Thesis title: Unconventional roles of epigenetic regulators in cellular division
Chromatin remodeling complexes are central regulators of nuclear architecture and gene expression. Beyond these canonical roles, specific subunits of the human SRCAP and TIP60/P400 complexes perform non-canonical functions during mitosis. In our previous studies, the ATPase SRCAP and other remodeling subunits were found to localize to key mitotic structures, including the spindle, centrosomes, and midbody. Their depletion led to the accumulation of mitotic defects, and SRCAP, TIP60, and BAF53a were shown to interact with cytokinesis regulators and tubulin in telophase-synchronized HeLa cells. These findings revealed a role for chromatin remodeling factors in chromosome segregation and cytokinesis, extending their functions beyond transcriptional regulation. In this study, we investigated the cellular consequences of SRCAP mutation in lymphoblastoid cells derived from a patient with Floating-Harbor Syndrome (FHS) carrying a frameshift mutation (Thr2512fs*5, SCKL1) and compared them with cells from a healthy donor. Patient-derived cells exhibited increased mitotic abnormalities, including chromosome misalignment, micronuclei formation, multinucleation, and elongated intercellular bridges. Analysis of telophase-synchronized cells revealed defective recruitment of key cytokinesis regulators—Anillin, Spastin, and MKLP2—at the midbody, supporting a structural and regulatory role of SRCAP in late mitosis. To further assess the impact of SRCAP variants on mitotic processes, Tet-inducible stable RPE-1 cell lines expressing either wild-type or truncated SRCAP were generated. While the wild-type protein localized predominantly to the nucleus, the truncated variant displayed a diffuse cytoplasmic distribution and induced mitotic defects, including persistent chromatin bridges. Unexpectedly, SRCAP was also detected at the Golgi apparatus, where it colocalized with Golgin-97 and was required for proper Golgi reassembly after Brefeldin A treatment, suggesting a moonlighting role in cytoplasmic organelle organization. Following the characterization of SRCAP functions in mitosis and cytokinesis, we extended our investigation to explore whether additional epigenetic regulators could cooperate with chromatin remodelers and other cytokinesis regulators in organizing the midbody. Increasing evidence indicates that RNA molecules, including long non-coding RNAs (lncRNAs), play active roles in cell division. They can act as scaffolds for protein complexes and contribute to the structural organization of cytoplasmic compartments. To test whether RNA molecules are involved in midbody assembly, we performed immunofluorescence analysis using an antibody against N6-methyladenosine (m6A), which revealed a distinct RNA signal at the midbody. The signal was abolished by RNase treatment, confirming its RNA-dependent nature. Importantly, RNase-mediated RNA depletion disrupted the midbody microtubule array, indicating that RNA contributes to the structural integrity of this organelle rather than being a passive component. Building on these observations, RNA-seq analysis of isolated midbodies identified a subset of lncRNAs selectively enriched at this structure. qRT-PCR validation confirmed the midbody localization of six candidate lncRNAs. Notably, several of these transcripts displayed altered expression in cancer datasets, suggesting a possible link between cytokinetic regulation and tumorigenesis. Together, these findings support the hypothesis that specific RNA species, including lncRNAs, act as structural and regulatory elements of the midbody, potentially cooperating with moonlighting factors such as SRCAP to ensure faithful cell division. These results establish SRCAP as a multifunctional regulator that coordinates chromatin remodeling, mitotic progression, cytokinesis, and Golgi organization. SRCAP dysfunction in FHS disrupts these processes, linking its non-canonical functions to mitotic failure, genomic instability, and potentially to disease pathogenesis.