Saudi Cultural Missions Theses & Dissertations
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Item Restricted THE ROLE OF USP51 IN REGULATION OF PRC2 IN OVARIAN CANCER(University at Buffalo, 2025-05) Alghanem, Meshal; Atanassov, BoykoBackground Despite advancements in ovarian cancer treatment, resistance to platinum-based chemotherapy remains a major clinical challenge and a key contributor to disease relapse. Understanding the molecular mechanisms driving this resistance is essential for identifying new therapeutic vulnerabilities. Recent studies have shown that USP51, a deubiquitinating enzyme (DUB) involved in transcriptional regulation, is downregulated in platinum-resistant ovarian cancer cells. To further investigate the role of USP51 in epigenetic regulation in ovarian cancer cells and its role in platinum resistance, we modulated its expression via knockdown and overexpression and assessed changes in drug resistance, histone modifications, and gene expression. Methods We used shRNA-mediated knockdown and ectopic overexpression of either wild-type or catalytically inactive mutant USP51 in ovarian cancer cell lines A1847 and OVCAR5 to assess changes in histone modifications. Histone extracts and whole-cell lysates were probed by western blotting, and histone levels were quantified using ImageJ. CUT&RUN was performed to evaluate genome-wide enrichment of H2AK119ub1 and H3K27me3. RNA expression levels of target genes were analyzed using RT-qPCR. Functional assays including colony formation and MTT assays were utilized to measure the sensitivity of the ovarian cancer cells to cisplatin and EHZ2 inhibitors. Statistical significance was determined using two-tailed t-tests and two-way ANOVA with post hoc analysis. Results Our study shows that USP51 removes H2AK119ub1, a repressive histone mark added by the Polycomb Repressive Complex 1 (PRC1). This mark facilitates the recruitment of PRC2, leading to the subsequent deposition of H3K27me3, both essential for chromatin-mediated gene silencing. USP51 depletion resulted in a significant increase in global levels of both H2AK119ub1 and H3K27me3, whereas overexpression led to their reduction. These changes occurred without affecting the expression levels of PRC2 core components (EZH2, SUZ12, and EED), suggesting that USP51 acts independently of PRC2 abundance to modulate epigenetic repression. By examining genome-wide changes in chromatin states, we observed a widespread accumulation of the H2AK119ub1 and H3K27me3 marks in cells depleted of USP51. There was significant co-occupancy of these marks at shared genomic loci. Enrichment and metaplot analyses confirmed the increased deposition of these marks, supporting a model in which the loss of USP51 enhances PRC2-mediated repression. Notably, genes that were enriched for H2AK119ub1 and H3K27me3 following USP51 knockdown—such as HOXD13, NRN1, CEACAM6, and BAMBI—were significantly downregulated. In contrast, the ablation of EZH2 resulted in their overexpression. Conclusions Our results demonstrate that USP51 plays a key role in regulating gene expression in ovarian cancer cells by modulating the deposition of H3K27me3, a repressive histone mark, at target loci. Its loss results in enhanced PRC2 activity, leading to gene silencing. However, altering the levels of USP51 expression did not affect the sensitivity of cancer cells to cisplatin treatment. This implies that the reduced expression of USP51 in platinum-resistant cells is a consequence of treatment adaptation, rather than a cause. The depletion of USP51, however, increases sensitivity to EZH2 inhibitors in ovarian cancer cell lines, indicating that the loss of USP51 represents a targetable epigenetic vulnerability. Our findings establish USP51 as a crucial regulator of the chromatin landscape in ovarian cancer. This presents an opportunity for patients with low USP51 expression to benefit from PRC2-targeted therapy, which may help overcome platinum resistance and enhance treatment outcomes.35 0Item Restricted HISTONE H3K27ME3 IN PREIMPLANTATION MOUSE EMBRYO DEVELOPMENT(Saudi Digital Library, 2023-06) Harasani, Ahlam; Pennings, Sari; Taylor, JaneDuring the developmental stages of the mouse preimplantation embryo, totipotent blastomeres generate the first three cell lineages of the embryo: trophectoderm (TE), epiblast (EPI) and primitive endoderm (PrE). The first cell fate decision consists of the separation of cells that will comprise the TE and the ICM (inner cell mass). Then, further segregation of ICM cells takes place into EPI-fated cells and PrE-fated cells. It is thought that this cell lineage specification is mediated in part by epigenetic processes, in which genes are regulated both temporally and spatially without any changes in the genome. Certain transcription factors such as Oct4, Sox2, Cdx2, Gata6, Nanog, and Fgf4 coordinate pluripotency and early cell fates during development. They are part of a network of pluripotency and associated genes that control each other’s expression as well as that of other major transcription factors through epigenetic mechanisms and simultaneous signalling pathways. Epigenetic mechanisms such as DNA methylation and post-translational histone modifications play a critical role in regulating gene expression in preimplantation embryos. During the embryonic development process, methylation of DNA is required for overall development. Similarly, trimethylation of lysine 27 on histone 3 (H3K27me3) is required to regulate developmental genes. This histone modification is facilitated by the polycomb repressive complex 2 (PRC2) subunit named enhancer of zeste homolog 1/2 (EZH1/2), an enzyme that epigenetically modulates chromatin structure and gene expression through H3K27me3 and may be involved in recruitment of DNA methyltransferases for gene silencing. The SET domain of EZH2 is thought to serve in the mechanism for transferring methyl groups from S-adenosylmethionine (SAM) to H3K27. EZH2 has a pivotal role to play in cancer progression and malignancy, but it also plays a vital role in the renewal, maintenance and differentiation of stem cells into specific cell lineages. Several recent studies have shown that blocking PRC2-EZH2 alone is not enough to suppress cancer-related genes. The dual inhibitors of both PRC2-EZH2 and PRC2-EZH1 were found to suppress H3K27me3 completely in cells after treatment. It is also suggested that Ezh1 may be involved in enhancing the maintenance of ES cell identity when EZH2 is not present. In this study, two small molecules, GSK-126 and EPZ-6438, have been used to inhibit the catalytic subunit of EZH2 in preimplantation mouse embryos in vitro to study the contribution of this enzyme through its inhibition in the embryos' developmental process. The objectives of this study are to determine the role of PRC2 complex methyltransferase EZH2 and the H3K27me3 epigenetic mark in the development of pluripotent ICM cells and other early cell fates in the mouse preimplantation embryo, based on morphology, epigenetic and cell fate markers and gene expression. This study was carried out using cultured preimplantation embryos from superovulated B6CBA F1 mice. Fertilised zygotes were collected at embryonic day 0.5 (E0.5). Embryos were cultured from age E0.5 (1-cell stage), to E1.5 (2-cell stage), E2.5 (8-cell stage) or until E4.5 (blastocyst stage) in the presence of either GSK-126 (1μM, 3μM), EPZ-6438 (0.5μM, 0.75μM, 1μM) or dimethyl sulfoxide (DMSO) as a vehicle. The effect of EZH2 inhibition was assessed based on morphology and using two techniques: reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for determining gene expression and immunocytochemistry (ICC) for measuring protein abundance. Quantitative analyses of the gene expression data of the relevant genes were conducted using the housekeeping genes GAPDH and H2Afz as endogenous controls. Indirect immunofluorescence staining was used in embryos, images were captured using confocal microscopy, and analyses were performed using MATLAB, IMARIS, and ImageJ software programs. The findings showed that inhibition of EZH2 reduced H3K27me3 levels in blastocysts. Also, the number of blastomeres was lower in the treated blastocysts than in control. EZH2 appears to influence developmental delay in the preimplantation mouse embryo. Furthermore, ICC results showed a decrease in the number of blastocyst cells expressing CDX2, the trophectoderm marker, compared with the control group. In conclusion, the results of this study will contribute to our understanding of the role H3K27me3 plays in early mammalian embryos during the specification and maintenance of cell fates.24 0