scholarly journals A genome-wide RNAi screen reveals multiple regulators of caspase activation

2007 ◽  
Vol 179 (4) ◽  
pp. 619-626 ◽  
Author(s):  
Caroline H. Yi ◽  
Dodzie K. Sogah ◽  
Michael Boyce ◽  
Alexei Degterev ◽  
Dana E. Christofferson ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammalian Cells ◽  
Caspase Activation ◽  
Functional Conservation ◽  
Cellular Processes ◽  
Regulatory Circuits ◽  
Genome Wide ◽  
A Genome ◽  
Dependent Cell ◽  
Functionally Diverse

Apoptosis is an evolutionally conserved cellular suicide mechanism that can be activated in response to a variety of stressful stimuli. Increasing evidence suggests that apoptotic regulation relies on specialized cell death signaling pathways and also integrates diverse signals from additional regulatory circuits, including those of cellular homeostasis. We present a genome-wide RNA interference screen to systematically identify regulators of apoptosis induced by DNA damage in Drosophila melanogaster cells. We identify 47 double- stranded RNA that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. Our data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death.

scholarly journals Chromatin architectural proteins regulate flowering time by precluding gene looping

2021 ◽  
Vol 7 (24) ◽  
pp. eabg3097
Author(s):  
Bo Zhao ◽  
Yanpeng Xi ◽  
Junghyun Kim ◽  
Sibum Sung
Keyword(s):  
Chromatin Structure ◽  
Cellular Processes ◽  
Genome Wide ◽  
A Genome ◽  
Evolutionarily Conserved ◽  
Architectural Proteins ◽  
Floral Repressor ◽  
Flanking Regions ◽  
Genome Wide Study

Chromatin structure is critical for gene expression and many other cellular processes. In Arabidopsis thaliana, the floral repressor FLC adopts a self-loop chromatin structure via bridging of its flanking regions. This local gene loop is necessary for active FLC expression. However, the molecular mechanism underlying the formation of this class of gene loops is unknown. Here, we report the characterization of a group of linker histone-like proteins, named the GH1-HMGA family in Arabidopsis, which act as chromatin architecture modulators. We demonstrate that these family members redundantly promote the floral transition through the repression of FLC. A genome-wide study revealed that this family preferentially binds to the 5′ and 3′ ends of gene bodies. The loss of this binding increases FLC expression by stabilizing the FLC 5′ to 3′ gene looping. Our study provides mechanistic insights into how a family of evolutionarily conserved proteins regulates the formation of local gene loops.

scholarly journals A genome-wide screen in the mouse liver reveals sex-specific and cell non-autonomous regulation of cell fitness

2021 ◽  
Author(s):  
Heather R. Keys ◽  
Kristin A. Knouse
Keyword(s):  
Functional Genomics ◽  
High Throughput ◽  
Mouse Liver ◽  
Ex Vivo ◽  
Screening Method ◽  
Living Organism ◽  
Cellular Processes ◽  
Autonomous Regulation ◽  
Genome Wide ◽  
A Genome

ABSTRACTOur ability to understand and modulate mammalian physiology and disease requires knowing how all genes contribute to any given phenotype in the organism. Genome-wide screening using CRISPR-Cas9 has emerged as a powerful method for the genetic dissection of cellular processes1,2, but the need to stably deliver single guide RNAs to millions of cells has restricted its implementation to ex vivo systems. These ex vivo systems cannot reproduce all of the cellular phenotypes observed in vivo nor can they recapitulate all of the factors that influence these phenotypes. There thus remains a pressing need for high-throughput functional genomics in a living organism. Here, we establish accessible genome-wide screening in the mouse liver and use this approach to uncover the complete regulation of cellular fitness in a living organism. We discover novel sex-specific and cell non-autonomous regulation of cell growth and viability. In particular, we find that the class I major histocompatibility complex is essential for preventing immune-mediated clearance of hepatocytes. Our approach provides the first comprehensive picture of cell fitness in a living organism and highlights the importance of investigating cellular phenomena in their native context. Our screening method is robust, scalable, and easily adapted to examine diverse cellular processes using any CRISPR application. We have hereby established a foundation for high-throughput functional genomics in a living mammal, enabling unprecedented insight into mammalian physiology and disease.

