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Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-α protein levels and activity

Abstract

Estrogen receptor-α (ERα) is a central transcription factor that regulates mammary gland physiology and a key driver in breast cancer. In the present study, we aimed to identify novel modulators of ERα-mediated transcriptional regulation via a custom-built siRNA library screen. This screen was directed against a variety of coregulators, transcription modifiers, signaling molecules and DNA damage response proteins. By utilizing a microscopy-based, multi-end point, estrogen responsive biosensor cell line platform, the primary screen identified a wide range of factors that altered ERα protein levels, chromatin remodeling and mRNA output. We then focused on UBR5, a ubiquitin ligase and known oncogene that modulates ERα protein levels and transcriptional output. Finally, we demonstrated that UBR5 also affects endogenous ERα target genes and E2-mediated cell proliferation in breast cancer cells. In conclusion, our multi-end point RNAi screen identified novel modulators of ERα levels and activity, and provided a robust systems level view of factors involved in mechanisms of nuclear receptor action and pathophysiology. Utilizing a high throughput RNAi screening approach we identified UBR5, a protein commonly amplified in breast cancer, as a novel regulator of ERα protein levels and transcriptional activity.

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Acknowledgements

We gratefully acknowledge ZD Sharp for aid in the development of the PRL-HeLA cell line, FJ Ashcroft for creation of the GFP-ERa:PRL-HeLa cells, and I Mikic, TJ Moran and JY Newberg for help in developing the automated high-content analysis tools used in this study. We also acknowledge R Lanz and C Stephan for curation, handling and upkeep of the RNAi library. We acknowledge NIEHS funding from 7RC2ES018789 (MAM), the Keck Foundation pre-doctoral fellowship and imaging/automation resource support from the John S Dunn Gulf Coast for Chemical Genomics (PJ Davies and MAM), Dan L Duncan Baylor Cancer Center (K Osborne), Center for Reproductive Biology (FJ Demayo), Keck Center NLM Training Program in Biomedical Informatics of the Gulf Coast Consortia National Library of Medicine (T15LM007093 to MB), and the Diana Helis Henry Medical Research Foundation (MAM) through its direct engagement in the continuous active conduct of medical research in conjunction with Baylor College of Medicine and the Cancer Program. This project was supported by the Integrated Microscopy Core at Baylor College of Medicine with funding from the NIH (HD007495, DK56338 and CA125123), the Dan L Duncan Cancer Center, and the John S Dunn Gulf Coast Consortium for Chemical Genomics.

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Bolt, M., Stossi, F., Callison, A. et al. Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-α protein levels and activity. Oncogene 34, 154–164 (2015). https://doi.org/10.1038/onc.2013.550

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Keywords

  • estrogen receptor
  • UBR5
  • high content screening
  • high throughput microscopy
  • ubiquitin ligase

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