Opi1-mediated transcriptional modulation orchestrates genotoxic stress response in budding yeast.
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.
View details for DOI 10.1093/genetics/iyad130
View details for PubMedID 37440469
Genome-wide CRISPR screen reveals v-ATPase as a drug target to lower levels of ALS protein ataxin-2.
2022; 41 (4): 111508
Mutations in the ataxin-2 gene (ATXN2) cause the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). A therapeutic strategy using antisense oligonucleotides targeting ATXN2 has entered clinical trial in humans. Additional ways to decrease ataxin-2 levels could lead to cheaper or less invasive therapies and elucidate how ataxin-2 is normally regulated. Here, we perform a genome-wide fluorescence-activated cell sorting (FACS)-based CRISPR-Cas9 screen in human cells and identify genes encoding components of the lysosomal vacuolar ATPase (v-ATPase) as modifiers of endogenous ataxin-2 protein levels. Multiple FDA-approved small molecule v-ATPase inhibitors lower ataxin-2 protein levels in mouse and human neurons, and oral administration of at least one of these drugs-etidronate-is sufficient to decrease ataxin-2 in the brains of mice. Together, we propose v-ATPase as a drug target for ALS and SCA2 and demonstrate the value of FACS-based screens in identifying genetic-and potentially druggable-modifiers of human disease proteins.
View details for DOI 10.1016/j.celrep.2022.111508
View details for PubMedID 36288714
ReporterSeq reveals genome-wide dynamic modulators of the heat shock response across diverse stressors.
Understanding cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single-cell isolation. We used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. Genome-wide HSR regulation in budding yeast was assessed across 15 stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.
View details for DOI 10.7554/eLife.57376
View details for PubMedID 34223816
ALS Genetics: Gains, Losses, and Implications for Future Therapies.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by the loss of motor neurons from the brain and spinal cord. The ALS community has made remarkable strides over three decades by identifying novel familial mutations, generating animal models, elucidating molecular mechanisms, and ultimately developing promising new therapeutic approaches. Some of these approaches reduce the expression of mutant genes and are in human clinical trials, highlighting the need to carefully consider the normal functions of these genes and potential contribution of gene loss-of-function to ALS. Here, we highlight known loss-of-function mechanisms underlying ALS, potential consequences of lowering levels of gene products, and the need to consider both gain and loss of function to develop safe and effective therapeutic strategies.
View details for DOI 10.1016/j.neuron.2020.08.022
View details for PubMedID 32931756