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

DeJesus, Rowena; Moretti, Francesca; McAllister, Gregory; Wang, Zuncai; Bergman, Philip S.; Liu, Shanming; Frias, Elizabeth; Alford, John; Reece‐Hoyes, John S.; Lindeman, Alicia; Kelliher, Jennifer; Russ, Carsten; Knehr, Judith; Carbone, Walter; Beibel, Martin; Roma, Guglielmo; Ng, Aylwin; Tallarico, John A.; Porter, Jeffery A.; Xavier, Ramnik J.; Mickanin, Craig; Murphy, Leon O.; Hoffman, Gregory R.; Nyfeler, Beat

doi:10.7554/elife.17290

Cited by 134 publications

(172 citation statements)

References 47 publications

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“…H4 Cas9 GFP-NCOA4 cells stably express Cas9 and the GFP-NCOA4 fusion protein and treatment with the v-ATPase inhibitor Bafilomycin A1 (BafA1) confirmed the lysosomal trafficking of the reporter (Figure 1B). The CRISPR-based screening paradigm has been previously applied to GFP-p62 and comprehensively mapped the machinery of mammalian autophagy, thereby validating the approach (DeJesus et al, 2016). We identified numerous components of the HOPS, GARP and ESCRT complexes as specific regulators of NCOA4 (Figure 1D), revealing an unexpected role for vacuolar protein sorting (VPS) components in ferritin turnover.…”

Section: Resultsmentioning

confidence: 99%

Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9

Goodwin¹,

Dowdle²,

DeJesus³

et al. 2017

Cell Reports

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149

126

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SUMMARY Iron is vital for many homeostatic processes and its liberation from ferritin nanocages occurs in the lysosome. Studies indicate that ferritin and its binding partner nuclear receptor coactivator-4 (NCOA4) are targeted to lysosomes by a form of selective autophagy. By using genome-scale functional screening we identify an alternative lysosomal transport pathway for ferritin that requires FIP200, ATG9A, VPS34 and TAX1BP1 but lacks involvement of the ATG8 lipidation machinery that constitutes classical macroautophagy. TAX1BP1 binds directly to NCOA4 and is required for lysosomal trafficking of ferritin under basal and iron depleted conditions. Under basal conditions ULK1/2-FIP200 controls ferritin turnover but its deletion leads to TAX1BP1-dependent activation of TBK1 which regulates redistribution of ATG9A to the Golgi enabling continued trafficking of ferritin. Cells expressing an Amyotrophic Lateral Sclerosis (ALS)-associated TBK1 allele are incapable of degrading ferritin suggesting a novel molecular mechanism that explains the presence of iron deposits in patient brain biopsies.

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Section: Resultsmentioning

confidence: 99%

Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9

Goodwin¹,

Dowdle²,

DeJesus³

et al. 2017

Cell Reports

Self Cite

149

126

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show abstract

“…Individual sgRNA constructs were cloned into pNGx_LV_g003 [27] with unique sequences corresponding to the target: (sgRNA-OGT_v1), (sgRNA-OGT_v2), (sgRNA-OGT_v3) and (sgRNA-CTRL).…”

Section: Methodsmentioning

confidence: 99%

Multidimensional pooled shRNA screens in human THP-1 cells identify candidate modulators of macrophage polarization

et al. 2017

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Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the ‘classical’ (M1) and ‘alternative’ (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.

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“…Similarly, a pooled approach can be used to find genes that either enhance or mitigate the effect of a selective pressure or stimuli (e.g., rescue from virus-induced cell death[13–16]). One can also use strategies that employ cell sorting to identify a desired phenotype from pooled format (e.g., gain or loss of a reporter protein)[17–19]. However, there are many assays that are not amenable to pooled approaches, including a variety of high-content assays.…”

Section: Introductionmentioning

confidence: 99%

Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

Tan¹,

Martin²

2016

PLoS ONE

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To date, lentiviral-based CRISPR-Cas9 screens have largely been conducted in pooled format. However, numerous assays are not amenable to pooled approaches, and lentiviral screening in arrayed format presents many challenges. We sought to examine synthetic CRISPR reagents in the context of arrayed screening. Experiments were performed using aberrant DNA replication as an assay. Using synthetic CRISPR RNAs targeting the known control gene GMNN in HCT-116 cells stably expressing Cas9, we observed statistically significant phenotype among the majority of transfected cells within 72 hours. Additional studies revealed near complete loss of GMNN protein and editing of GMNN DNA. We next conducted a screen of synthetic CRISPR RNAs directed against 640 ubiquitin-related genes. Screening identified known and novel DNA replication regulators that were also supported by siRNA gene knockdown. Notably, CRISPR screening identified more statistically significant hits than corresponding siRNA screens run in parallel. These results highlight the possibility of using synthetic CRISPR reagents as an arrayed screening tool.

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

Cited by 134 publications

References 47 publications

Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9

Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9

Multidimensional pooled shRNA screens in human THP-1 cells identify candidate modulators of macrophage polarization

Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening

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