Transcriptional Regulation of Autophagy.

Macroautophagy (hereafter autophagy) is a cellular recycling process through which cytoplasmic contents are delivered into the lysosome/vacuole for degradation by double-membrane organelles, autophagosomes. Autophagy is essential for cell survival under stress; however, too much autophagy can also be detrimental. Autophagy activity can be regulated by modulating the size or the number of autophagosomes. Although there are more than 40 autophagy-related (ATG) genes that have been identified, it is not fully understood how most of these genes contribute to these aspects of regulation. Autophagy is highly conserved among eukaryotic cells, and its molecular machinery has been best characterized in the budding yeast Saccharomyces cerevisiae. In my thesis studies, I use budding yeast as the model organism, taking advantage of its utility in genetic/genomic screening; in addition, high-throughput sequencing and powerful autophagy assays have been developed uniquely in the yeast system, to explore how autophagy is modulated through transcriptional regulation. When I joined the Klionsky lab, I became involved in the study of a negative transcriptional regulator of autophagy, Ume6. Deletion of the UME6 gene results in an increase in the size, but not the number, of autophagosomes by increasing the expression of Atg8. From a subsequent genetic screen for autophagy modulators, I identified another transcription repressor of autophagy, Pho23. Intriguingly, Pho23 ended up being characterized as a specific regulator of the number, but not the size, of autophagosomes, or it can be viewed as controlling the rate of autophagosome formation by regulating Atg9 expression. These studies support a model whereby the size and numbers of autophagosomes are independently regulated through precise transcriptional regulation of different ATG genes. Collaborating with Amélie Bernard, a postdoc in the lab, to further explore the transcriptional regulation network of autophagy, we analyzed 139 yeast strains each deleted for a single gene encoding a transcription factor; we profiled the transcription of several ATG genes in each strain. Through this screen we identified Gcn4, Slf1, Gat1 and Gln3 as transcriptional activators, and Spt10, Fyv5, and Rph1 as transcriptional repressors of autophagy. We also further investigated the detailed molecular mechanisms of the regulation of autophagy by Rph1.PhDMolecular, Cellular and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133444/1/jmeiyan_1.pd

Role of autophagy in yeast cell adaptation

Autophagy is an evolutionarily conserved degradative pathway. Autophagy occurs constitutively at a basal level and it is involved in the recycling and turnover of damaged or superfluous organelles and proteins. It has a critical role in cellular homeostasis. Autophagy can be induced in response to starvation or other types of stress. Induction of autophagy during these conditions has a major role in protection and adaptation of the cell. Autophagy needs to be properly regulated. A wide range of diseases is associated with dysregulation of autophagy. Better understanding of autophagy mechanisms can help to develop strategies designed to modulate autophagic responses occuring in a number of diseases. This work is focused on current knowledge of main types of autophagy and how autophagy helps yeast cells to adapt. Key words: autophagy, yeast, degradative pathway, adaptation, TORC1Autofágie je evolučně konzervovaná degradativní dráha. V buňkách je běžně udržována na nízké hladině, kdy degraduje nadbytečné či poškozené organely a proteiny a tím se zásadně podílí na udržování buněčné homeostázy. Pokud jsou buňky vystavené nepříznivým podmínkám, například nedostatku živin nebo jiným druhům stresu, hladina autofágie se zvýší. V této fázi má především ochranou roli a pomáhá buňce v adaptaci na změnu podmínek. Autofágie je přísně regulována, její dysfuknce je spojována s mnoha lidskými onemocněními. Podrobné porozumnění regulačním mechanismům autofágie může mít v budoucnu vliv při vývoji léčebných postupů chorob s ní spojených. Tato práce shrnuje poznatky o základních typech autofágie u kvasinek a popisuje, jak autofágie pomáhá adaptaci buňky na nepříznivé podmínky. Klíčová slova: autofágie, kvasinky, degradační dráha, adaptace, TORC1Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePřírodovědecká fakult

Investigation of platinum drug mode of action and resistance using yeast and human neuroblastoma cells as model systems

