In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream activation sequences (UASINO). Upon genotoxic stress, cells activate the DNA Damage Response (DDR) to coordinate a complex network of signaling pathways aimed at preserving genomic integrity. Here, we reveal that Opi1 is important to modulate transcription in response to genotoxic stress. We find that cells lacking Opi1 exhibit hypersensitivity to genotoxins, along with a delayed G1 to S-phase transition and decreased gamma-H2A levels. Transcriptome analysis using RNA-seq reveals that Opi1 plays a central role in modulating essential biological processes during MMS-associated stress, including repression of phospholipid biosynthesis and transduction of mating signaling. Moreover, Opi1 induces sulfate assimilation and amino acid metabolic processes, such as arginine and histidine biosynthesis and glycine catabolism. Furthermore, we observe increased mitochondrial DNA instability in opi1Δ cells upon MMS treatment. Notably, we show that constitutive activation of the transcription factors Ino2-Ino4 is responsible for genotoxin sensitivity in Opi1-deficient cells, and the production of inositol pyrophosphates by Kcs1 counteracts Opi1 function specifically during MMS-induced stress. Overall, our findings highlight Opi1 as a critical sensor of genotoxic stress in budding yeast, orchestrating gene expression to facilitate appropriate stress responses.
Inositol is a six-carbon sugar that functions as a precursor for signaling molecules such as phosphoinositides and inositol polyphosphates, which are involved in the regulation of important biological processes such as energy metabolism, environmental stress response, phosphate signaling, among others. Given its role in a myriad of signaling pathways, regulation of inositol synthesis is essential for cellular homeostasis. In budding yeast, transcription of genes involved in inositol metabolism is regulated by the transcriptional repressor Opi1, which repress transcription of genes containing cis-acting inositol-sensitive upstream activation sequences (UASINO). Upon genotoxic stress, cells activate the DNA Damage Response (DDR) to coordinate DNA repair and cell cycle progression. It has been proposed that inositol containing molecules might act as modulators of the DDR, but evidences are still scarce. Herein, we report thatopi1cells fail to downregulate the inositol phosphate pathway leading to sensitivity to genotoxins and replication defects. Moreover, cells lacking Opi1 show decreased gamma-H2A levels which might indicate that Opi1 contributes to the activation of the DDR kinases Mec1/Tel1 (ATR/ATM in mammals). Importantly, we show that deletion of the inositol pyrophosphate kinase Kcs1 (IP6K1/2/3 in mammals), which leads to inhibition of inositol pyrophosphate synthesis, rescues the MMS sensitivity and replication defects ofopi1cells. Further, overexpression of Kcs1 recapitulates the MMS sensitivity of cells lacking Opi1. Therefore, we propose that cells must downregulate inositol pyrophosphate synthesis during replication stress in order to trigger an effective DNA Damage Response.
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