A gene-expression screen identifies a non-toxic sumoylation inhibitor that mimics SUMO-less human LRH-1 in liver

Abstract

SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation.DOI: http://dx.doi.org/10.7554/eLife.09003.001

LRH-1 regulates hepatic lipid homeostasis and maintains arachidonoyl phospholipid pools critical for phospholipid diversity

“…Distinct structural features at the mouth of the ligand-binding pocket allow human LRH-1 (hLRH-1) to efficiently bind phospholipid ligands compared with mouse LRH-1 (mLRH-1) (23,43). To test whether hLRH-1 rescues hepatic phenotypes observed in Lrh-1 AAV8-Cre mice, we humanized mouse livers by simultaneously infecting mice with AAV8-Cre-and AAV8-FLAG-tagged hLRH-1 (Lrh-1 AAV8-Cre+hLrh1 ), which eliminates mLRH-1, while introducing epitope-tagged hLRH-1 in approximately 80% of hepatocytes (44), as shown in Figure 6A. Known targets, such as Cyp8b1 and Shp, as well as targets identified in this study respond appropriately after expression of hLRH-1 ( Figure 6B and Supplemental Figure 7).…”

“…Adult KO of mLRH-1 and expression of hLRH-1 in liver was accomplished using viral vectors acquired or generated at the University of Pennsylvania Gene Therapy Vector Core. Briefly, adenoassociated virus 8-TBG vectors (AAV8-TBG) expressing wild-type hLRH-1 (AAV8-hLRH-1) or a ligand-binding deficient mutant (AAV8-hPM) were generated as previously described (44). Six-week-old Lrh-1 fl/fl male mice were infected with either AAV8-GFP (Lrh-1 AAV8-GFP ) or AAV8-Cre (Lrh-1 AAV8-Cre ) at a concentration of 1 × 10 11 genome copies/ml via retro-orbital injection.…”

“…Primary hepatocyte analyses. Primary hepatocytes were isolated from nonfasted mice as previously described (44). Hepatocytes were seeded at 4.5 × 10 5 cells per well on type 1 collagen-coated 6-well plates.…”

“…DNAse-treated (1 Ug) total RNA was used to generate cDNA using the High-Capacity cDNA Reverse Transcription kit (Life Technologies). RT-qPCR was performed using SYBR green, High ROX (Quanta), and data were analyzed as described previously (44). For Western blotting, livers were lysed in Tris-SDS buffer (2% SDS, 0.6 M Tris-Cl, pH 8.0, and 0.1 M DTT) supplemented with protease inhibitors (Calbiochem) and 20 mM N-ethylmaleimide (MilliporeSigma) and then sonicated using a Diagenode Bioruptor.…”

Small-Molecule Inhibitors Targeting Protein SUMOylation as Novel Anticancer Compounds

“…Among these natural products, ginkgolic acid is the most widely used and commercially available chemical regent that targets global SUMOylation, but its inhibitory effects on cells can vary greatly depending on the assay and a measured substrate. Tannic acid has been identified as targeting human liver receptor homolog-1 as a general nontoxic SUMOylation inhibitor via cell-based screening (Suzawa et al, 2015).…”