The platinum-based cytotoxic chemotherapeutics cisplatin, carboplatin, and oxaliplatin are widely used as anti-cancer drugs, but their efficacy is limited by the occurrence of resistance. The mechanisms of actions of these drugs as well as the mechanisms underlying resistance (formation) to platinum drugs remain incompletely understood. More knowledge is required to develop more effective therapies. In this study, we used yeast cells and neuroblastoma cell lines to investigate the mode of action and resistance to platinum drugs. First, we screened a Saccharomyces cerevisiae transcription factor heterozygous gene deletion library to identify genes that when deleted result in enhanced sensitivity to cisplatin, carboplatin and/or oxaliplatin. In addition, data on platinum drug sensitivity in yeast, derived from screening a yeast whole-genome homozygous deletion library, was extracted from the Yeast Fitness Database (http://fitdb.stanford.edu/). There was a substantial overlap in the genes (and related pathways) that determine sensitivity to the individual platinum drugs, but also considerable differences. Notably, cisplatin and carboplatin are anticipated to be more similar in their mode of action compared to oxaliplatin, but the yeast data did not entirely support this notion. Amoung the genes involved in response to platinum drugs, BDF1 (Bromodomain-containing factor 1) was identified as a novel gene which, when deleted, resulted in increased sensitivity to all three platinum drugs. Its re-expression reversed platinum drug sensitivity, confirming its role in determining the yeast cell response to platinum drugs. Notably, BET proteins (the human equivalents of Bdf1) are increasingly recognised as potential anti-cancer drug targets. Our data suggest that they may have a role in sensitising cancer cells to platinum drugs. Next, we investigated a unique panel of cell lines consisting of neuroblastoma cell lines UKF-NB-3 and UKF-NB-6 and their sub-lines with acquired resistance to cisplatin (UKF-NB-3rCDDP1000, UKF-NB-6rCDDP2000), oxaliplatin (UKF-NB-3rOXALI2000, UKF-NB6rOXALI4000), or carboplatin (UKF-NB3rCARBO2000, UKF-NB-6rCARBO2000). Adaption to platinum drugs was associated with changes in doubling times and cell morphology but there were no consistent patterns. This suggests that resistance mechanisms are complex and heterogeneous. The resistance phenotype was stable after cultivation of the resistant sub-lines for three months in the absence of drugs, indicating that resistance was not a consequence of the reversible enrichment of a pre-existing sub-population of cells, but due to a permanent, irreversible genomic change. This notion was supported by the determination of sensitivity profiles to cisplatin, carboplatin and oxaliplatin in the cell line panel. Both UKF-NB-3 and UKF-NB-6 parental lines exhibited sensitivity to all three platinum drugs, and the platinum drug-adapted parental cells displayed generally increased resistance, not just to the drug to which they were adapted, but to all three platinum drugs. However cisplatin- and carboplatin- resistant UKF-NB-3 cells displayed no cross-resistance to oxaliplatin, and oxaliplatin-resistant UKF-NB-3 cells displayed none to cisplatin and carboplatin. In contrast to the yeast data, this supports the notion that cisplatin and carboplatin are more similar in their mode of action than oxaliplatin. Finally, in a proteomics study, we compared the UKF-NB-3 and UKF-NB-6 cells with their cisplatin-resistant sub-lines to study acquired cisplatin resistance, and also investigated their responses to acute cisplatin treatment. The resulting data, together with previous proteomics studies that investigated acquired resistance in cisplatin-adapted neuroblastoma cell lines, suggested that, despite overlaps, the resistance mechanisms are heterogeneic and cell line-specific. This was also the case, comparing our cell lines only, for the acute cisplatin responses. In conclusion our data demonstrate that resistance formation to cisplatin is a complex and individual/cell line specific process. Further research will be required to enable a systems-level understanding of cisplatin resistance that can be translated into improved therapeutic approaches

Transcriptional Regulation of Metabolic Genes by the Basic Leucine Zipper Transcription Factor Hac1ip and Nutrient Stimuli

