Not Simply the Induction of Alternative Oxidase: The AOD2 and AOD5 Transcription Factors Play Roles in Regulation of Metabolism and Energy Production in Neurospora crassa

• Author / Creator

  Qi, Zhigang

• Mitochondria are important organelles in most eukaryotic organisms as they supply the majority of the ATP needed for cellular functions. Although mitochondria contain their own DNA, the vast majority of mitochondrial proteins are encoded in the nucleus. Thus, when mitochondria experience stress they signal the nucleus to alter gene expression and provide a response to the stress. In Neurospora crassa, one model for studying mitochondrial signaling is the induction of alternative oxidase (AOX). This enzyme is a mitochondrial protein encoded by the nuclear aod-1 gene and serves as an alternative terminal oxidase in the electron transport chain. Under normal growth conditions, AOX is not expressed. However, induction of the AOX occurs when cells are grown in the presence of drugs that decrease the function of the standard electron transport chain or when they carry mutations affecting the standard electron transport chain. The induction of AOX requires the function of two Zn(II)Cys6 transcription factors named AOD2 and AOD5. In this study, I demonstrated that both AOD2 and AOD5 localize to the nucleus regardless of whether or not cells are grown under conditions that induce AOX. AOD2 and AOD5 were also shown to exist as a heterodimer in vivo. The orthologues of AOD2 and AOD5 in other fungi have been shown to regulate the expression of genes required for gluconeogenesis. Here, I have shown that the growth of N. crassa on poor carbon sources was severely impaired in the absence of AOD2 or AOD5. Moreover, the expression of the gene encoding phosphoenolpyruvate carboxylase (PEPCK), which is required for gluconeogenesis, was greatly reduced in the absence of AOD2. However, no AOX was detected in cells grown in poor carbon sources, suggesting that the action of AOD2 and AOD5 in regulating gluconeogenesis and AOX is complex and might occur via response to different signals. To investigate whether AOD2 and AOD5 are involved in other cellular functions, I performed chromatin immunoprecipitations followed by next generation sequencing (ChIP-seq) to investigate the global binding of AOD2 and AOD5 in cells grown in the presence and absence of chloramphenicol, a drug that inhibits mitochondrial protein synthesis and, thus, indirectly affects the function of the standard electron transport chain. I focused on the 65 ChIP-seq peaks that exhibited more than four-fold enrichment from strains expressing tagged versions of AOD2 and AOD5 that could be immunoprecipitated with commercial antibodies as compared to a wild-type control strain with no tagged proteins. ChIP-seq was done following growth in both AOX inducing and non-inducing conditions. I found that the 5’ regions of aod-1 and PEPCK are constitutively bound by AOD2 and AOD5. Furthermore, AOD2 and AOD5 also bind to the 5’ regions of many other genes. Most of the binding sites for the 65 peaks contain at least one AOX induction motif, which was previously shown to bind AOD2 and AOD5 using in vitro assays. Several of the genes associated with the 65 peaks were validated as requiring AOD2 and AOD5 for normal expression using RT-qPCR. This study suggests that not only do AOD2 and AOD5 regulate the AOX induction pathway and gluconeogenic pathway, but they also appear to play larger roles in energy homeostasis and reprogramming of metabolism under oxidative stress.

• Subjects / Keywords

  • Zinc cluster transcription factors
  • Alternative oxidase
  • Neurospora-crassa
  • ChIP-seq

• Graduation date

  Fall 2016

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3JS9HD4P

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.