scholarly journals Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation

2018 ◽  
Vol 217 (11) ◽  
pp. 3817-3828 ◽  
Author(s):  
Keigo Morita ◽  
Yutaro Hama ◽  
Tamaki Izume ◽  
Norito Tamura ◽  
Toshihide Ueno ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Degradation Process ◽  
Autophagic Flux ◽  
Autophagosome Formation ◽  
Early Step ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

Macroautophagy is an intracellular degradation process that requires multiple autophagy-related (ATG) genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified TMEM41B as a novel ATG gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of TMEM41B blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in TMEM41B-knockout (KO) cells. The phenotype of TMEM41B-KO cells resembled those of VMP1-KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in TMEM41B-KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.

scholarly journals Genome-wide CRISPR Screen to Identify Genes that Suppress Transformation in the Presence of Endogenous KrasG12D

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jianguo Huang ◽  
Mark Chen ◽  
Eric S. Xu ◽  
Lixia Luo ◽  
Yan Ma ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Gene Mutations ◽  
Soft Agar ◽  
Reading Frame ◽  
Immunodeficient Mice ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

AbstractCooperating gene mutations are typically required to transform normal cells enabling growth in soft agar or in immunodeficient mice. For example, mutations in Kras and transformation-related protein 53 (Trp53) are known to transform a variety of mesenchymal and epithelial cells in vitro and in vivo. Identifying other genes that can cooperate with oncogenic Kras and substitute for Trp53 mutation has the potential to lead to new insights into mechanisms of carcinogenesis. Here, we applied a genome-wide CRISPR/Cas9 knockout screen in KrasG12D immortalized mouse embryonic fibroblasts (MEFs) to search for genes that when mutated cooperate with oncogenic Kras to induce transformation. We also tested if mutation of the identified candidate genes could cooperate with KrasG12D to generate primary sarcomas in mice. In addition to identifying the well-known tumor suppressor cyclin dependent kinase inhibitor 2A (Cdkn2a), whose alternative reading frame product p19 activates Trp53, we also identified other putative tumor suppressors, such as F-box/WD repeat-containing protein 7 (Fbxw7) and solute carrier family 9 member 3 (Slc9a3). Remarkably, the TCGA database indicates that both FBXW7 and SLC9A3 are commonly co-mutated with KRAS in human cancers. However, we found that only mutation of Trp53 or Cdkn2a, but not Fbxw7 or Slc9a3 can cooperate with KrasG12D to generate primary sarcomas in mice. These results show that mutations in oncogenic Kras and either Fbxw7 or Slc9a3 are sufficient for transformation in vitro, but not for in vivo sarcomagenesis.

A Whole Genome In Vivo Crispr Screen in Primary ALL Predicts Leukaemic Relapse

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2619-2619
Author(s):  
Katherine Dormon ◽  
Elda S Latif ◽  
Matthew Bashton ◽  
Deepali Pal ◽  
Matthew Selby ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Whole Genome ◽  
Competitive Situation ◽  
Nsg Mice ◽  
A Genome ◽  
Crispr Screen

