Studying the role of chromatin remodelers during cell division

The genome of eukaryotic cells is organized in a nucleoproteic structure defined chromatin, consisting of DNA, RNA and associated proteins. A dynamic balance is needed between chromatin packaging and genome access to promote essential functions, such as DNA replication and repair, transcription. In these processes, multiple molecules are involved, including histone chaperones, chromatin remodelers and enzymes that modify histones and DNA. The structural characteristic of the remodelling enzymes belonging to a family of remodelling complex, INO80, is the ATPase domain interspersed with a long insertion. The SRCAP (SNF2-Related CBP Activator Protein) and Tip60 (HIV1 Tat Interacting Protein, 60 kDa) remodelling complexes belong to the INO80 family (subfamily Swr1) and they are conserved from yeast to human. These complexes share some subunits. Evolutionary speaking, these two complexes derive from duplication of Drosophila dTip60 complex. The main function is to promote the exchange of the canonical histone H2A with the histone variant. Previous studies suggested that chromatin and remodelling proteins might also play roles in cell division, yet evidence remains sporadic. In past years, in our lab my colleagues found that subunits of the human SRCAP and Tip60/p400 complexes, besides the canonical localization in the interphase chromatin, at different times during cell division target different sites of the mitotic apparatus (centrosomes, spindle and midbody, the bridge connecting the daughter cells at the finale step of mitosis). So, my PhD project aimed at studying the involvement of chromatin remodelling complex in ensuring proper cell division in both human and Drosophila cell lines, two species separated by 700 million years of evolution. First, I found that RNAi-mediated depletion of SRCAP, Tip60, Gas41, YL1 and MRG15 subunits in human cells and DOM-A, Tip60, MRG15 and Yeti in Drosophila cells affect cell division. In particular, cells have shown aberrant spindle morphology, chromosome misalignments, long intercellular bridges (LIBs) connecting the daughter cells and multinucleation. Defects found are consistent with their localization in both human and Drosophila cells. Then, to study how these remodelers are recruited, I performed interaction assays with already known cell division players. Co-immunolocalization and co-immunoprecipitation assays performed thus far on HeLa telophase extracts suggested that the aforementioned players contribute to the relocation of the Tip60 subunit from chromatin to the mitotic apparatus. Moreover, The inhibition of kinase activity of Aurora B, a well-known cell division regulator, caused mislocalization of TIP60, SRCAP and BAF53a remodelers. Finally, RNAi-mediated depletion of SRCAP, Tip60, Gas41, YL1 and MRG15 caused mislocalization of key components of the midbody, such as Aurora B, MKLP2, CIT-K and PLK1. In conclusion, we found that subunits of ATP-dependent remodelling complexes: i) are recruited to the mitotic apparatus; ii) interact in telophase with main cell division regulators (Aurora B, Cit-K, etc.); iii) prevent the failure of cell division and genome instability. Together, our findings suggest a cross-talk between SRCAP and TIP60 complexes and the main regulators of cytokinesis and highlight the existence of a previously unexplored and evolutionarily conserved phenomenon where chromatin remodelling factors translocate from the interphase nucleus to the mitotic apparatus in order to ensure faithful cell division in both human and Drosophila cells.