Genome-wide CRISPR screen for PARKIN regulators PNAS PLUS reveals transcriptional repression as a determinant of mitophagy Christoph Pottinga, Christophe Crochemorea, Francesca Morettia, Florian Nigscha, Isabel Schmidta, Carole Mannevillea, Walter Carbonea, Judith Knehra, Rowena DeJesusb, Alicia Lindemanb, Rob Maherb, Carsten Russb, Gregory McAllisterb, John S. Reece-Hoyesb, Gregory R. Hoffmanb, Guglielmo Romaa, Matthias Müllera, Andreas W. Sailera, and Stephen B. Helliwella,1 aNovartis Institutes for BioMedical Research, Basel CH 4002, Switzerland; and bNovartis Institutes for BioMedical Research, Cambridge, MA 02139 Edited by Beth Levine, The University of Texas Southwestern, Dallas, TX, and approved November 29, 2017 (received for review June 20, 2017) PARKIN, an E3 ligase mutated in familial Parkinson’s disease, promotes how these proteins are themselves regulated, is important for mitophagy by ubiquitinating mitochondrial proteins for efficient the further understanding of mitophagy and the pathogenesis engagement of the autophagy machinery. Specifically, PARKIN- of Parkinson’s disease. synthesized ubiquitin chains represent targets for the PINK1 kinase Previous studies identified regulators of PINK1/PARKIN- generating phosphoS65-ubiquitin (pUb), which constitutes the mitoph- mediated mitophagy employing RNAi screens with damage- agy signal. Physiological regulation of PARKIN abundance, however, induced mitochondrial translocation of overexpressed GFP- and the impact on pUb accumulation are poorly understood. Using PARKIN as a mitophagy proxy (12–15). These efforts pro- cells designed to discover physiological regulators of PARKIN abun- foundly advanced the understanding of mitophagy regulation; dance, we performed a pooled genome-wide CRISPR/Cas9 knockout however, little is known about how cells set the threshold for screen. Testing identified genes individually resulted in a list of mitophagy to proceed. In this regard, cellular regulation of 53 positive and negative regulators. A transcriptional repressor net- PARKIN abundance is of particular interest as it may represent work including THAP11 was identified and negatively regulates en- a mechanism to tune the progression of mitophagy by impacting CELL BIOLOGY dogenous PARKIN abundance. RNAseq analysis revealed the pUb accumulation to adapt to physiological state changes. PARKIN-encoding locus as a prime THAP11 target, and THAP11 CRISPR The CRISPR/Cas9 gene-editing technology as a screening knockout in multiple cell types enhanced pUb accumulation. Thus, our tool appears to be superior to RNAi in most cases of lethality screens work demonstrates the critical role of PARKIN abundance, identifies (16–18) and in phenotypic screens (19). Using cells expressing a regulating genes, and reveals a link between transcriptional repres- PARKIN reporter protein from the endogenous PARK2 pro- sion and mitophagy, which is also apparent in human induced plurip- moter and in which steady state PARKIN levels dictate the otent stem cell-derived neurons, a disease-relevant cell type. kinetics of pUb accumulation, a phenotypic genome-wide CRISPR/Cas9 pooled screen was performed and resulted in a PARKIN | mitophagy | phosphoubiquitin | THAP11 | genome-wide screen list of 53 positive and negative regulators. We show that transcriptional ysfunction of mitochondria is implicated in aging and hu- Significance Dman disease, including Parkinson’s disease. Mitochondrial functionality is assured by a hierarchical system of interdepen- In mitophagy, damaged mitochondria are targeted for disposal dent cellular quality-control mechanisms acting at molecular or by the autophagy machinery. PARKIN promotes signaling of organellar levels to allow rapid adaptation to mitochondrial stress PARK2 mitochondrial damage to the autophagy machinery for en- and damage (1). The E3 ligase PARKIN (encoded by the gagement, and PARKIN mutations cause Parkinson’s disease, gene) takes on the role of a major sentinel that integrates multiple possibly because damaged mitochondria accumulate in neu- quality-control mechanisms ranging from facilitating proteasomal rons. Because regulation of PARKIN abundance and the impact degradation of mitochondrial proteins to suppressing mitochon- on signaling are poorly understood, we performed a genetic drial antigen presentation (2, 3). screen to identify PARKIN abundance regulators. Both positive ’ PARKIN s best-characterized role in mitochondrial quality and negative regulators were identified and will help us to control is in PINK1/PARKIN-mediated mitophagy where PARKIN further understand mitophagy and Parkinson’s disease. We acts in concert with the PINK1 kinase in the signaling of mitochon- show that some of the identified genes negatively regulate