Abstract Although paediatric acute lymphoblastic leukaemia (ALL) has a favourable prognosis, a number of cases will invariably relapse. One of the major problems associated with relapse is drug resistance, in particular to glucocorticoids, the mainstay of ALL treatment. Examining the underlying mechanisms is complicated by clonal heterogeneity within a patient and the potential impact of the leukaemic niche. To address mechanisms of drug resistance in a patient-relevant setting, we performed a genome-wide in vivo CRISPR screen in primary ALL material. To that end, we took advantage of primografted material from patient L707, who initially presented with a Dexamethasone (DEX) sensitive t(17;19) ALL, but relapsed 5 months after initial diagnosis. We transduced DEX sensitive presentation cells with the full genome GeCKOv2 CRISPR library, before transplantation into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were subsequently treated with DEX by oral gavage (15mg/kg for 5 weeks, 10mg/kg thereafter). DNA from several engrafted sites in the mouse was extracted and PCR amplified before being sequenced on the Illumina HiSeq2500. Changes in pool complexity were analysed using MaGEcK software to determine which sgRNAs were significantly enriched or depleted. By far the most significantly enriched sgRNAs were those targeting NR3C1, the gene encoding the glucocorticoid receptor. In addition, two of the top five significantly depleted sgRNAs targeted the Plexins, PLXNA1 and PLXND1. Whilst PLXNA1 is expressed at low levels, PLXND1 is highly expressed and has been linked to dexamethasone resistance. Notably, the matched relapsed material from L707 was highly DEX resistant both in tissue culture and when transplanted into NSG mice. SNP 6.0 analysis revealed a 5q deletion in the relapse, spanning 5 genes including NR3C1. Whole genome sequencing showed this was comprised of 2 deletions both targeting NR3C1, with different breakpoints for each allele. The differential gene expression between the L707 presentation and relapse established that NR3C1 was the most significant of all the genes lost at relapse, based on gene set enrichment analysis (GSEA). This contrasts with many ALL cases, where one of the downstream effectors of apoptosis is lost as opposed to NR3C1. Growth of the relapse material in vivo and in vitro was slower than the presentation in a competitive situation, but with DEX treatment the relapse phenotype began to emerge with a small percentage of cells showing a heterozygous deletion of NR3C1. These combined data strongly suggest that the NR3C1 deletion is the main driver of DEX resistance in the L707 relapse. Moreover, it proves that our in vivo CRISPR screen predicted the leukaemic relapse. These results confirm NR3C1 deletion as a driver in glucocorticoid resistance and demonstrate the power of in vivo CRISPR screens to predict mechanisms of gain of drug resistance and subsequent relapse. The parallels that can be drawn between the relapse and the CRISPR screen are striking, giving the indication that the progression from presentation to relapse may follow the same path in a patient derived xenograft setting as it did in the patient. Disclosures No relevant conflicts of interest to declare.

scholarly journals In vivo CRISPR/Cas9 knockout screen: TCEAL1 silencing enhances docetaxel efficacy in prostate cancer

2020 ◽  
Vol 3 (12) ◽  
pp. e202000770 ◽  
Author(s):  
Linda K Rushworth ◽  
Victoria Harle ◽  
Peter Repiscak ◽  
William Clark ◽  
Robin Shaw ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Elongation Factor ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Genome Wide ◽  
A Genome ◽  
Docetaxel Treatment ◽  
Crispr Screen ◽  
Altered Cell

Docetaxel chemotherapy in metastatic prostate cancer offers only a modest survival benefit because of emerging resistance. To identify candidate therapeutic gene targets, we applied a murine prostate cancer orthograft model that recapitulates clinical invasive prostate cancer in a genome-wide CRISPR/Cas9 screen under docetaxel treatment pressure. We identified 17 candidate genes whose suppression may enhance the efficacy of docetaxel, with transcription elongation factor A–like 1 (Tceal1) as the top candidate. TCEAL1 function is not fully characterised; it may modulate transcription in a promoter dependent fashion. Suppressed TCEAL1 expression in multiple human prostate cancer cell lines enhanced therapeutic response to docetaxel. Based on gene set enrichment analysis from transcriptomic data and flow cytometry, we confirmed that loss of TCEAL1 in combination with docetaxel leads to an altered cell cycle profile compared with docetaxel alone, with increased subG1 cell death and increased polyploidy. Here, we report the first in vivo genome-wide treatment sensitisation CRISPR screen in prostate cancer, and present proof of concept data on TCEAL1 as a candidate for a combinational strategy with the use of docetaxel.

scholarly journals Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Spencer Hill ◽  
Kurt Reichermeier ◽  
Daniel C Scott ◽  
Lorena Samentar ◽  
Jasmin Coulombe-Huntington ◽  
...  
Keyword(s):  
Chain Extension ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Extension Activity ◽  
The Stability ◽  
Genetic Buffering ◽  
Ligase Function ◽  
In Cells

The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.

scholarly journals PICALM modulates autophagy activity and tau accumulation

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Kevin Moreau ◽  
Angeleen Fleming ◽  
Sara Imarisio ◽  
Ana Lopez Ramirez ◽  
Jacob L. Mercer ◽  
...  
Keyword(s):  
Genetic Risk ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Transgenic Models ◽  
Autophagosome Formation ◽  
Genome Wide ◽  
Autophagy Pathway ◽  
Autophagy Activity

