CENP-A amino terminalinin epigenetik fonksiyonlarının ın vivo araştırılması

Centromeric Protein A (CENP-A) is a histone H3 variant. During mitosis, it drives kinetochore formation with other centromere-specific DNA binding proteins. Furthermore, CENP-A serves as an epigenetic marker that determines the identification of centromeres. Phosphorylation of the CENP-A N-terminal has drawn a lot of attention since it differs greatly from other H3 variants. Phosphorylation of human CENP-A serine 7 (S7p) is of particular interest because it resembles H3 S10p, a marker of mitotic entry. There are conflicting findings related to the purpose of S7 phosphorylation during cell division. Besides the lack of consensus on human CENP-A S7 phosphorylation, the function of corresponding phosphorylation sites in other species needs to be investigated. In a recent publication, the first three serines (S15, S16, and S22) of the murine CENP-A were proposed to be phosphorylation sites with an analogous role to human CENP-A S7p during mitosis. However, no in vivo study has been reported addressing S15 and S16 functions. In order to understand the purpose of murine CENP-A N-terminal phosphorylation, we decided to create a knockin mouse model expressing S15A and S16A non-phosphorylatable residue substitutions. We used CRISPR-EZ technology (CRISPR ribonucleoprotein Electroporation of Zygotes) to genetically engineer mice. Firstly, we edited the mouse Tyr locus which has published targeting and genotyping protocols, in order to try and establish our own protocols. After successfully deriving the albino Tyr knock-out mouse model, we moved forward to create the CENP-A knockin mouse model. Homozygous knockin mice were viable and fertile suggesting that CENP-A N-terminal phosphorylation does not have a crucial role for chromosome segregation. Detailed studies will need to be carried out to characterize more subtle epigenetic functions of N-terminal phosphorylation.