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A Nucleolar Link to Pluripotency and Reprogramming?

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The study first utilised the P19 mouse embryonic carcinoma (EC) cell line to screen for factors which could affect the expression of a luciferase reporter gene under the control of the Nanog gene, a major regulator of pluripotency. This initial screening uncovered 90 activating and 14 inhibitory gene hits from the cDNA libraries, with the threshold set at a 2-fold change in luciferase activity. Functional classification of these hits revealed their involvement in functions associated with ESC biology/reprogramming and lineage specification. Subsequent analysis of these gene hits in mouse ESC showed that chromatin profile and expression status indicated their involvement in either the maintenance of pluripotency or lineage specification, whilst around 50 of the genes were bound by one of Nanog, Oct4, Sox2, c-Myc, or Klf4.

Further analysis carried out with 16 activating and 4 inhibitory genes revealed that three novel activator genes, Timp2, Hig2 and Mki67ip, promoted ES self-renewal when episomally expressed in ESC, whilst the inhibitor genes PU.1/Spi1, Prkaca and Jun induced differentiation. shRNA analysis demonstrated that knock-down of 4 activating genes (Timp2, Mki67ip, Esrrg and Dusp7) promoted ESC differentiation; with Timp2 and Mki67ip having the most pronounced phenotype, and causing a significant reduction in Nanog, Oct4, Sox2 and Rex1 (Zfp42). Conversely, knock-down of the inhibitory gene PU.1/Spi1 inhibited differentiation and induced Oct4, Sox2 and Rex1 expression. The activator gene Mki67ip, not previously linked to ESC-related functions, was found to interact with Npm1, a gene implicated in the cell cycle. Both genes are localised to the nucleolus are highly expressed in ESC and during early development with significant down-regulation during differentiation. Further analysis demonstrated co-localisation and physical interaction of these proteins, suggesting a functional interaction in mouse ESC, most likely acting to enhance the self-renewal properties of these cells.

The major highlights of this paper lie in the revelation of novel genes and new interactions which all contribute to the current information network pertaining to pluripotency/self-renewal and lineage specification. This study also enforces the significance of the nucleolus in pluripotency, involving Mki67ip and Npm1, as well as the previously studied nucleolar protein Nucleostemin (Gnl3), implicated in self-renewal. Both Mki67ip and Npm1 have previously been implicated in ribosome biogenesis in the nucleolus (Schlosser et al, Boon et al and Zeller et al), suggesting that a high level of ribosome biogenesis is required for the retention of self-renewal capability in pluripotent cells. Indeed both genes are also targets for the c-Myc gene, generally required for iPSC generation and implicated in tumourigenesis, strengthening the link between nucleolar function, ribosome generation and enhanced self-renewal.

References

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