Abstract Genome-wide association studies have identified several loci associated with Alzheimer’s disease (AD), including proteins involved in endocytic trafficking such as PICALM/CALM (phosphatidylinositol binding clathrin assembly protein). It is unclear how these loci may contribute to AD pathology. Here we show that CALM modulates autophagy and alters clearance of tau, a protein which is a known autophagy substrate and which is causatively linked to AD, both in vitro and in vivo. Furthermore, altered CALM expression exacerbates tau-mediated toxicity in zebrafish transgenic models. CALM influences autophagy by regulating the endocytosis of SNAREs, such as VAMP2, VAMP3 and VAMP8, which have diverse effects on different stages of the autophagy pathway, from autophagosome formation to autophagosome degradation. This study suggests that the AD genetic risk factor CALM modulates autophagy, and this may affect disease in a number of ways including modulation of tau turnover.

Identification of putative markers of triclabendazole resistance by a genome-wide analysis of genetically recombinant Fasciola hepatica

Parasitology ◽  
2013 ◽  
Vol 140 (12) ◽  
pp. 1523-1533 ◽  
Author(s):  
J. HODGKINSON ◽  
K. CWIKLINSKI ◽  
N. J. BEESLEY ◽  
S. PATERSON ◽  
D. J. L. WILLIAMS
Keyword(s):  
Genetic Resources ◽  
Fasciola Hepatica ◽  
Genome Wide Analysis ◽  
Extensive Analysis ◽  
Genome Wide ◽  
A Genome ◽  
Genetic Mechanisms ◽  
Triclabendazole Resistance

SUMMARYDespite years of investigation into triclabendazole (TCBZ) resistance in Fasciola hepatica, the genetic mechanisms responsible remain unknown. Extensive analysis of multiple triclabendazole-susceptible and -resistant isolates using a combination of experimental in vivo and in vitro approaches has been carried out, yet few, if any, genes have been demonstrated experimentally to be associated with resistance phenotypes in the field. In this review we summarize the current understanding of TCBZ resistance from the approaches employed to date. We report the current genomic and genetic resources for F. hepatica that are available to facilitate novel functional genomics and genetic experiments for this parasite in the future. Finally, we describe our own non-biased approach to mapping the major genetic loci involved in conferring TCBZ resistance in F. hepatica.

scholarly journals A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress

2021 ◽  
Author(s):  
Hans M. Dalton ◽  
Raghuvir Viswanatha ◽  
Ricky Brathwaite ◽  
Jae Sophia Zuno ◽  
Stephanie E. Mohr ◽  
...  
Keyword(s):  
Er Stress ◽  
Therapeutic Targets ◽  
Therapeutic Benefit ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Fructose Metabolism ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

AbstractPartial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually cause CDGs. While both in vivo models ostensibly cause ER stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.

scholarly journals CAPN1 (Calpain1)-Mediated Impairment of Autophagic Flux Contributes to Cerebral Ischemia-Induced Neuronal Damage

Stroke ◽  
2021 ◽  
Author(s):  
Yueyang Liu ◽  
Xiaohang Che ◽  
Haotian Zhang ◽  
Xiaoxiao Fu ◽  
Yang Yao ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Kinase Inhibitor ◽  
Neuronal Damage ◽  
Neuronal Cell ◽  
In Vivo Model ◽  
Autophagic Flux ◽  
Autophagosome Formation

Background and Purpose: CAPN1 (calpain1)—an intracellular Ca 2+ -regulated cysteine protease—can be activated under cerebral ischemia. However, the mechanisms by which CAPN1 activation promotes cerebral ischemic injury are not defined. Methods: In the present study, we used adeno-associated virus-mediated genetic knockdown and pharmacological blockade (MDL-28170) of CAPN1 to investigate the role of CAPN1 in the regulation of the autophagy-lysosomal pathway and neuronal damage in 2 models, rat permanent middle cerebral occlusion in vivo model and oxygen-glucose–deprived primary neuron in vitro model. Results: CAPN1 was activated in the cortex of permanent middle cerebral occlusion–operated rats and oxygen-glucose deprivation–exposed neurons. Genetic and pharmacological inhibition of CAPN1 significantly attenuated ischemia-induced lysosomal membrane permeabilization and subsequent accumulation of autophagic substrates in vivo and in vitro. Moreover, inhibition of CAPN1 increased autophagosome formation by decreasing the cleavage of the autophagy regulators BECN1 (Beclin1) and ATG (autophagy-related gene) 5. Importantly, the neuron-protective effect of MDL-28170 on ischemic insult was reversed by cotreatment with either class III-PI3K (phosphatidylinositol 3-kinase) inhibitor 3-methyladenine or lysosomal inhibitor chloroquine (chloroquine), suggesting that CAPN1 activation-mediated impairment of autophagic flux is crucial for cerebral ischemia-induced neuronal damage. Conclusions: The present study demonstrates for the first time that ischemia-induced CAPN1 activation impairs lysosomal function and suppresses autophagosome formation, which contribute to the accumulation of substrates and aggravate the ischemia-induced neuronal cell damage. Our work highlights the vital role of CAPN1 in the regulation of cerebral ischemia–mediated autophagy-lysosomal pathway defects and neuronal damage.

