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Phosphorylation of Sox2 Cooperates in Reprogramming to Pluripotent Stem Cells

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From the December Issue of Stem Cells

By Stuart P Atkinson

Modifications of histone proteins have been well studied in ESCs and iPSCs and we are beginning to understand the importance that these modifications have in relation to chromatin structure and gene regulation and indeed the enzymes which mediate these modifications. However, research into modifications of non-histone proteins in pluripotent cells has perhaps lagged behind. Studies in 2009 (Saxe et al, Swaney et al and Van Hoof et al) have provided an excellent insight into the global picture of protein phosphorylation in ECs and ESCs, which included the modification of both OCT4 and SOX2, and recently Nanog has been shown to be regulated by phosphorylation (Moretto-Zita et al). Now, in the December issue of Stem Cells, the lab of Zigang Dong from The Hormel Institute at the University of Minnesota provides data from an in depth analysis of the specific role of Sox2 phosphorylation in mouse ESC (Jeong et al), supporting a potential role in pluripotency and reprogramming.

LIF (Leukaemia Inhibitory Factor) signalling is involved in regulating pluripotency in mESC through Jak-Stat3 and PI3K-Akt signalling. Jeong et al uncovered that Akt1 interacts with and phosphorylates Sox2 at a highly conserved threonine residue in mESC growing under self-renewing conditions. This modification led to enhanced transcriptional activity, an increase in Sox2 protein abundance and induced the accumulation of Sox2 in the nucleus, due to an increased stability of the protein by antagonization of ubiquitin-mediated proteolysis. Interestingly, further work demonstrated that over-expression of a wild type Sox2, but not a mutant with a mutated phosphorylation site, could delay LIF-withdrawal-mediated differentiation, seemingly by blocking endoderm and mesoderm differentiation and enhancing pluripotency.

Next, the role of Sox2 phosphorylation was studied in the context of iPSC, since Sox2 is one of the “classic” reprogramming factors. If the phosphorylation site mutant is utilised instead of the wild type Sox2, reprogramming efficiency was decreased, when included alongside three other factors; Oct4, Klf4 and Nanog. Furthermore, in 2-factor reprogramming experiments, use of the wild type Sox2 alongside only Oct4 led to a greater numbers of iPSC colonies, when compared to the use of the mutant Sox2. However, when iPSC generated using wild type and mutant Sox2 where compared at later passages, no differences were observed at any level studied, suggesting that Sox2 phosphorylation plays an early role in reprogramming or establishment of pluripotency and is not required for the maintenance of iPSCs.

Another level of regulation in ESC and iPSC is now becoming increasingly appreciated and studied and this report represents an important step. It is vital that we understand signalling pathways in order to fully understand pluripotent cells, and understanding phosphorylation and the kinases/phosphatases which control this modification are equally important. Sox2 phosphorylation seems to represent an important level of control and perhaps upstream modulation of signalling to Sox2 could be utilised to further enhance reprogramming and control differentiation. This would fall into line with the current quest to remove the need for exogenous viral transgenes and replace them with small molecule drugs. However, there still exists a plethora of other modifications, methylation, acetylation, sumoylation and ubiquitination to name but a few, to be studied and full understanding of the dynamic changes of these modifications and how they are controlled may give us further insight into the nature of the pluripotent state of ESC and the mechanisms of reprogramming of somatic cells. From decades of research in other cell types we understand many of these pathways already, so application of this knowledge to pluripotent cells may vastly increase our knowledge of their nature.

References

Post-translational regulation of Oct4 transcriptional activity.
Saxe JP, Tomilin A, Schöler HR, Plath K, Huang J.
PLoS One. 2009;4(2):e4467

Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry.
Swaney DL, Wenger CD, Thomson JA, Coon JJ.
Proc Natl Acad Sci U S A. 2009 Jan 27;106(4):995-1000

Phosphorylation dynamics during early differentiation of human embryonic stem cells.
Van Hoof D, Muñoz J, Braam SR, Pinkse MW, Linding R, Heck AJ, Mummery CL, Krijgsveld J.
Cell Stem Cell. 2009 Aug 7;5(2):214-26.

Phosphorylation stabilizes Nanog by promoting its interaction with Pin1.
Moretto-Zita M, Jin H, Shen Z, Zhao T, Briggs SP, Xu Y.
Proc Natl Acad Sci U S A. 2010 Jul 27;107(30):13312-7.

Chul-Ho Jeong, Yong-Yeon Cho, Myoung-Ok Kim, Sung-Hyun Kim, Eunjin Cho, Sung-Young Lee, Young-Jin Jeon, Kun Yeong Lee, Ke Yao, Young-Sam Keum, Ann M. Bode and Zigang Dong
Stem Cells 2010 Accepted manuscript online