG genes are substantially increased in response to autophagy induction. This prompt upregulation upon nutrient or energy stimuli is critical for optimal autophagy efficiency and energy usage, while preventing excess autophagy that would otherwise be deleterious.

At present, a line of studies using the yeast Saccharomyces cerevisiae illustrate that several transcriptional regulators participate in regulation of the expression of ATG genes. One of the well-known transcription complexes, called the Rpd3 large (Rpd3L) complex, which is comprised of multiple subunits including Ume6, Sin3 and Rpd3, is capable of repressing ATG8 gene expression under nutrient-rich conditions. Ume6 is a DNA-binding protein that directly binds the ATG8 promoter via a consensus URS1 binding site. During nitrogen starvation, Ume6 undergoes Rim15-dependent phosphorylation that leads to the derepression of ATG8, thus promoting ATG8 transcription [1]. This transition is critical for the autophagic process as the amount of Atg8 correlates with the size of autophagosomes [2]. Of note, mammalian SIN3 (Ume6 is not conserved in more complex eukaryotes) plays a similar role in regulating the expression of the Atg8 homolog MAP1LC3B.

Pho23 is another negative regulator of autophagy activity in yeast. Unlike deletion of genes encoding other subunits of the Rpd3L complex described above, deletion of PHO23 leads to the upregulation of multiple ATG transcripts [3]. Particularly, Pho23 represses ATG9 expression under nutrient-rich conditions, whereas under nitrogen starvation conditions increased expression of ATG9 allows for a faster rate of autophagosome formation and hence a larger number of autophagosomes, thereby facilitating an increase in the magnitude of autophagy activity [3].

More recently, the histone demethylase Rph1 was discovered as a master transcriptional repressor to control the expression of a subset of ATG genes [4]. However, the role of Rph1 in autophagy is independent of its demethylase activity. It has been shown that deletion of RPH1 promotes autophagy, whereas overexpressing RPH1 blocks autophagy activity and autophagosome formation even under conditions of nitrogen starvation. It turns out that Rph1 directly binds the ATG7 promoter, and this association represses expression of ATG7 as well as a subset of ATG genes required for proper autophagy induction in rich conditions. When cells are starved for nitrogen, the Rim15 kinase phosphorylates Rph1 to release its repression of ATG gene expression. More importantly, like Rph1, the inhibition of KDM4A, a mammalian homolog of Rph1, also shows an enhanced autophagy activity, suggesting its conserved role in autophagy regulation [4].

Besides these negative regulators of ATG genes, several positive transcriptional regulators have also been identified. By analyzing the regulation of the expression of ATG genes from 139 DNA-binding protein deletion mutants, the transcription factor Gcn4 was identified to promote nonselective autophagy activity through ATG gene transcription under nutrient starvation conditions [5]. In addition, this screen also found that Gln3 and Gat1, two GATA-type transcription factors, positively regulate nonselective autophagy by targeting multiple ATG genes during starvation. These results strongly suggest that transcriptional control of autophagy is a critical aspect of autophagy regulation.

Very recently, two evolutionary conserved multisubunit complexes, which tightly associate with RNA polymerase II (RNAP II) and function in transcription elongation, also have been demonstrated to be important for autophagy regulation. The Spt4-Spt5 complex was initially shown to promote transcription elongation in yeast. However, the homolog of the Spt4-Spt5 complex in higher eukaryotes, DRB sensitivity-inducible factor (DSIF), works with the negative elongation factor (NELF) to transiently halt POLR2/RNAP II processing by pausing it downstream of the transcription start site [6]. Although no clear evidence demonstrates the presence of a NELF homolog in budding yeast, the Spt4-Spt5 complex may have a negative effect on transcription elongation. Upon deletion of SPT4, Wen et al. found an increase in both mRNA and protein levels of ATG8/Atg8 and ATG41/Atg41 in nutrient-rich conditions. Moreover, cells lacking Spt4 display increased autophagy activity, suggesting Spt4 negatively regulates autophagy [7].

How does Spt4 regulate autophagy activity? Wen et al. showed that the release of Spt4-dependent negative regulation during starvation is critical for autophagy induction. Mechanistically, Spt5, the partner of Spt4 in this complex, can be phosphorylated by the Bur1-Sgv1/Bur2 kinase complex after starvation or in an spt4∆ strain, and the phosphorylated state of Spt5 is required for optimal ATG41 expression and autophagy activity. Supporting this result, a phospho-deficient mutant of Spt5 shows a reduced autophagy activity, whereas a phospho-mimetic form of this protein exhibits upregulated ATG41 expression in nutrient-rich conditions. Furthermore, both Spt4 and Spt5 can bind to the promoter-proximal region of the ATG41 gene, and the presence of Spt4 allows the accumulation of Spt5 at this region, further confirming the integral function of the Spt4-Spt5 complex. Surprisingly, in contrast to SPT4 depletion, an inducible Spt5 degradation strain displayed a significant decrease in ATG41/Atg41 expression and an autophagy deficiency, suggesting that as an essential gene SPT5 alone may play other distinct roles in regulating autophagy, including a requirement as a positive factor for transcription elongation. Overall, this recent report for the first time showed that the RNAP II-associated Spt4-Spt5 complex plays a negative role in regulating ATG41 expression and bulk autophagy activity.