Controlling quality and amount of mitochondria by mitophagy: insights into the role of ubiquitination and deubiquitination

2016 ◽  
Vol 397 (7) ◽  
pp. 637-647 ◽  
Author(s):  
Tao Tan ◽  
Marcel Zimmermann ◽  
Andreas S. Reichert
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Fission ◽  
Baker’S Yeast ◽  
Negative Regulator ◽  
Mitochondrial Outer Membrane ◽  
Baker's Yeast ◽  
Genome Wide ◽  
A Genome ◽  
Autophagy Pathway ◽  
Quantity Control

Abstract Mitophagy is a selective autophagy pathway conserved in eukaryotes and plays an essential role in mitochondrial quality and quantity control. Mitochondrial fission and fusion cycles maintain a certain amount of healthy mitochondria and allow the isolation of damaged mitochondria for their elimination by mitophagy. Mitophagy can be classified into receptor-dependent and ubiquitin-dependent pathways. The mitochondrial outer membrane protein Atg32 is identified as the only known receptor for mitophagy in baker’s yeast, whereas mitochondrial proteins FUNDC1, NIX/BNIP3L, BNIP3 and Bcl2L13 are recognized as mitophagy receptors in mammalian cells. Earlier studies showed that ubiquitination and deubiquitination occurs in yeast, yet there is no direct evidence for an ubiquitin-dependent mitophagy pathway in this organism. In contrast, a ubiquitin-/PINK1-/Parkin-dependent mitophagy pathway was unraveled and was extensively characterized in mammals in recent years. Recently, a quantitative method termed synthetic quantitative array (SQA) technology was developed to identify modulators of mitophagy in baker’s yeast on a genome-wide level. The Ubp3-Bre5 deubiquitination complex was found as a negative regulator of mitophagy while promoting other autophagic pathways. Here we discuss how ubiquitination and deubiquitination regulates mitophagy and other selective forms of autophagy and what argues for using baker’s yeast as a model to study the ubiquitin-dependent mitophagy pathway.

scholarly journals SEC24A identified as an essential mediator of thapsigargin-induced cell death in a genome-wide CRISPR/Cas9 screen

2018 ◽  
Vol 4 (1) ◽  
Author(s):  
Tamutenda Chidawanyika ◽  
Elizabeth Sergison ◽  
Michael Cole ◽  
Kenneth Mark ◽  
Surachai Supattapone
Keyword(s):  
Cell Death ◽  
Genome Wide ◽  
A Genome

scholarly journals Intracellular Staphylococcus aureus Perturbs the Host Cell Ca2+ Homeostasis To Promote Cell Death

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e02250-20
Author(s):  
Kathrin Stelzner ◽  
Ann-Cathrin Winkler ◽  
Chunguang Liang ◽  
Aziza Boyny ◽  
Carsten P. Ade ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Death ◽  
Host Cell ◽  
Intracellular Bacterium ◽  
Tissue Destruction ◽  
Content Type ◽  
Host Cell Death ◽  
Genome Wide ◽  
A Genome ◽  
Spread Of Infection

ABSTRACTThe opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca2+ increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca2+ concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca2+ rise led to an increase in mitochondrial Ca2+ concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca2+ homeostasis and induces cytoplasmic Ca2+ overload, which results in both apoptotic and necrotic cell death in parallel or succession.IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca2+ overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca2+ homeostasis.