drial damage to the autophagy machinery. Herein, PINK1 accumu- PARKIN gene expression, which impacts signaling of mito- lates on the surface of dysfunctional mitochondria and recruits and chondrial damage in mitophagy. This link between transcrip- activates cytosolic PARKIN. Preformed ubiquitin chains on multiple tional repression and mitophagy is also apparent in neurons in mitochondrial surface proteins are extended by activated PARKIN culture, bearing implications for disease. and S65-phosphorylated by PINK1 (4–7). The accumulation of S65- phosphorylated ubiquitin (pUb) on mitochondria constitutes the Author contributions: C.P. and S.B.H. designed research; C.P., C.C., F.M., F.N., I.S., C.M., signaling mechanism to engage the autophagy machinery for selective W.C., J.K., R.D., A.L., and R.M. performed research; C.P. and F.N. contributed new re- agents/analytic tools; C.P., C.C., F.M., F.N., W.C., J.K., C.R., G.M., J.S.R.-H., G.R.H., G.R., clearance of dysfunctional mitochondria (8). The efficiency of M.M., A.W.S., and S.B.H. analyzed data; and C.P. and S.B.H. wrote the paper. mitophagy appears to depend on the pUb signal: while PINK1 Conflict of interest statement: All authors are employees of, and may own shares of, generated pUb from preformed chains in the absence of PARKIN Novartis Pharma AG. can induce some mitophagy, the presence of PARKIN amplifies the This article is a PNAS Direct Submission. accumulation of pUb via a feed-forward mechanism and enhances This open access article is distributed under Creative Commons Attribution-NonCommercial- mitophagy (8, 9). Mutations in both PINK1 and PARKIN are a cause NoDerivatives License 4.0 (CC BY-NC-ND). of familial Parkinson’s disease, suggesting that compromised 1To whom correspondence should be addressed. Email: [email protected]. mitophagy is an underlying feature (10, 11). Thus, the thorough This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. understanding of how these proteins regulate mitophagy, and 1073/pnas.1711023115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1711023115 PNAS Early Edition | 1of10 Downloaded by guest on September 28, 2021 repression negatively regulates endogenous PARKIN steady-state contains enhancer and repressor sites for physiological control of level. In particular, THAP11 depletion affects both PARKIN pro- expression (20) (Fig. S1B). We selected a clone showing the lowest tein levels and pUb accumulation in multiple cell types. Finally, discrete GFP-signal in flow cytometry (endoGFP-PARKIN cells) human induced pluripotent stem cell (iPSC)-derived inducible (Fig. 1A). The fluorescence signal is below the detection threshold Neurogenin 2 (iNGN2) neurons in which THAP11 was targeted of conventional fluorescence microscopy, but is GFP-PARKIN– by CRISPR/Cas9 display de-repression of PARK2 transcription dependent because PARKIN-directed single guide RNAs (sgRNAs) and enhanced pUb accumulation, demonstrating the impact of resulted in the concomitant loss of GFP-PARKIN protein and GFP- PARKIN-level regulation in a relevant cell type. fluorescence (Fig. 1 B and C). While endogenous PARKIN protein levels in the parental cells were near the detection threshold, GFP- Results PARKIN was readily detectable, likely reflecting multiple transgene PARKIN Levels Dictate Kinetics of pUb Accumulation. To assess the insertions (Fig. 1D). effects of cellular PARKIN abundance on downstream processes, We then examined the downstream effects of PARKIN-level we generated cellular models with different levels of PARKIN ex- alterations by assessing the accumulation of pUb upon mito- pression. HEK293-based JumpIN TI 293 cells expressing endoge- chondrial damage in parental and endoGFP-PARKIN cells, nous PARKIN were infected with lentiviruses for stable integration additionally infected with either a PARK2-specific or a control of Cas9 to enable gene editing and PARKIN depletion (parental [scrambled (SCR)] sgRNA. After treatment of parental cells cells) (Fig. S1A). Next, we aimed at generating cells with mildly transduced with control viruses with the mitochondria-damaging elevated PARKIN expression additionally amenable to functional agent carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for screening for physiological regulators of PARKIN. Thus, parental 6h,α-pUb signals appeared that were absent in DMSO-treated cells were transfected with a transgene encoding GFP-PARKIN samples (Fig. 1E), indicating the initiation of mitophagy. pUb under the control of a large PARK2 promoter fragment, which accumulation in endoGFP-PARKIN cells was robustly induced endo AB C DGFP- parental PARKIN endoGFP-PARKIN sgRNA vs. SCR endoGFP-PARKIN GFP-negative parental PARK2 #i PARK2 #ii Day 8 Day 15 300 250 SCR PARK2#i SCR PARK2#i Day 8