It has been identified that many transcriptional regulators affect the expression of core ATG genes and autophagy, but little is known with regard to transcription factors targeting selective autophagy pathways. Our recent work shed light on the transcriptional regulation of mitophagy, in which a selective degradation process eliminates damaged and/or superfluous mitochondria. Mitophagy is mediated by the outer mitochondria membrane receptor Atg32 under rich conditions in yeast, which links targeted mitochondria to the autophagic machinery. Atg32 interacts with Atg8 and Atg11 to recruit mitochondria to phagophores (the precursors to autophagosomes) for sequestration [8]. Therefore, the expression level of Atg32 is essential to mitophagic efficiency.

It has been reported that ATG32 transcription is inhibited by the Rpd3L complex. Intriguingly, although disruption of SIN3 or RPD3 significantly increases ATG32 expression, it does not lead to mitophagy induction during nitrogen starvation, implying the presence of another regulatory mechanism [9]. By analyzing gene expression patterns from 165 strains depleted of individual epigenetic or transcriptional regulators, we unveiled the Paf1 complex (Paf1C) as a transcriptional repressor of ATG genes [10]. The Paf1C/PAF1C consists of five subunits conserved from yeast to mammals. Consistently, we observed that deletion of the genes encoding each subunit of Paf1C results in upregulation of both mRNA and protein levels of ATG11/Atg11 and ATG32/Atg32 under rich conditions, suggesting the potential role of this complex in mitophagy induction.

Next, we examined whether Paf1C could directly regulate mitophagy induction. In yeast, mitophagy can be induced by growth in the presence of a non-fermentable carbon source for a prolonged period of time. Consistent with glucose starvation-induced autophagy, PAF1-defective cells display upregulation of mitophagy activity in medium with glycerol as the sole carbon source. Moreover, we observed an accumulation of mitochondria in the vacuole when Paf1C is disrupted under glucose depletion conditions. Finally, the mitophagy activity assay further confirms that Paf1C specifically regulates mitophagy but not nonselective autophagy. Together, these results support the hypothesis that Paf1C regulates mitophagy.

How does Paf1C affect ATG32 expression to regulate mitophagy activity? We found that Paf1C represses ATG32 expression independent of H2B ubiquitination, but through direct binding at its gene locus. Using a chromatin immunoprecipitation assay, we showed that Paf1C is enriched at the ATG32 promoter, but not for example at the ATG39 locus, indicating the specificity of this complex. Upon glucose depletion, we observed that Paf1C is dissociated from the ATG32 promoter, thereby facilitating gene expression and mitophagy induction.

Given that Paf1C is conserved in more complex eukaryotes, we also examined its mitophagic role in mammalian cells. Several mitochondria receptors have been identified in mammals [8]. Intriguingly, we only observed that a reduction of the PAF1 or CTR9 level is correlated with an increase of OPTN (optineurin) mRNA level, but has no effect on other mammalian mitochondrial receptors, including BCL2L13 (BCL2 like 13), which is reported to be a mammalian homolog of the yeast mitophagy receptor Atg32. However, as expected, PAF1 knockdown cells treated with a mitophagy inducer display more degradation of mitochondrial proteins, whereas overexpression of PAF1 retards the degradation of those proteins. Therefore, we conclude that PAF1C regulates mitophagy likely through regulating OPTN expression.

To summarize, these two recent works showed that two highly conserved RNAP II-associated transcription factors, the Spt4-Spt5 complex and the Paf1 complex, repress ATG gene expression under nutrient-rich conditions. Upon nitrogen starvation, phosphorylation of Spt5 by the Bur1-Sgv1/Bur2 kinase complex releases the inhibitory effect of Spt4 on ATG41 transcription, which allows for optimal autophagy induction. By contrast, the release of the inhibitory effect of Paf1C on ATG11 and ATG32 is dependent on the activation of the AMPK pathway under glucose-starvation conditions, which specifically regulates selective mitophagy induction. These studies further expand our knowledge that many “old” transcription factors may have new roles in regulating autophagy, particularly those that may target selective autophagy pathways, which awaits further investigation. In addition, these two studies point out that the conserved factors likely function in a similar manner in all eukaryotes. The current studies in the yeast Saccharomyces cerevisiae will be helpful for understanding the transcriptional regulation of the epigenetic and transcription factors that modulate autophagy in more complex eukaryotes, which may provide important clues for therapeutic strategies aimed at treating various diseases.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Key R&D Program of China [2019YFA0802501]; National Natural Science Foundation of China [31971231]; Wuhan University [2042019kf018] and the National Institutes of Health [GM131919].