Author response: Genome-wide CRISPR screen identifies noncanonical NF-κB signaling as a regulator of density-dependent proliferation

Epithelial cells possess intrinsic mechanisms to maintain an appropriate cell density for normal tissue morphogenesis and homeostasis. Defects in such mechanisms likely contribute to hyperplasia and cancer initiation. To identify genes that regulate the density-dependent proliferation of murine mammary epithelial cells, we developed a fluorescence-activated cell sorting assay based on fluorescence ubiquitination cell cycle indicator, which marks different stages of the cell cycle with distinct fluorophores. Using this powerful assay, we performed a genome-wide CRISPR/Cas9 knockout screen, selecting for cells that proliferate normally at low density but continue to divide at high density. Unexpectedly, one top hit was Traf3, a negative regulator of NF-κB signaling that has never previously been linked to density-dependent proliferation. We demonstrate that loss of Traf3 specifically activates noncanonical NF-κB signaling. This in turn triggers an innate immune response and drives cell division independently of known density-dependent proliferation mechanisms, including YAP/TAZ signaling and cyclin-dependent kinase inhibitors, by blocking entry into quiescence.

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Decision letter: Genome-wide CRISPR screen identifies noncanonical NF-κB signaling as a regulator of density-dependent proliferation

10.7554/elife.63603.sa1 ◽

2020 ◽

Keyword(s):

Density Dependent ◽

Genome Wide ◽

Crispr Screen

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Genome-Wide CRISPR Screen Identifies Non-Canonical NF-κB Signaling as a Potent Regulator of Density-dependent Proliferation

10.1101/2020.10.02.324301 ◽

2020 ◽

Author(s):

Maria Fomicheva ◽

Ian G. Macara

Keyword(s):

Epithelial Cells ◽

Kinase Inhibitors ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Negative Regulator ◽

Density Dependent ◽

Genome Wide ◽

A Genome ◽

Cancer Initiation ◽

Crispr Screen

ABSTRACTEpithelial cells possess intrinsic mechanisms to maintain an appropriate cell density for normal tissue morphogenesis and homeostasis. Defects in such mechanisms likely contribute to hyperplasia and cancer initiation. To identify genes that regulate the density-dependent proliferation of murine mammary epithelial cells, we developed a fluorescence-activated cell sorting assay based on FUCCI, which marks different stages of the cell cycle with distinct fluorophores. Using this powerful assay, we performed a genome-wide CRISPR/Cas9 knockout screen, selecting for cells that proliferate normally at low density but continue to divide at high density. Unexpectedly, one top hit was Traf3, a negative regulator of NF-κB signaling that has never previously been linked to density-dependent proliferation. We demonstrate that loss of Traf3 specifically activates non-canonical NF-κB signaling. This in turn triggers an innate immune response and drives cell division independently of known density-dependent proliferation mechanisms, including YAP/TAZ signaling and cyclin kinase inhibitors, by blocking entry into quiescence.

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A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection

Cell Reports ◽

10.1016/j.celrep.2021.108859 ◽

2021 ◽

Vol 34 (11) ◽

pp. 108859

Author(s):

Jessie Kulsuptrakul ◽

Ruofan Wang ◽

Nathan L. Meyers ◽

Melanie Ott ◽

Andreas S. Puschnik

Keyword(s):

Virus Infection ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Host Factors ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

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A Genome-wide CRISPR Screen Identifies CDC25A as a Determinant of Sensitivity to ATR Inhibitors

Molecular Cell ◽

10.1016/j.molcel.2016.03.006 ◽

2016 ◽

Vol 62 (2) ◽

pp. 307-313 ◽

Cited By ~ 87

Author(s):

Sergio Ruiz ◽

Cristina Mayor-Ruiz ◽

Vanesa Lafarga ◽

Matilde Murga ◽

Maria Vega-Sendino ◽

...

Keyword(s):

Genome Wide ◽

A Genome ◽

Crispr Screen

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Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62

eLife ◽

10.7554/elife.17290 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 78

Author(s):

Rowena DeJesus ◽

Francesca Moretti ◽

Gregory McAllister ◽

Zuncai Wang ◽

Phil Bergman ◽

...

Keyword(s):

Adaptor Protein ◽

Er Stress Response ◽

Genome Wide ◽

A Genome ◽

Crispr Screen ◽

Selection Step ◽

A Cell ◽

Cell Type Specific ◽

Cellular Pathways ◽

Mtor Signalling

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.

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Author response: Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

10.7554/elife.60498.sa2 ◽

2020 ◽

Author(s):

Hajnalka L Pálinkás ◽

Angéla Békési ◽

Gergely Róna ◽

Lőrinc Pongor ◽

Gábor Papp ◽

...

Keyword(s):

Distribution Patterns ◽

Author Response ◽

Human Dna ◽

Genome Wide

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Genome-wide CRISPR screen identifies regulators of MAPK and MTOR pathways mediating sorafenib resistance in acute myeloid leukemia

Haematologica ◽

10.3324/haematol.2020.257964 ◽

2020 ◽

Author(s):

Alisa Damnernsawad ◽

Daniel Bottomly ◽

Stephen E. Kurtz ◽

Christopher A. Eide ◽

Shannon K. McWeeney ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Ex Vivo ◽

Mek Inhibitors ◽

Genome Wide ◽

A Genome ◽

Flt3 Mutations ◽

Flt3 Inhibitors ◽

Crispr Screen ◽

Acute Myeloid

Drug resistance impedes the long-term effect of targeted therapies in acute myeloid leukemia (AML), necessitating the identification of mechanisms underlying resistance. Approximately 25% of AML patients carry FLT3 mutations and develop post-treatment insensitivity to FLT3 inhibitors, including sorafenib. Using a genome-wide CRISPR screen, we identified LZTR1, NF1, TSC1 or TSC2, negative regulators of the MAPK and MTOR pathways, as mediators of sorafenib resistance. Analyses of ex vivo drug sensitivity assays in FLT3-ITD AML patient samples revealed lower expression of LZTR1, NF1, and TSC2 correlated with sorafenib sensitivity. Importantly, MAPK and/or MTOR complex1 (MTORC1) activity were upregulated in AML cells made resistant to several FLT3 inhibitors, including crenolanib, quizartinib, or sorafenib. These cells were sensitive to MEK inhibitors, and the combination of FLT3 and MEK inhibitors showed enhanced efficacy, suggesting its effectiveness in AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors.

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