100 Use of a Genome-wide Loss-of-function Screen to Identify Novel Mechanisms of Resistance to TRAIL Induced Apoptosis

AbstractFosfomycin is an antibiotic which has seen a revival in use due to its unique mechanism of action and resulting efficacy against isolates resistant to many other antibiotics. Mechanisms of resistance have been elucidated and loss of function mutations within the genes encoding the sugar importers, GlpT and UhpT are commonly selected for by fosfomycin exposure in E. coli. There has however not been a genome wide analysis of the basis for fosfomycin sensitivity reported to date. Here we used ‘TraDIS-Xpress’ a high-density transposon mutagenesis approach to assay the role of all genes in E. coli in fosfomycin sensitivity. The data confirmed known mechanisms of action and resistance as well as identifying a set of novel loci involved in fosfomycin sensitivity. The assay was able to identify sub domains within genes of importance and also revealed essential genes with roles in fosfomycin sensitivity based on expression changes. Novel genes identified included those involved in glucose metabolism, the phosphonate import and breakdown system, phnC-M and the phosphate importer, pstSACB. The impact of these genes in fosfomycin sensitivity was validated by measuring the susceptibility of defined inactivation mutants. This work reveals a wider set of genes contribute to fosfomycin sensitivity including core sugar metabolism genes and two transport systems previously unrecognised as having a role in fosfomycin sensitivity. The work also suggests new routes by which drugs with a phosphonate moiety may be transported across the inner membrane of Gram-negative bacteria.ImportanceThe emergence and spread of antibiotic resistant bacteria had resulted in increased use of alternative drugs which retain efficacy against isolates resistant to other classes of drugs. One example is fosfomycin; an old drug which has found greatly increased use in recent years. We studied the mechanisms of fosfomycin resistance by applying a genome wide screen based on comparing the fitness of a massive library of transposon mutants in the presence of fosfomycin. This approach identified the previously known mechanisms of resistance but also identified a number of new pathways which contribute to fosfomycin sensitivity including two importer systems. This information advances our knowledge about an increasingly important antibiotic and identifies new potential routes to resistance.

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A Nonsense Variant in Hephaestin Like 1 (HEPHL1) Is Responsible for Congenital Hypotrichosis in Belted Galloway Cattle

Genes ◽

10.3390/genes12050643 ◽

2021 ◽

Vol 12 (5) ◽

pp. 643

Author(s):

Thibaud Kuca ◽

Brandy M. Marron ◽

Joana G. P. Jacinto ◽

Julia M. Paris ◽

Christian Gerspach ◽

...

Keyword(s):

Genome Wide Association Study ◽

Homozygosity Mapping ◽

Mendelian Inheritance ◽

Large Animal Model ◽

Large Animal ◽

Loss Of Function ◽

Protein Coding ◽

Positional Candidate ◽

Genome Wide ◽

A Genome

Genodermatosis such as hair disorders mostly follow a monogenic mode of inheritance. Congenital hypotrichosis (HY) belong to this group of disorders and is characterized by abnormally reduced hair since birth. The purpose of this study was to characterize the clinical phenotype of a breed-specific non-syndromic form of HY in Belted Galloway cattle and to identify the causative genetic variant for this recessive disorder. An affected calf born in Switzerland presented with multiple small to large areas of alopecia on the limbs and on the dorsal part of the head, neck, and back. A genome-wide association study using Swiss and US Belted Galloway cattle encompassing 12 cases and 61 controls revealed an association signal on chromosome 29. Homozygosity mapping in a subset of cases refined the HY locus to a 1.5 Mb critical interval and subsequent Sanger sequencing of protein-coding exons of positional candidate genes revealed a stop gain variant in the HEPHL1 gene that encodes a multi-copper ferroxidase protein so-called hephaestin like 1 (c.1684A>T; p.Lys562*). A perfect concordance between the homozygous presence of this most likely pathogenic loss-of-function variant and the HY phenotype was found. Genotyping of more than 700 purebred Swiss and US Belted Galloway cattle showed the global spread of the mutation. This study provides a molecular test that will permit the avoidance of risk matings by systematic genotyping of relevant breeding animals. This rare recessive HEPHL1-related form of hypotrichosis provides a novel large animal model for similar human conditions. The results have been incorporated in the Online Mendelian Inheritance in Animals (OMIA) database (OMIA 002230-9913).

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Generation and Transcriptome Profiling of Slr1-d7 and Slr1-d8 Mutant Lines with a New Semi-Dominant Dwarf Allele of SLR1 Using the CRISPR/Cas9 System in Rice

International Journal of Molecular Sciences ◽

10.3390/ijms21155492 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5492 ◽

Cited By ~ 1

Author(s):

Yu Jin Jung ◽

Jong Hee Kim ◽

Hyo Ju Lee ◽

Dong Hyun Kim ◽

Jihyeon Yu ◽

...

