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

Author:

Karanika, Eleftheria

Soupsana, Katerina

Christogianni, Anastasia

Stellas,Dimitris

Klinakis, Apostolos

Politou, Anastasia S

Georgatos, Spyros [ORCID]
Author Address

Stem Cell and Chromatin Group, Institute of Molecular Biology and Biotechnology, Biomedical Division, FORTH-ITE, Ioannina, Greece., Laboratory of Biology, University of Ioannina, Faculty of Medicine, Ioannina, Greece., Laboratory of Biological Chemistry, University of Ioannina, Faculty of Medicine, Ioannina, Greece., Biomedical Research Foundation, Academy of Athens, Athens, Greece., Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, USA., Stem Cell and Chromatin Group, Institute of Molecular Biology and Biotechnology, Biomedical Division, FORTH-ITE, Ioannina, Greece. sgeorgat@uoi.gr., Laboratory of Biology, University of Ioannina, Faculty of Medicine, Ioannina, Greece. sgeorgat@uoi.gr.,

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.

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