Investigating Haspin-dependent phosphorylation of histones during mitosis
|Abstract:||Haspin is a conserved mitotic serine/threonine kinase that is known to function through histone H3 phosphorylation (H3pT3). Despite this, H3T3 is the only well-known substrate for Haspin, H3pT3 may not be enough to explain all Haspin functions during mitosis. Interestingly, homology of H3 N-terminus with H2B C-terminus suggests that H2BT119 is a strong potential candidate to be a major substrate of Haspin. Thus, the aim was to investigate Haspin-dependent phosphorylation of H2BT119 during mitosis. Phosphorylation of recombinant H2B at T119 by Haspin was confirmed by a radioactivity-based kinase assay. Also, H2BpT119 signal on recombinant H2B was detected by an anti-H2BpT119 antibody, confirming Haspin-dependent phosphorylation of H2B at this site in the in vitro kinase assay. Also, an upshift of H2B was observed following the kinase assay. Results from in vivo experiments, including Western blot analysis of mitotic histone extract and immunofluorescence microscopy with the anti-H2BpT119, support phosphorylation of T119 in H2B, which also was found to depend on Haspin activity. However, due to the potential anti-H2BpT119 cross-reactivity with H3pT3, while exploring H2BpT119 signal during mitosis, tagged H2B was used. The tag increases H2B molecular size and helps to distinguish H2BpT119 from H3pT3. Several tagging systems was used, but all attempts failed, because of the low level of the exogenous tagged H2B. However, the results of this project suggest H2BpT119 signal during mitosis that may reveal a novel Haspindependent mechanism for chromosome segregation regulation. Therefore, it remains important to study this histone mark during mitosis.|
|Document Type:||Thèse de doctorat|
|Open Access Date:||28 September 2020|
|Collection:||Thèses et mémoires|
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