Introduction Protein post translational modifications (PTMs) play an important role in many cellular processes including; transcription, apoptosis, cell cycle regulation and cytoskeleton organisation. Sumoylation is a particular PTM which affects cellular activities such as chromatin organisation, protein localisation and cell cycle regulation. SUMO is a small ubiquitin like molecule. Typically it is covalently attached to lysine residues in the ψKxE consensus sequence (K- lysine, E - glutamic acid) although ∼40% of SUMOylated proteins are modified on non-consensus sequences.
Aim SUMO modification has been shown to be important in hESC self-renewal where one of the master regulators, Oct4, is stabilised and its degradation is inhibited by SUMOylation (Zhang et al, 2007). We hypothesised that SUMO modification may not only regulate hESC self-renewal, but may also be required for efficient hESC differentiation. We therefore interrogated the role of SUMOylation in hESC differentiation to hepatic endoderm (HE). hESC were differentiated using our established and efficient model (Hay et al, 2008).
Method Cell lysates were collected at different time points throughout the differentiation and analysed by Western blotting for changes to the levels of key proteins involved in the conjugation and de conjugation of SUMO.
Results We demonstrate that peak levels of SUMOylation were detectable in hESC populations during cellular differentiation to definitive endoderm (DE). Following commitment to DE we observed a decrease in the level of SUMO modified proteins as the cells in culture developed a hepatic fate. This corresponded with an increase in SENP 1, a SUMO specific protease. We also detected reduced levels of HNF4α, a critical regulator of hepatic status and metabolic function, as SUMOylation decreased. As a result we investigated the role of SUMO modification in HNF4α metabolism and if this process was involved in modulating HNF4α's critical role in HE.
Conclusion In conclusion, SUMO modification and deconjugation at critical points during cellular differentiation may regulate protein stability enhancing transcriptional activity and/or modulating subcellular localisation. As a consequence this may improve HE differentiation, viability and maturity, which are essential to the generation of high fidelity human models in culture and maybe directly applicable to other stem cell populations for example iPSCs.
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