A high throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway
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A high throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway. / Singh, Sonia R.; Meyer-Jens, Moritz; Alizoti, Erda; Bacon, W Clark; Davis, Gregory; Osinska, Hanna; Gulick, James; Reischmann-Düsener, Silke; Orthey, Ellen; McLendon, Patrick M.; Molkentin, Jeffery D; Schlossarek, Saskia; Robbins, Jeffrey; Carrier, Lucie.
In: AUTOPHAGY, 2020.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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T1 - A high throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway
AU - Singh, Sonia R.
AU - Meyer-Jens, Moritz
AU - Alizoti, Erda
AU - Bacon, W Clark
AU - Davis, Gregory
AU - Osinska, Hanna
AU - Gulick, James
AU - Reischmann-Düsener, Silke
AU - Orthey, Ellen
AU - McLendon, Patrick M.
AU - Molkentin, Jeffery D
AU - Schlossarek, Saskia
AU - Robbins, Jeffrey
AU - Carrier, Lucie
PY - 2020
Y1 - 2020
N2 - The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Initially considered as two independent pathways, there is emerging evidence that they can work in concert. As alterations of UPS and ALP function can contribute to neurodegenerative disorders, cancer and cardiac disease, there is great interest in finding targets that modulate these catabolic processes. We undertook an unbiased, total genome high-throughput screen to identify novel effectors that regulate both UPS and ALP. We generated a stable HEK293 cell line expressing a UPS reporter (UbG76V-mCherry) and an ALP reporter (GFP-LC3) and screened for genes for which knockdown increased both UbG76V-mCherry intensity and GFP-LC3 puncta. With stringent selection, we isolated 80 candidates, including the transcription factor ZNF418 (ZFP418 in rodents). After screen validation with Zfp418 overexpression in HEK293 cells, we evaluated Zfp418 knockdown and overexpression in neonatal rat ventricular myocytes (NRVMs). Endogenous and overexpressed ZFP418 were localized in the nucleus. Subsequent experiments showed that ZFP418 negatively regulates UPS and positively regulates ALP activity in NRVMs. RNA-seq from Zfp418 knockdown revealed altered gene expression of numerous ubiquitinating and deubiquitinating enzymes, decreased expression of autophagy activators and initiators and increased expression of autophagy inhibitors. We found that ZPF418 activates the promoters of Dapk2 and Fyco1, which are involved in autophagy. RNA-seq from Zfp418 knockdown revealed accumulation of several genes involved in cardiac development and/or hypertrophy. In conclusion, our study provides evidence that ZNF418 activates the ALP, inhibits the UPS and regulates genes associated with cardiomyocyte structure/function.
AB - The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Initially considered as two independent pathways, there is emerging evidence that they can work in concert. As alterations of UPS and ALP function can contribute to neurodegenerative disorders, cancer and cardiac disease, there is great interest in finding targets that modulate these catabolic processes. We undertook an unbiased, total genome high-throughput screen to identify novel effectors that regulate both UPS and ALP. We generated a stable HEK293 cell line expressing a UPS reporter (UbG76V-mCherry) and an ALP reporter (GFP-LC3) and screened for genes for which knockdown increased both UbG76V-mCherry intensity and GFP-LC3 puncta. With stringent selection, we isolated 80 candidates, including the transcription factor ZNF418 (ZFP418 in rodents). After screen validation with Zfp418 overexpression in HEK293 cells, we evaluated Zfp418 knockdown and overexpression in neonatal rat ventricular myocytes (NRVMs). Endogenous and overexpressed ZFP418 were localized in the nucleus. Subsequent experiments showed that ZFP418 negatively regulates UPS and positively regulates ALP activity in NRVMs. RNA-seq from Zfp418 knockdown revealed altered gene expression of numerous ubiquitinating and deubiquitinating enzymes, decreased expression of autophagy activators and initiators and increased expression of autophagy inhibitors. We found that ZPF418 activates the promoters of Dapk2 and Fyco1, which are involved in autophagy. RNA-seq from Zfp418 knockdown revealed accumulation of several genes involved in cardiac development and/or hypertrophy. In conclusion, our study provides evidence that ZNF418 activates the ALP, inhibits the UPS and regulates genes associated with cardiomyocyte structure/function.
U2 - 10.1080/15548627.2020.1856493
DO - 10.1080/15548627.2020.1856493
M3 - SCORING: Journal article
JO - AUTOPHAGY
JF - AUTOPHAGY
SN - 1554-8627
ER -