A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

2021 ◽  
Author(s):  
María Mariner-Faulí
Keyword(s):  
Epigenetic Silencing ◽  
Knock Out ◽  
Cell State ◽  
Genome Wide ◽  
A Genome ◽  
Germline Genes

scholarly journals A genome-wide knock-out screen for actors of epigenetic silencing reveals new regulators of germline genes and 2-cell like cell state

2021 ◽  
Author(s):  
Nikhil Gupta ◽  
Lounis Yakhou ◽  
Julien Richard Albert ◽  
Fumihito Miura ◽  
Laure Ferry ◽  
...  
Keyword(s):  
Es Cells ◽  
Epigenetic Mechanisms ◽  
Mammalian Development ◽  
Reporter System ◽  
Transcriptional Responses ◽  
Knock Out ◽  
Mouse Es Cells ◽  
Genome Wide ◽  
A Genome ◽  
Germline Genes

Epigenetic mechanisms are essential to establish and safeguard cellular identities in mammals. They dynamically regulate the expression of genes, transposable elements, and higher-order chromatin structures. Expectedly, these chromatin marks are indispensable for mammalian development and alterations often lead to diseases such as cancer. Molecularly, epigenetic mechanisms rely on factors to establish patterns, interpret them into a transcriptional output, and maintain them across cell divisions. A global picture of these phenomena has started to emerge over the years, yet many of the molecular actors remain to be discovered. In this context, we have developed a reporter system sensitive to epigenetic perturbations to report on repressive pathways based on Dazl, which is normally repressed in mouse ES cells. We used this system for a genome-wide CRISPR knock-out screen, which yielded expected hits (DNMT1, UHRF1, MGA), as well as novel candidates. We prioritized the candidates by secondary screens, and led further experiments on 6 of them: ZBTB14, KDM5C, SPOP, MCM3AP, BEND3, and KMT2D. Our results show that all 6 candidates regulate the expression of germline genes. In addition, we find that removal of ZBTB14, KDM5C, SPOP and MCM3AP led to similar transcriptional responses, including a reactivation of the 2-cell like cell (2CLC) signature. Therefore, our genetic screen has identified new regulators of key cellular states.

BIOM-32. ENDOPLASMIC RETICULUM PROTEIN SSR3 DETERMINES AND PREDICTS RESPONSE TO PACLITAXEL IN BREAST CANCER AND GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi17-vi18
Author(s):  
Crismita Dmello ◽  
Aarón Sonabend ◽  
Víctor Arrieta ◽  
Daniel Zhang ◽  
Deepak Kanojia ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Endoplasmic Reticulum ◽  
Glioma Cells ◽  
Predictive Biomarkers ◽  
Predictive Biomarker ◽  
Precision Oncology ◽  
Patient Response ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome

Abstract Paclitaxel (PTX) is one the most potent and commonly used chemotherapies for breast and pancreatic cancer. Given the potency of this drug for glioblastomas (GBM) several ongoing clinical trials are investigating means of enhancing delivery of PTX across the blood-brain barrier for this disease. In spite of the efficacy of PTX, individual tumors exhibit variable susceptibility to this drug, with response rate in the range of 30%-60%. To identify predictive biomarkers for response to PTX, we performed a genome-wide CRISPR knock-out screen using human glioma cells. The most enriched genes in the CRISPR screen underwent further selection based on their correlation with survival in the breast cancer patient cohorts treated with PTX and not in patients treated with other chemotherapies, a finding that was validated on a second independent patient cohort. This led to the discovery of endoplasmic reticulum (ER) protein SSR3 as a putative predictive biomarker for PTX. SSR3 protein levels showed positive correlation with response to PTX in breast cancer cells, glioma cells, in multiple intracranial glioma xenografts and in GBM patient derived explant cultures. Knockout of SSR3 turned the cells resistant to PTX while its overexpression sensitized the cells to PTX. In gliomas, SSR3-mediated susceptibility to PTX relates to modulation of phosphorylation of ER stress sensor IRE1α. Thus, by using genome-wide screen combined with patient response data, we discovered a biomarker that demonstrates causal and correlative relationship with response to PTX in breast cancer and GBM. Prospective validation of this biomarker is warranted for its broad implementation for precision oncology.

scholarly journals Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma

2019 ◽  
Vol 294 (25) ◽  
pp. 9734-9745 ◽  
Author(s):  
Chong Wang ◽  
Sizun Jiang ◽  
Liangru Ke ◽  
Luyao Zhang ◽  
Difei Li ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Advanced Disease ◽  
Gene Knockout ◽  
Histone Acetyl Transferase ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome ◽  
Cellular Factors ◽  
New Treatments ◽  
Specific Symptoms

Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients respond poorly to treatment. New treatments are therefore needed to improve the outcome of NPC. To better understand the molecular pathogenesis of NPC, here we used an NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways essential for NPC (i.e. dependence factors). This screen identified the Moz, Ybf2/Sas3, Sas2, Tip60 histone acetyl transferase complex, NF-κB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene as NPC dependence factors/pathways. Using gene knock out, complementary DNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.

scholarly journals A genome-wide CRISPR/Cas phenotypic screen for modulators of DUX4 cytotoxicity reveals screen complications

2020 ◽  
Author(s):  
Ator Ashoti ◽  
Francesco Limone ◽  
Melissa van Kranenburg ◽  
Anna Alemany ◽  
Mirna Baak ◽  
...  
Keyword(s):  
Treatment Options ◽  
Underlying Mechanism ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Knock Out ◽  
Genome Wide ◽  
Phenotypic Screen ◽  
A Genome ◽  
The Face ◽  
Muscle Disorder ◽  
Pioneer Transcription Factor

AbstractFacioscapulohumeral muscular dystrophy (FHSD), a fundamentally complex muscle disorder that thus far remains untreatable. As the name implies, FSHD starts in the muscles of the face and shoulder gridle. The main perturbator of the disease is the pioneer transcription factor DUX4, which is misexpressed in affected tissues due to a failure in epigenetic repressive mechanisms. In pursuit of unraveling the underlying mechanism of FSHD and finding potential therapeutic targets or treatment options, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of DUX4 cytotoxicity. We found no key effectors other than DUX4 itself, suggesting treatment efforts in FSHD should be directed towards its direct modulation.The screen did however reveal some rare and unexpected Cas9-induced genomic events, that may provide important considerations for planning future CRISPR/Cas9 knock-out screens.

scholarly journals Morc3 silences endogenous retroviruses by enabling Daxx-mediated H3.3 incorporation

2020 ◽  
Author(s):  
Sophia Groh ◽  
Anna Viktoria Milton ◽  
Lisa Marinelli ◽  
Cara V. Sickinger ◽  
Heike Bollig ◽  
...  
Keyword(s):  
Binding Sites ◽  
Atp Hydrolysis ◽  
Chromatin Accessibility ◽  
Endogenous Retroviruses ◽  
Chromatin Modifications ◽  
Interaction Domain ◽  
Knock Out ◽  
Genome Wide ◽  
A Genome ◽  
Mammalian Genomes

ABSTRACTEndogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although, specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1 mediated H3K9me3 and DNA methylation. However, the protein network which regulates deposition of these chromatin modifications is still incompletely understood. Here, we performed a genome-wide sgRNA screen for genes involved in ERV silencing and identified the GHKL ATPase protein Morc3 as top scoring hit. Morc3 knock-out cells display de-repression, reduced H3K9me3 and increased chromatin accessibility of distinct ERV classes. We found that the GHKL ATPase domain of Morc3 is critical for ERV silencing, since mutants which cannot bind ATP, or which are defective in ATP hydrolysis cannot rescue the Morc3 ko phenotype. Proteomic analysis revealed that Morc3 mutant protein which cannot bind ATP fails to interact with the H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation, as Daxx lacking the SUMO interaction domain shows reduced association with Morc3. Notably, in Morc3 ko cells, we observed strongly reduced H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as critical regulator of Daxx-mediated H3.3 incorporation to ERV regions.

scholarly journals New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast

2011 ◽  
Vol 286 (12) ◽  
pp. 10744-10754 ◽  
Author(s):  
D. Christian Martin ◽  
Hyemin Kim ◽  
Nancy A. Mackin ◽  
Lymarie Maldonado-Báez ◽  
Carlos C. Evangelista ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Membrane Trafficking ◽  
Knock Out ◽  
Voltage Gated ◽  
High Affinity ◽  
Voltage Gated Calcium Channels ◽  
Genome Wide ◽  
A Genome ◽  
Target Membrane ◽  
Α Subunits

The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca2+ influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca2+ after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis.