Keyword(s):

Gene Expression Analysis ◽

Transcriptome Profiling ◽

Rna Seq ◽

Loss Of Function ◽

Amino Acid Motif ◽

Dwarf Phenotype ◽

Genome Wide ◽

A Genome ◽

Mutant Lines ◽

Genome Wide Gene Expression

The rice SLR1 gene encodes the DELLA protein (protein with DELLA amino acid motif), and a loss-of-function mutation is dwarfed by inhibiting plant growth. We generate slr1-d mutants with a semi-dominant dwarf phenotype to target mutations of the DELLA/TVHYNP domain using CRISPR/Cas9 genome editing in rice. Sixteen genetic edited lines out of 31 transgenic plants were generated. Deep sequencing results showed that the mutants had six different mutation types at the target site of the TVHYNP domain of the SLR1 gene. The homo-edited plants selected individuals without DNA (T-DNA) transcribed by segregation in the T1 generation. The slr1-d7 and slr1-d8 plants caused a gibberellin (GA)-insensitive dwarf phenotype with shrunken leaves and shortened internodes. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two GA-related genes, GA20OX2 (Gibberellin oxidase) and GA3OX2, were increased in the edited mutant plants, suggesting that GA20OX2 acts as a convert of GA12 signaling. These mutant plants are required by altering GA responses, at least partially by a defect in the phytohormone signaling system process and prevented cell elongation. The new mutants, namely, the slr1-d7 and slr1-d8 lines, are valuable semi-dominant dwarf alleles with potential application value for molecule breeding using the CRISPR/Cas9 system in rice.

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

Science Translational Medicine ◽

10.1126/scitranslmed.aay0271 ◽

2020 ◽

Vol 12 (536) ◽

pp. eaay0271 ◽

Cited By ~ 6

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.

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Genome-wide Loss-of-Function Screen Reveals an Important Role for the Proteasome in HDAC Inhibitor-Induced Apoptosis

Cancer Cell ◽

10.1016/j.ccr.2008.12.001 ◽

2009 ◽

Vol 15 (1) ◽

pp. 57-66 ◽

Cited By ~ 97

Author(s):

Susan Fotheringham ◽

Mirjam T. Epping ◽

Lindsay Stimson ◽

Omar Khan ◽

Victoria Wood ◽

...

Keyword(s):

Hdac Inhibitor ◽

Loss Of Function ◽

Genome Wide ◽

Induced Apoptosis

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CUX2 mutations in temporal lobe epilepsy; increased kainate susceptibility and excitatory input to hippocampus in deficient mice

10.1101/2021.09.17.460803 ◽

2021 ◽

Author(s):

Toshimitsu Suzuki ◽

Tetsuya Tatsukawa ◽

Genki Sudo ◽

Caroline Delandre ◽

Yun Jin Pai ◽

...

Keyword(s):

Synaptic Transmission ◽

Temporal Lobe Epilepsy ◽

Entorhinal Cortex ◽

Temporal Lobe ◽

Genome Wide Association Study ◽

De Novo ◽

Cell Number ◽

Loss Of Function ◽

Genome Wide ◽

A Genome

CUX2 gene encodes a transcription factor that controls neuronal proliferation, dendrite branching and synapse formation, locating at the epilepsy-associated chromosomal region 12q24 that we previously identified by a genome-wide association study (GWAS) in Japanese population. A CUX2 recurrent de novo variant p.E590K has been described in patients with rare epileptic encephalopathies and the gene is a candidate for the locus, however the mutation may not be enough to generate the genome-wide significance in the GWAS and whether CUX2 variants appear in other types of epilepsies and physiopathological mechanisms are remained to be investigated. Here in this study, we conducted targeted sequencings of CUX2, a paralog CUX1 and its short isoform CASP harboring a unique C-terminus on 271 Japanese patients with a variety of epilepsies, and found that multiple CUX2 missense variants, other than the p.E590K, and some CASP variants including a deletion, predominantly appeared in patients with temporal lobe epilepsy (TLE). Human cell culture and fly dendritic arborization analyses revealed loss-of- function properties for the CUX2 variants. Cux2- and Casp-specific knockout mice both showed high susceptibility to kainate, increased excitatory cell number in the entorhinal cortex, and significant enhancement in glutamatergic synaptic transmission to the hippocampus. CASP and CUX2 proteins physiologically bound to each other and co-expressed in excitatory neurons in brain regions including the entorhinal cortex. These results suggest that CUX2 and CASP variants contribute to the TLE pathology through a facilitation of excitatory synaptic transmission from entorhinal cortex to hippocampus.

