No abstract

The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.

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 Haspinknockout 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.The metazoan genomes encode >500 typical, atypical and pseudo-protein kinases 1,2 . Of these, about 100 are directly or indirectly involved in cell division 3,4 . One kinase that appears to play important role in mitosis is Haspin, a protein originally identified in mouse testes and now known to be expressed in all somatic cells 5-8 . It is encoded by a single-copy gene (Gsg2) and is highly conserved in eukaryotic organisms, from humans to yeast 9 .Haspin possesses distinct N-terminal and C-terminal domains. The C-terminal domain (residues 452-789 in the human protein) folds autonomously as a bilobar structure and has measurable kinase activity. However, 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 finally, 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 specifically the N-terminal "tail" of histone H3 at Thr3 (H3T3ph) 10,11 . In somatic cells, H3T3ph is detected at the centromeric area of mitotic chromosomes 12-14 and serves as a binding site for Survivin, a subunit of the Chromosomal Passenger Complex (CPC) [15][16][17] . CPC deposits the master kinase Aurora B near the sites of microtubule-chromosome contact, allowing correction of improper attachments and activation of the Spindle Assembly Checkpoint (SAC) in the event of...