OR53-6 Identifying Small Molecules and Signaling Events That Control NR5As Sumoylation

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: OR53-Transcriptional Regulators & Epigenetic Control
Tuesday, June 18, 2013: 9:15 AM-10:45 AM
Presentation Start Time: 10:30 AM
Room 133 (Moscone Center)
Miyuki Suzawa*, Emily J Faivre, Kean-Hooi Ang, Michelle R Arkin and Holly A Ingraham
University of California San Francisco, San Francisco, CA
Sumoylation is generally considered a repressive mark for many transcription factors. In the last several years, we asked how sumoylation affect the activity of NR5A receptors, SF-1 (NR5A1) and LRH-1 (NR5A2). We previously reported that eliminating SF-1 sumoylation in mice (Sf-1(K119R, K194R, or 2KR)) failed to phenocopy a simple gain of SF-1 function, but instead triggers activation of a new set of target genes. Both SF-1 and LRH-1 are extremely good substrates for sumoylation in vitro and in vivo. However, it remains unclear what molecular entities mediate NR5As sumoylation. To identify endogenous and synthetic mediators of NR5As sumoylation, we undertook an siRNA high-throughput screening (HTS) approach directed against the human kinome and with a small library of bioactive molecules. This gene-expression based screen was carried out in JEG-3 cells, using three highly SUMO-sensitive targets to 3KR LRH-1, Muc1, ApoC3 and Ass1. After optimizing screening conditions, kinome siRNAs or drugs were added to cells and mRNA purified from cell lysates using Oligo-(dT) coated plates, followed by cDNA synthesis; qPCR was then performed to assess expression of SUMO-sensitive genes. All steps were performed in a 384 well format. Hits include sphingosine kinase 1 (SPHK1) as well as three drugs. Indeed, SPHK1 knock-down inhibited LRH-1 sumoylation, validating our screen. Experiments are currently underway to modify this assay for more relevant cellular systems and for higher-throughput. Such HTS screens have the potential to provide new chemical tools that can be used to modulate NR5A activity, independent of classic ligand-dependent activation.

Nothing to Disclose: MS, EJF, KHA, MRA, HAI

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

Sources of Research Support: NIH Grant RO1 DK070024NIH Grant R21 ES017136UCSF-Roche Extending Innovation Network Program/UCSF Program for BreakthroughBiomedical Research
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