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Genome-wide synthetic lethal CRISPR screen identifies FIS1 as a genetic interactor of ALS-linked C9ORF72

10.1101/778118 ◽

2019 ◽

Author(s):

Noori Chai ◽

Michael S. Haney ◽

Julien Couthouis ◽

David W. Morgens ◽

Alyssa Benjamin ◽

...

Keyword(s):

Genetic Interaction ◽

Mitochondrial Fission ◽

Loss Of Function ◽

Synthetic Lethal ◽

Genome Wide ◽

A Genome ◽

Crispr Screen ◽

Insight Into ◽

Mitochondrial Membrane Protein ◽

Lateral Sclerosis

AbstractMutations in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis (ALS). Both toxic gain of function and loss of function pathogenic mechanisms have been proposed. Accruing evidence from mouse knockout studies point to a role for C9ORF72 as a regulator of immune function. To provide further insight into its cellular function, we performed a genome-wide synthetic lethal CRISPR screen in human myeloid cells lacking C9ORF72. We discovered a strong synthetic lethal genetic interaction between C9ORF72 and FIS1, which encodes a mitochondrial membrane protein involved in mitochondrial fission and mitophagy. Mass spectrometry experiments revealed that in C9ORF72 knockout cells, FIS1 strongly bound to a class of immune regulators that activate the receptor for advanced glycation end (RAGE) products and trigger inflammatory cascades. These findings present a novel genetic interactor for C9ORF72 and suggest a compensatory role for FIS1 in suppressing inflammatory signaling in the absence of C9ORF72.

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NRF2 Activation Confers Resistance to eIF4A Inhibitors in Cancer Therapy

Cancers ◽

10.3390/cancers13040639 ◽

2021 ◽

Vol 13 (4) ◽

pp. 639

Author(s):

Viraj R. Sanghvi ◽

Prathibha Mohan ◽

Kamini Singh ◽

Linlin Cao ◽

Marjan Berishaj ◽

...

Keyword(s):

Protein Production ◽

Mechanisms Of Resistance ◽

New Class ◽

Translation Factors ◽

Genome Wide ◽

A Genome ◽

Nrf2 Activation ◽

Anti Cancer ◽

New Strategy ◽

Related Compounds

Inhibition of the eIF4A RNA helicase with silvestrol and related compounds is emerging as a powerful anti-cancer strategy. We find that a synthetic silvestrol analogue (CR-1-31 B) has nanomolar activity across many cancer cell lines. It is especially active against aggressive MYC+/BCL2+ B cell lymphomas and this likely reflects the eIF4A-dependent translation of both MYC and BCL2. We performed a genome-wide CRISPR/Cas9 screen and identified mechanisms of resistance to this new class of therapeutics. We identify three negative NRF2 regulators (KEAP1, CUL3, CAND1) whose inactivation is sufficient to cause CR1-31-B resistance. NRF2 is known to alter the oxidation state of translation factors and cause a broad increase in protein production. We find that NRF2 activation particularly increases the translation of some eIF4A-dependent mRNAs and restores MYC and BCL2 production. We know that NRF2 functions depend on removal of sugar adducts by the frutosamine-3-kinase (FN3K). Accordingly, loss of FN3K results in NRF2 hyper-glycation and inactivation and resensitizes cancer cells to eIF4A inhibition. Together, our findings implicate NRF2 in the translation of eIF4A-dependent mRNAs and point to FN3K inhibition as a new strategy to block NRF2 functions in cancer.

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A genome-wide DNA methylation signature for SETD1B-related syndrome

Clinical Epigenetics ◽

10.1186/s13148-019-0749-3 ◽

2019 ◽

Vol 11 (1) ◽

Cited By ~ 12

Author(s):

I. M. Krzyzewska ◽

S. M. Maas ◽

P. Henneman ◽

K. v. d. Lip ◽

A. Venema ◽

...

Keyword(s):

Clinical Utility ◽

De Novo ◽

Histone H3 ◽

Missense Mutations ◽

Loss Of Function ◽

Variant Of Uncertain Significance ◽

Epigenetic Marker ◽

Genome Wide ◽

A Genome ◽

Uncertain Significance

Abstract SETD1B is a component of a histone methyltransferase complex that specifically methylates Lys-4 of histone H3 (H3K4) and is responsible for the epigenetic control of chromatin structure and gene expression. De novo microdeletions encompassing this gene as well as de novo missense mutations were previously linked to syndromic intellectual disability (ID). Here, we identify a specific hypermethylation signature associated with loss of function mutations in the SETD1B gene which may be used as an epigenetic marker supporting the diagnosis of syndromic SETD1B-related diseases. We demonstrate the clinical utility of this unique epi-signature by reclassifying previously identified SETD1B VUS (variant of uncertain significance) in two patients.

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