A Genome-Wide Search Identifies Epigenetic Silencing of Somatostatin, Tachykinin-1, and 5 Other Genes in Colon Cancer

2006 ◽  
Vol 131 (3) ◽  
pp. 797-808 ◽  
Author(s):  
Yuriko Mori ◽  
Kun Cai ◽  
Yulan Cheng ◽  
Suna Wang ◽  
Bogdan Paun ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Epigenetic Silencing ◽  
Genome Wide ◽  
A Genome ◽  
Genome Wide Search

Escherichia coli glycogen metabolism is controlled by the PhoP-PhoQ regulatory system at submillimolar environmental Mg2+ concentrations, and is highly interconnected with a wide variety of cellular processes

2009 ◽  
Vol 424 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Manuel Montero ◽  
Gustavo Eydallin ◽  
Alejandro M. Viale ◽  
Goizeder Almagro ◽  
Francisco J. Muñoz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Responses ◽  
Regulatory System ◽  
Glycogen Metabolism ◽  
Metabolic Model ◽  
Knock Out ◽  
Cellular Processes ◽  
Genome Wide ◽  
A Genome ◽  
Keio Collection

Using the Keio collection of gene-disrupted mutants of Escherichia coli, we have recently carried out a genome-wide screening of the genes affecting glycogen metabolism. Among the mutants identified in the study, ΔmgtA, ΔphoP and ΔphoQ cells, all lacking genes that are induced under low extracellular Mg2+ conditions, displayed glycogen-deficient phenotypes. In this work we show that these mutants accumulated normal glycogen levels when the culture medium was supplemented with submillimolar Mg2+ concentrations. Expression analyses conducted in wild-type, ΔphoP and ΔphoQ cells showed that the glgCAP operon is under PhoP-PhoQ control in the submillimolar Mg2+ concentration range. Subsequent screening of the Keio collection under non-limiting Mg2+ allowed the identification of 183 knock-out mutants with altered glycogen levels. The stringent and general stress responses, end-turnover of tRNA, intracellular AMP levels, and metabolism of amino acids, iron, carbon and sulfur were major determinants of glycogen levels. glgC::lacZY expression analyses using mutants representing different functional categories revealed that the glgCAP operon belongs to the RelA regulon. We propose an integrated metabolic model wherein glycogen metabolism is (a) tightly controlled by the energy and nutritional status of the cell and (b) finely regulated by changes in environmental Mg2+ occurring at the submillimolar concentration range.

scholarly journals Morc3 silences endogenous retroviruses by enabling Daxx-mediated histone H3.3 incorporation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sophia Groh ◽  
Anna Viktoria Milton ◽  
Lisa Katherina Marinelli ◽  
Cara V. Sickinger ◽  
Angela Russo ◽  
...  
Keyword(s):  
Chromatin Accessibility ◽  
Endogenous Retroviruses ◽  
Chromatin Modifications ◽  
Knock Out ◽  
Mutant Proteins ◽  
Guide Rna ◽  
Histone H3.3 ◽  
Genome Wide ◽  
A Genome ◽  
Mammalian Genomes

AbstractEndogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1-mediated H3K9me3 and DNA methylation. However, the protein network which regulates the deposition of these chromatin modifications is still incompletely understood. Here, we perform a genome-wide single guide RNA (sgRNA) screen for genes involved in ERV silencing and identify the GHKL ATPase protein Morc3 as a top-scoring hit. Morc3 knock-out (ko) cells display de-repression, reduced H3K9me3, and increased chromatin accessibility of distinct ERV families. We find that the Morc3 ATPase cycle and Morc3 SUMOylation are important for ERV chromatin regulation. Proteomic analyses reveal that Morc3 mutant proteins fail to interact with the histone H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation and Daxx SUMO binding. Notably, in Morc3 ko cells, we observe strongly reduced histone H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as a critical regulator of Daxx-mediated histone H3.3 incorporation to ERV regions.

scholarly journals A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors

2021 ◽  
Vol 12 ◽  
Author(s):  
Klara Klein ◽  
Angelique Hölzemer ◽  
Tim Wang ◽  
Tae-Eun Kim ◽  
Haley L. Dugan ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Cell Lines ◽  
Nk Cell ◽  
Killer Cell ◽  
Heparan Sulfate Proteoglycans ◽  
Knock Out ◽  
Cell Receptors ◽  
Nk Cell Receptors ◽  
Genome Wide ◽  
A Genome

While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed via surface plasmon resonance, and removal of heparan sulfate proteoglycans from cell surfaces abolished KIR3DS1-Fc binding. Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain (KIR3DS1, KIR3DL1, KIR3DL2, KIR2DL4, and KIR2DL5) bound to heparan sulfate, while those without a D0 domain (KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4) did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition.

Sign in / Sign up

Export Citation Format

Share Document