Browsing by Author "Nikolic, Ana"

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    Epigenetic Investigation of PF(A) Ependymoma
    (2024-09-13) Hasheminasabgorji, Elham; Gallo, Marco; Nikolic, Ana; Bahlis, Nizar; Chan, Jennifer
    Posterior Fossa type A (PFA) ependymoma in infants presents therapeutic challenges with a 60% survival rate and significant treatment-associated neurocognitive sequalae. Our research revealed a distinctive 3D genomic architecture — Type B Ultra-Long Interactions in PFA (TULIPs) - universally present in PFA samples and occurring at recurrent regions of the genome. Here, we investigate the molecular origins of TULIPs in PFA ependymoma. We hypothesized that EZHIP expression may underlie TULIP formation, given its widespread expression in virtually all PFAs. To test this hypothesis, EZHIP was expressed in human neural progenitor cell (hNPC) cultures, and we validated the models using immunofluorescence and western blot assays. Our results demonstrated the nuclear localization of EZHIP and its association with chromatin. Moreover, the expression of EZHIP induced profound alterations in the conformation and localization of heterochromatin in the cells, consistent with the induction of TULIPs. Additionally, functional EdU incorporation assays provided evidence that the expression of EZHIP positively influences cell proliferation. Based on our immunofluorescence results, TULIPs appear as large H3K9me3-rich foci in the nucleus of cells. In various cell types, regions of heterochromatin marked by H3K9me3 are typically associated with HP1 proteins. Considering that HP1 proteins are involved in maintaining the liquid-liquid phase separation (LLPS) state of H3K9me3, we assessed whether TULIPs were in fact condensates. hNPCs expressing EZHIP were treated with 1,6-hexanediol, a compound widely used to disrupt liquid condensates. Our results indicated that 1,6-hexanediol treatment caused disruption of the H3K9me3 configuration, suggesting that TULIPs are in fact in LLPS states. In conclusion, our study provides valuable insights into the molecular origins of TULIPs in PFA ependymoma, elucidating the role of EZHIP expression and its impact on chromatin conformation and cell proliferation. While our data suggest that TULIPs may exist in an LLPS state, further investigation is required to confirm this. These findings enrich our understanding of the molecular landscape of PFA ependymomas and offer potential therapeutic targets.
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    Interrogating the Epigenetic Determinants of State Heterogeneity in Adult Glioblastoma
    (2023-01-24) Nikolic, Ana; Gallo, Marco; Chan, Jennifer; Weiss, Samuel; Goodarzi, Aaron; Hawkins, Cynthia
    Glioblastoma is the most common malignant brain tumour in adults, and prognosis remains poor, with most patients dying in a year or two despite aggressive treatment. These tumours have high degrees of intratumoral heterogeneity, with a subpopulation of cells having stem cell properties. The drivers of this stem cell phenotype are complex, and include a large number of epigenetic and genetic factors. In this thesis, I explore glioblastoma heterogeneity in two distinct ways: I use single-cell chromatin accessibility data to explore influences of copy number alterations on the glioblastoma epigenome, and I apply functional experimental techniques and genomics to characterize macroH2A2, an epigenetic regulator in this disease. In the first part of my thesis, I develop a tool for calling copy number alterations in single-cell ATAC-seq data, called CopyscAT, and show that glioblastoma tumours have limited intratumoral genetic heterogeneity. In addition, genetic subclones share similar epigenetic profiles, but with slightly different predispositions to acquiring distinct cellular states. In the second section of the thesis, I focus on the role of macroH2A2, a histone variant involved in development and cancer. I find that expression of macroH2A2 is associated with improved prognosis in glioblastoma patients, especially in patients receiving treatment. Examining existing datasets, I find that macroH2A2 expression is enriched in neural progenitor-like cells and repressed at the leading edge of tumours, and confirm this in primary patient specimens. MacroH2A2 is associated with reduced self-renewal in vitro and in vivo and knockdown of the protein increases self-renewal markers, reduces the proportion of CD44-positive cells, and shortens mouse survival. Genome-wide, macroH2A2 knockdown results in altered accessibility, with loss of early response (FOS/AP-1) motifs, which I confirm in patient-derived single-cell ATAC-seq data. Chromatin immunoprecipitation confirms that direct effects of macroH2A2 are largely repressive in nature. Lastly, a screen of epigenetic drugs identifies a compound, MI-3, capable of increasing macroH2A2 levels, and shows that macroH2A2 repression impairs sensitivity to this compound. This work contributes novel ideas to the regulation of cell states and fates in glioblastoma.