Abstract 360: Loss of Endothelial CXCR7 Exacerbates Wire-injury Induced Neointimal Formation

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Huifeng Hao ◽  
Sheng Hu ◽  
Dawei Bu ◽  
Xiaogang Sun ◽  
Miao Wang
Keyword(s):  
Vascular Remodeling ◽  
Tamoxifen Treatment ◽  
Neointimal Formation ◽  
Genome Wide ◽  
Endothelial Repair ◽  
A Genome ◽  
Artery Disease

CXCR7 is a non-classical chemokine receptor for CXCL12, whose gene represents a genome-wide association locus for coronary artery disease. Global deletion of CXCR7 increased experimentally induced neointimal formation and atherosclerosis in hyperlipidemic mice, with evidence that CXCR7 modified cholesterol uptake to adipose tissue. We found that CXCR7 was expressed in endothelial cells of mouse neointima and human aortic lesions. To examine a role of endothelial CXCR7 in vascular remodeling, endothelial CXCR7 inducible knockout mice were studied for their vascular response to wire injury in femoral arteries. Tamoxifen treatment of mice harboring floxed CXCR7 and Cdh5 -promoter driven CreERT2 , essentially abolished endothelial CXCR7 expression in vitro and in vivo. Postnatal deletion of endothelial CXCR7 exacerbated neointimal formation on normalipidemic background, four weeks after injury. Mechanistically, this was attributable to attenuated endothelial repair following endothelial injury. Collectively, endothelial CXCR7 is a key regulator of vascular remodeling, independent of lipid traits.

scholarly journals The tumor suppressor MIR139 is silenced by POLR2M to promote AML oncogenesis

Leukemia ◽  
2021 ◽  
Author(s):  
Christiaan J. Stavast ◽  
Iris van Zuijen ◽  
Elena Karkoulia ◽  
Arman Özçelik ◽  
Antoinette van Hoven-Beijen ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Regulatory Factor ◽  
Molecular Control ◽  
Polycomb Repressive Complex 2 ◽  
Genome Wide ◽  
A Genome

AbstractMIR139 is a tumor suppressor and is commonly silenced in acute myeloid leukemia (AML). However, the tumor-suppressing activities of miR-139 and molecular mechanisms of MIR139-silencing remain largely unknown. Here, we studied the poorly prognostic MLL-AF9 fusion protein-expressing AML. We show that MLL-AF9 expression in hematopoietic precursors caused epigenetic silencing of MIR139, whereas overexpression of MIR139 inhibited in vitro and in vivo AML outgrowth. We identified novel miR-139 targets that mediate the tumor-suppressing activities of miR-139 in MLL-AF9 AML. We revealed that two enhancer regions control MIR139 expression and found that the polycomb repressive complex 2 (PRC2) downstream of MLL-AF9 epigenetically silenced MIR139 in AML. Finally, a genome-wide CRISPR-Cas9 knockout screen revealed RNA Polymerase 2 Subunit M (POLR2M) as a novel MIR139-regulatory factor. Our findings elucidate the molecular control of tumor suppressor MIR139 and reveal a role for POLR2M in the MIR139-silencing mechanism, downstream of MLL-AF9 and PRC2 in AML. In addition, we confirmed these findings in human AML cell lines with different oncogenic aberrations, suggesting that this is a more common oncogenic mechanism in AML. Our results may pave the way for new targeted therapy in AML.

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