Identification of SUMO Targets Associated With the Pluripotent State in Human Stem Cells

Abstract

To investigate the role of SUMO modification in the maintenance of pluripotent stem cells, we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 resulted in the loss of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with C-terminal TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created three networks of proteins with functions in regulation of gene expression, ribosome biogenesis, and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The rest have roles in transcription and the regulation of chromatin structure. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centered on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins, there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO influences the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells.

Keywords: SUMO; SUMO site proteomics; TRIM28; human stem cells; pluripotency; retroviral elements; transcriptional repression.