eLife (Jun 2016)
Functional CRISPR screening identifies the ufmylation pathway as a regulator of SQSTM1/p62
- Rowena DeJesus,
- Francesca Moretti,
- Gregory McAllister,
- Zuncai Wang,
- Phil Bergman,
- Shanming Liu,
- Elizabeth Frias,
- John Alford,
- John S Reece-Hoyes,
- Alicia Lindeman,
- Jennifer Kelliher,
- Carsten Russ,
- Judith Knehr,
- Walter Carbone,
- Martin Beibel,
- Guglielmo Roma,
- Aylwin Ng,
- John A Tallarico,
- Jeffery A Porter,
- Ramnik J Xavier,
- Craig Mickanin,
- Leon O Murphy,
- Gregory R Hoffman,
- Beat Nyfeler
Affiliations
- Rowena DeJesus
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Francesca Moretti
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland
- Gregory McAllister
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Zuncai Wang
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Phil Bergman
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Shanming Liu
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Elizabeth Frias
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- John Alford
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- John S Reece-Hoyes
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Alicia Lindeman
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Jennifer Kelliher
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Carsten Russ
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Judith Knehr
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland
- Walter Carbone
- ORCiD
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland
- Martin Beibel
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland
- Guglielmo Roma
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland
- Aylwin Ng
- Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, United States
- John A Tallarico
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Jeffery A Porter
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Ramnik J Xavier
- Gastrointestinal Unit and Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, Harvard Medical School, Boston, United States
- Craig Mickanin
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Leon O Murphy
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Gregory R Hoffman
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, United States
- Beat Nyfeler
- ORCiD
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland
- DOI
- https://doi.org/10.7554/eLife.17290
- Journal volume & issue
-
Vol. 5
Abstract
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.
Keywords