scholarly journals VPS37A directs ESCRT recruitment for phagophore closure

2019 ◽  
Vol 218 (10) ◽  
pp. 3336-3354 ◽  
Author(s):  
Yoshinori Takahashi ◽  
Xinwen Liang ◽  
Tatsuya Hattori ◽  
Zhenyuan Tang ◽  
Haiyan He ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Growth Factor Receptor ◽  
Multivesicular Body ◽  
Regulatory Mechanisms ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Escrt Machinery ◽  
Genome Wide ◽  
A Genome ◽  
Epidermal Growth

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

scholarly journals An inducible CRISPR interference library for genetic interrogation of Saccharomyces cerevisiae biology

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Amir Momen-Roknabadi ◽  
Panos Oikonomou ◽  
Maxwell Zegans ◽  
Saeed Tavazoie
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Repression ◽  
Nucleosome Occupancy ◽  
Design Parameters ◽  
Experimental Conditions ◽  
Crispr Interference ◽  
Cellular Processes ◽  
Genome Wide ◽  
Reliable Assessment ◽  
A Genome

AbstractGenome-scale CRISPR interference (CRISPRi) is widely utilized to study cellular processes in a variety of organisms. Despite the dominance of Saccharomyces cerevisiae as a model eukaryote, an inducible genome-wide CRISPRi library in yeast has not yet been presented. Here, we present a genome-wide, inducible CRISPRi library, based on spacer design rules optimized for S. cerevisiae. We have validated this library for genome-wide interrogation of gene function across a variety of applications, including accurate discovery of haploinsufficient genes and identification of enzymatic and regulatory genes involved in adenine and arginine biosynthesis. The comprehensive nature of the library also revealed refined spacer design parameters for transcriptional repression, including location, nucleosome occupancy and nucleotide features. CRISPRi screens using this library can identify genes and pathways with high precision and a low false discovery rate across a variety of experimental conditions, enabling rapid and reliable assessment of genetic function and interactions in S. cerevisiae.

scholarly journals Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy

2020 ◽  
Vol 12 (536) ◽  
pp. eaay0271 ◽  
Author(s):  
Angela Lek ◽  
Yuanfan Zhang ◽  
Keryn G. Woodman ◽  
Shushu Huang ◽  
Alec M. DeSimone ◽  
...  
Keyword(s):  
Cell Death ◽  
Muscular Dystrophy ◽  
Phenotypic Selection ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Loss Of Function ◽  
Hypoxia Response ◽  
Genome Wide ◽  
A Genome ◽  
Structural And Functional Properties ◽  
Cellular Hypoxia

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of DUX4, the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.

scholarly journals The Antisense Transcriptomes of Human Cells

Science ◽  
2008 ◽  
Vol 322 (5909) ◽  
pp. 1855-1857 ◽  
Author(s):  
Yiping He ◽  
Bert Vogelstein ◽  
Victor E. Velculescu ◽  
Nickolas Papadopoulos ◽  
Kenneth W. Kinzler
Keyword(s):  
Mammalian Cells ◽  
Cell Types ◽  
Human Cells ◽  
Antisense Transcripts ◽  
Genome Wide ◽  
Human Genes ◽  
A Genome ◽  
Dna Strand ◽  
The Relationship ◽  
Rna Transcript

Transcription in mammalian cells can be assessed at a genome-wide level, but it has been difficult to reliably determine whether individual transcripts are derived from the plus or minus strands of chromosomes. This distinction can be critical for understanding the relationship between known transcripts (sense) and the complementary antisense transcripts that may regulate them. Here, we describe a technique that can be used to (i) identify the DNA strand of origin for any particular RNA transcript, and (ii) quantify the number of sense and antisense transcripts from expressed genes at a global level. We examined five different human cell types and in each case found evidence for antisense transcripts in 2900 to 6400 human genes. The distribution of antisense transcripts was distinct from that of sense transcripts, was nonrandom across the genome, and differed among cell types. Antisense transcripts thus appear to be a pervasive feature of human cells, which suggests that they are a fundamental component of gene regulation.

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