br Results br Discussion PSC

Results
Discussion PSC offer great opportunities for human health, allowing the derivation of patient-specific tissues and providing disease models for drug screening. Thus, investigation of factors and mechanisms that regulate pluripotency constitutes a rapidly growing area of cutting-edge research. In addition to their value for regenerative medicine, pluripotency factors are promising targets to study cancer stem cells, since oncogenic transformation and cellular reprogramming share common properties (Goding et al., 2014; Hadjimichael et al., 2015). In this report we addressed the functions of the pleiotropic regulator PML in embryonic and induced PSC. PML is highly expressed in undifferentiated ESC and its ablation induces significant changes in ESC morphology, global gene expression profile, and lineage specification decision. Specifically, PML physically interacts and regulates the expression of crucial mediators of pluripotency and contributes to the preservation of self-renewal. Furthermore, PML influences mESC cell-cycle profile, since its depletion leads to prolonged G1 phase, resembling that of differentiated cells. This effect can be attributed to the inhibition of the pro-proliferative functions of LIF/STAT3 and Myc-elicited mechanisms that we observed in lack of PML expression. In addition, GO analysis of our microarrays data highlighted PI3K pathway-related genes as being strongly downregulated in PML-deficient ESC. PI3K signaling is a well-documented mediator of proliferation, and its reduced activity in PML loss is in agreement with the effect on the ESC cell cycle. In somatic pim inhibitor PML inhibits cell proliferation through activation of pRB and p53, and this function highly contributes to its role as a tumor suppressor (Guan and Kao, 2015). These effects are mainly due to the regulation of post-translational modifications including phosphorylation and dephosphorylation by PML bodies. In mESC, pRB is constitutively inactivated by phosphorylation (Aksoy et al., 2007; Coronado et al., 2013; White and Dalton, 2005) and the p53 pathway is not active (Lee et al., 2012). Our data showed that PML is involved in maintaining pRB in an inactive state, and that PML loss impairs cell-cycle progression and favors advance to the primed-like state. The effect of PML disruption on ESC transition from the naive to the EpiSC pluripotent state is also underpinned by transcription factor expression, specific signaling pathway deregulation, and metabolic alterations. Our transcriptomic analysis revealed a downregulation of genes encoding enzymes of FAO and OXPHOS. Thus, PML loss results in decreased respiration and mitochondrial function, another indication for exit from the naive state and progression to the EpiSC state. In addition, Pml−/− cells sustain propagation in F/A culture conditions and proliferate normally in the presence of Jaki, reinforcing the idea that they move on toward a primed-like stem cell state. Moreover, PML overexpression delays the exit from the naive state by either permitting long-term existence of a small fraction of undifferentiated naive ESC or allowing a fraction of EpiSC to regain the naive pluripotent state. These data propose that PML safeguards the naive pluripotent state. PML is also implicated in the differentiation process. PML ablation stirred ESC toward mesodermal rather than endodermal lineage commitment through Tbx3 repression. Tbx3, well-known downstream target of the PI3K signaling pathway, is one of the most significantly repressed genes upon PML KD. Forced expression of TBX3 in PML KD ESC rescued the differentiation changes caused by PML loss. Physical interaction and reciprocal inhibition between TBX2/3 and PML was previously shown in human fibroblasts where the pro-senescence activity of PML counteracted cell proliferation driven by TBX (Martin et al., 2012). However, in ESC, TBX3 is positively regulated by PML and determines cell differentiation choices.