Saccharomyces cerevisiae cells respond to nutrients in their environment by altering their metabolic and transcriptional state in order to optimise the use of available nutrients and decide which of the several developmental pathways to pursue. In the yeast S. cerevisiae, meiosis and pseudohyphal growth are two major differentiation outcomes in response to nitrogen starvation. A central component of unfolded protein response pathway, the bZIP transcription factor Hac1ip, negatively regulates meiosis and pseudohyphal growth. The present study investigates this negative regulatory mechanism at early meiotic genes by Hac1ip in nitrogen-rich conditions. Regulation of transcription by Ume6p transcriptional regulator, Rpd3p-Sin3p histone deacetylase complex and Isw2p-Itc1p chromatin remodelling complex at URS1 was also investigated here. We also tested for induction of pseudohyphal growth in diploids from SK1 genetic background in response to nitrogen starvation conditions known to induce meiosis. I constructed destabilised β-galactosidase reporters expressed from URS1- CYC1-Ub-X-lacZ reporters to analyze transcriptional activity at URS1 site of early meiotic genes in nutrient rich conditions. The data presented here successfully demonstrates Hac1ip-mediated repression at URS1 sites in nitrogen-rich conditions. URS1-CYC1-Ub-X-lacZ reporters were expressed in mitotic repression machinery mutants (ume6Δ, rpd3Δ, sin3Δ, isw2Δ and itc1Δ) under nitrogen rich conditions. The data presented here from these experiments not only corroborates their known role in repression at URS1 but also suggested regulation at additional sites in the minimal CYC1 promoter. Deletion of Sin3p suggested independent repression function separable from Rpd3p. Isw2p also acts independently of Itc1p at sites other than URS1. We also show that pseudohyphal growth was stimulated by non-fermentable carbon sources in sporulation efficient SK1 genetic background. The data also indicates that stimulation of pseudohyphal growth by non-fermentable carbon sources does not require respiration function or functional mitochondrial RTG pathway

The transcription factor Spt4-Spt5 complex regulates the expression of ATG8 and ATG41

Macroautophagy/autophagy, a highly conserved dynamic process, is one of the major degradative pathways in cells. So far, over 40 autophagy-related (ATG) genes have been identified in Saccharomyces cerevisiae, most of which have homologs in more complex eukaryotes. Autophagy plays a crucial role in cell survival and maintenance, and its dysfunction is related to various diseases, indicating that the proper regulation of autophagy is important. Although the overall process of autophagy has been extensively studied, in particular with regard to the function of the Atg proteins, relatively little is known about the regulatory mechanisms that control autophagy activity. Spt5 is one of the transcriptional factors that is universally conserved across all domains. This protein can form a complex with Spt4, together playing a central role in transcription. In complex eukaryotic cells, the Spt4-Spt5 complex plays a dual role in gene regulation, acting both to delay transcription through promoter-proximal pausing, and to facilitate transcriptional elongation. In contrast, in S. cerevisiae, only the positive function of the Spt4-Spt5 complex has been identified. Here, we show for the first time that the Spt4-Spt5 transcription factor complex negatively regulates ATG genes in S. cerevisiae, inhibiting autophagy activity during active growth. Under autophagy-inducing conditions, the repression is released by Spt5 phosphorylation, allowing an upregulation of autophagy activity

Med13 Degradation Defines a New Receptor-Mediated Autophagy Pathway Activated by Nutrient Deprivation

Cells are exposed to an enormous amount of diverse extracellular cues but have a limited arsenal of weapons for protecting and maintaining homeostasis. To overcome these restrictions, nature has engineered proteins that have multiple functions. The pleiotropy of using one protein to carry out a variety of functions allows cells to rapidly execute tailored responses to a diverse set of signals. The Cdk8 kinase module (CKM) is a conserved detachable unit of the Mediator complex predominantly known for its role in transcriptional regulation. The CKM is composed of four proteins, the scaffolding proteins Med13 and Med12, as well as the non-canonical cyclin, cyclin C, and its cognate kinase, Cdk8. Previously it has been shown that cyclin C is a multifunctional protein that performs transcriptional and stress-induced roles at the mitochondria. The localization, post-translational modifications, and different functional domains of cyclin C regulate these separate functions. Here we show that Med13 also has dual roles in regulating stress response following nutrient depletion. In physiological conditions, Med13 works within the CKM to negatively regulate the expression of autophagy genes (ATG). Following starvation, this repression is relieved by Snx4-assisted autophagy of Med13. Moreover, we identified Ksp1 to be the autophagic receptor protein for this novel autophagy pathway. Structural analysis by others showed that Med13 has an RNA binding region. Consistent with this, we showed that once in the cytosol, Med13 localizes to ribonucleoprotein granules known as processing bodies (P-bodies) which function in mRNA silencing, decay, and storage. In addition, we show that Med13, together with Ksp1 and Snx4, are required for the autophagic degradation of conserved P-body proteins following stress. These results illustrate the day and night jobs of Med13 in response to starvation stress. Lastly, we illustrate that the regulation of autophagy by the CKM is evolutionarily conserved. Here we show that cyclin C promotes autophagy and proteasome activity in the murine pancreatic cancer model. Collectively, these studies demonstrate the multifunctionality and conservation of the CKM in stress response

