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.
In humans, the SRCAP and P400/TIP60 chromatin remodeling complexes, belonging to the INO80 family, play a crucial role in multiple cellular processes [1].
The main function of the SRCAP remodeling complex is to promote the exchange of the canonical histone H2A with the variant H2A.Z [2]. The Tip60 complex in humans consists of 16 evolutionarily conserved subunits [3, 4] and is involved in multiple cellular processes including transcriptional regulation, DNA repair, chromatin remodeling and cell migration.
Consequently, the role of chromatin remodelers in cell cycle control and its evolutionary conservation is unexpected and could open new horizons of investigation.
In particular, an increasing number of investigations have indicated that acetylation plays an important role in tumor metabolism [5,6]. KAT5 acetyltransferase has been found to acetylate Aurora B kinase at kinetochores to ensure accurate chromosome segregation [7]. Therefore, as their physical interaction is persisting in telophase, addressing its acetyltransferase function in cytokinesis is crucial in view of deeper investigations on the study of the relationships between chromatin remodeling, cell cycle control and tumorigenesis.
Bibliografia:
[1] - Clapier, C.R., and Cairns, B.R., The biology of chromatin remodeling complexes. Annu Rev Biochem 2009. 78, 273-304.
[2] - Monroy, M. A., et al., Regulation of cAMP-responsive element-binding protein-mediated transcription by the SNF2/SWI-related protein, SRCAP. J Biol Chem 2001. 276, 40721-6.
[3] - Prozzillo, Y., et al., The true story of Yeti, the `abominable¿ heterochromatic gene of Drosophila melanogaster. Frontiers in physiology 2019. 10, 1093
[4] - Messina, G., Atterrato M. T., Prozzillo Y, Piacentini L., Losada A., Dimitri P., The human Cranio Facial Development Protein 1 (Cfdp1) gene encodes a protein required for the maintenance of higher-order chromatin organization. Scientific reports 2017. 7 (1), 1-10
[5] - Di Martile M, Del Bufalo D, Trisciuoglio D (2016) The multifaceted role of lysine acetylation in cancer: prognostic biomarker and therapeutic target. Oncotarget 7: 55789-55810
[6] - Zhao D, Li FL, Cheng ZL, Lei QY (2014) Impact of acetylation on tumor metabolism. Molecular & cellular oncology 1: e963452
[7] - Mo F, Zhuang X, Liu X, Yao PY, Qin B, Su Z, Zang J, Wang Z, Zhang J, Dou Z, Tian C, Teng M, Niu L, Hill DL, Fang G, Ding X, Fu C, Yao X (2016) Acetylation of Aurora B by TIP60 ensures accurate chromosomal segregation. Nature chemical biology 12: 226-232