Ricerc@Sapienza

This project aims to utilize a range of cutting-edge tools for unveiling mechanisms underpinning the mitotic function of KAT5 acetyltransferase, a subunit P400/TIP60 chromatin remodeling complex, in hTERT Retinal Pigmental Epithelial (hereafter RPE-1) human cell line.
In addition to its role in exchanging H2A.Z/H2A.X with the canonical H2A histone to regulate chromatin structure, gene expression and DNA damage repair, the P400/TIP60 complex has been shown to be involved in cell cycle regulation.
In particular, the KAT5 (Tip60) acetyltransferase relocates from chromatin to the mitotic apparatus (centrosomes, spindle and midbody), interacts with key cytokinesis regulators and its depletion causes mitosis and cytokinesis failure. Similar results have been found for the Drosophila Tip60. Thus, the relocation of KAT5 remodeling factor from nucleus to the mitotic apparatus appears to be an evolutionary conserved and highly regulated phenomenon in which this acetyltransferase may play the crucial roles in cell division that is worth elucidating.
To investigate more in deep the roles of KAT5 in cell division, I will exploit the CRISPR/Cas9 technology to specifically tag the endogenous Kat5 gene with the dTAG degrader-tagged version for rapid targeted degradation. I will use nontumoral RPE-1 cell line as experimental system because of its genomically stable diploid karyotype, easiness to grow and amenable to transfection and synchronization. These characteristics make them widely used in cell biology especially for in vivo time-lapse microscopy to depict chromosome segregation and mitotic defects. These experiments will allow me to test a direct involvement of KAT5 in mitosis and cytokinesis, independently of its chromatin remodeling functions.
The approaches proposed are state-of-the art and the infrastructure and support are exceptionally strong, so there will be excellent career development opportunities at the end of the grant.