Dejesus, Rowena, Moretti, Francesca, Mcallister, Gregory, Wang, Zuncai, Bergman, Phil, Liu, Shanming, Frias, Elizabeth, Alford, John, Reece-Hoyes, John, Lindeman, Alicia, Kelliher, Jennifer, Russ, Carsten, Knehr, Judith, Carbone, Walter, Beibel, Martin, Roma, Guglielmo, Aylwin, Ng, Ramnik, Xavier, Mickanin, Craig, Murphy, Leon, Hoffman, Gregory and Nyfeler, Beat (2016) Functional CRISPR screening identifies the protein ufmylation pathway as a regulator of SQSTM1/p62. eLife, 5 (NA). NA-NA. ISSN 2050-084X

Abstract

SQSTM1 (also known as p62) integrates multiple cellular signaling pathways and is tightly regulated at the transcriptional as well as post-translational level. To identify cellular pathways and novel modulators of SQSTM1, we developed a forward genetic screening paradigm exploiting CRISPR-Cas9-mediated genome editing coupled to cell selection by fluorescence activated cell sorting (FACS). Through the systematic comparison of pooled libraries, we show that CRISPR outperforms RNA interference (RNAi) in identifying known modulators of SQSTM1. A genome-wide CRISPR screen exposed MTOR complex 1 and macroautophagy as key regulators of SQSTM1 and identified several novel candidates including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. Using autophagy flux and gene expression assays, we show that protein ufmylation regulates SQSTM1 in an autophagy-independent manner by eliciting a cell type-specific ER stress response inducing SQSTM1 expression. In summary, this study validates pooled CRISPR screening as powerful methodology to map cellular pathways using a FACS-based readout and provides a resource of candidates to expand our understanding of the regulation of SQSTM1.

Item Type:	Article
Date Deposited:	16 Aug 2016 00:45
Last Modified:	16 Aug 2016 00:45
URI:	https://oak.novartis.com/id/eprint/29114