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

Eleftheria Karanika,Anastasia Christogianni,Spyros Georgatos,Dimitris Stellas,Anastasia S Politou,Apostolos Klinakis,Katerina Soupsana

doi:10.1038/s41598-019-54350-4

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.

Highlights

The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC)
Targeting of the CPC at the centromere is critical for correct chromosome alignment, at this point it is unclear whether Haspin-mediated phosphorylation of histone H3 is essential for cell division
CPC targeting to sites of microtubule-chromosome contact can be accomplished by Bub1-mediated phosphorylation of histone H2A at Thr[120] (H2AT120ph)

Summary

Introduction

The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). By comparison to other eukaryotic protein kinases, Haspin[452–789] seems to possess atypical features: an α-helical extension preceding the G-loop and two insertions further downstream bury the N-terminal lobe almost entirely; the A-loop is shorter than usual and starts with a DYT, instead of a DFG, motif; there is an additional insertion between the β7 and the β8 loops; and a deletion in the C-terminal lobe removes the αG helix, which is conspicuous in other members of the kinase superfamily[10,11] Despite these structural differences, Haspin[452–789] phosphorylates the N-terminal “tail” of histone H3 at Thr[3] (H3T3ph)[10,11]. It has been used as a tool for investigating the role of Haspin in a variety of cellular systems[26,27,28,29]

Methods

Results

Conclusion