The Paf1 complex transcriptionally regulates the mitochondrial-anchored protein Atg32 leading to activation of mitophagy

Mitophagy is a critical process that safeguards mitochondrial quality control in order to maintain proper cellular homeostasis. Although the mitochondrial-anchored receptor Atg32-mediated cargo-recognition system has been well characterized to be essential for this process, the signaling pathway modulating its expression as a contribution of governing the mitophagy process remains largely unknown. Here, bioinformatics analyses of epigenetic or transcriptional regulators modulating gene expression allow us to identify the Paf1 complex (the polymerase-associated factor 1 complex, Paf1C,) as a transcriptional repressor of ATG genes. We show that Paf1C suppresses glucose starvation-induced autophagy, but does not affect nitrogen starvation- or rapamycin-induced autophagy. Moreover, we show that Paf1C specifically regulates mitophagy through modulating ATG32 expression. Deletion of the genes encoding two core subunits of Paf1C, Paf1 and Ctr9, increases ATG32 and ATG11 expression and facilitates mitophagy activity. Although Paf1C is required for many histone modifications and gene activation, we show that Paf1C regulates mitophagy independent of its positive regulatory role in other processes. More importantly, we also demonstrate the mitophagic role of PAF1C in mammals. Overall, we conclude that Paf1C maintains mitophagy at a low level through binding the promoter of the ATG32 gene in glucose-rich conditions. Dissociation of Paf1C from ATG32 leads to the increased expression of this gene, and mitophagy induction upon glucose starvation. Thus, we uncover a new role of Paf1C in modulating the mitophagy process at the transcriptional level

On the edge of degradation: Autophagy regulation by RNA decay

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149292/1/wrna1522_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149292/2/wrna1522.pd

Short and long-term regulation of autophagy

Autophagy is a conserved catabolic pathway triggered by stress conditions in which portions of the cytoplasm, damage organelles, misfolded proteins and intracellular bacteria are delivered and degraded in the lysosome/vacuoles. Thus, an efficient induction and completion of the process is required to ensure a proper homeostasis of the cell. Autophagy has been considered a cytoplasmic event where the role of the nucleus on the regulation of this pathway was underestimated. However, recent findings elicited the role of histone modifying enzymes on the transcriptional regulation of autophagy-related (ATG) genes. In line with those results, we focused on the role of the two histone modifying enzymes regulating the histone 3 lysine 36 (H3K36) trimethylation, Rph1/KDM4A and Set2/SETD2, on the regulation of autophagy. In paper I, we investigated the function of the histone demethylase, Rph1/KDM4 as a negative regulator of autophagy, whereas in paper II we uncovered the role of the histone methyltransferase, Set2/SETD2, as a positive transcriptional regulator of ATG genes, as the impact on the differential expression of ATG14 splice isoforms that results on the inhibition of the autophagosome-lysosome fusion. Moreover, in paper III, we identify that SETD2 inactivating mutations on clear cell renal cell carcinomas (ccRCC) lead to an aberrant ATG12-containing complexes and accumulation of free ATG12, which is associated with a differential expression of ATG12 isoforms and reduced autophagic flux. Whereas the previous studies report the involvement of histone modifying enzymes and on the short-term regulation of autophagy, we also aimed to decipher the epigenetic mechanism responsible for the long-lasting effects of autophagy. In paper IV, we found that short autophagy stimulus is associated with an upregulation of de novo DNA methyltransferase 3A (DNMT3A) responsible of an increase of DNA methylation on selected ATG genes. Eventually, this epigenetic memory involves a persistent decrease of baseline autophagy. Moreover, in paper V, we uncovered the mechanism upstream on the regulation of DNMT3A expression by ULK3-mediated phosphorylation and activation of GLI1. Overall, these insights bring light on novel mechanisms and signaling pathways controlling short and long-term transcriptional regulation of autophagy by histone modifying enzymes, alternative splicing and DNA methylation