Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen

Lina Schukur,Scott Gleim,Giorgio G Galli,Melivoia Rapti,Angelica Mendiola,Britta Knapp,Tamara Zimmermann,Claudio R Thoma,Xiaoyou Liang,Ole Niewoehner,Ines Barbosa,Beatrice Bauer-Probst,John Reece-Hoyes,Grainne Kerr,Markus Reschke,Thomas Radimerski

doi:10.1038/s41598-020-76960-z

Abstract

MYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.

Highlights

Protein abundance is regulated by transcriptional and translational processes and by the regulation of protein degradation, an important process that acts at the posttranslational level in order to maintain homeostasis[1]
Changes in MYC protein level were monitored by measuring GFP versus mCHERRY ratios using FACSbased readouts
Upon ubiquitination and subsequent degradation of MYC-GFP, the GFP signal is reduced in comparison to mCHERRY

Summary

Introduction

Protein abundance is regulated by transcriptional and translational processes and by the regulation of protein degradation, an important process that acts at the posttranslational level in order to maintain homeostasis[1]. We validated these findings in various cell lines, and could show that depletion of UBR5 reduces cellular viability in cell lines with high MYC expression level through the increase of MYC protein level.

Results

Conclusion