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Haspin-dependent and independent effects of the kinase inhibitor 5-Iodotubercidin on self-renewal and differentiation.

ABSTRACT: The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting "off-target" effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the "off-target" effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.

ORGANISM(S): Mus musculus

PROVIDER: GSE119737 | GEO | 2020/01/16

REPOSITORIES: GEO

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Project description:Embryonic stem cells studies have generated a great interest due to their ability to form a wide variety of matured cells. However, there remains a poor understanding of mechanisms regulating the cell state of ESCs and of the genes they express during early differentiation. Gene expression analysis may be a valuable tool to elucidate either the molecular pathways involved in self-renewal and pluripotency or early differentiation and to identify potential molecular therapy targets. The aim of this study was to characterize at molecular level the undifferentiated mouse ESCs state and the early development towards embryoid bodies. To attemp this issue we performed CodeLink Mouse Uniset I 20K bioarrays in a well-characterized mouse ESC line, MES3, 3- and 7 day-old embryoid bodies and we compared our findings with those in adult tissue cells. Gene expression results were subsequently validated in a commercial stem cell line, CGR8 (ATCC). SAM analysis was used to identify statistically significant changes in microarray data. We identified 3664 genes expressed at significantly greater levels in MES3 stem cells than in adult tissue cells, which included 611 with 3-fold higher gene expression level versus the adult cells. We also investigated the gene expression profile during early embryoid body formation, identifying 2040 and 2243 genes that were up-regulated in 3- and 7-day-old EBs, respectively. Our gene expression results in MES3 cells were partially confirmed in CGR8 cells, showing numerous genes that are expressed in both mouse stem cells. In conclusion, our results suggest that commonly expressed genes may be strong candidates for involvement in maintenance of a pluripotent and undifferentiated phenotype and in early development. This study characterizes at molecular level the undifferentiated mouse ESCs state and the early development towards embryoid bodies. We identified numerous genes not been previously reported, which may offer novel insights into the behaviour of mouse ESC, the maintenance of their undifferentiated state and the mechanisms underlying their pluripotency, self-renewal and early differentiation.