<b>INVESTIGATING THE ROLES OF FUMARASE IN CELLULAR RESPONSES TO DNA REPLICATION STRESS AND DEVELOPING NOVEL CHROMATIN-BASED STRATEGIES FOR OPTIMIZING EXPRESSION OF TRANSGENES</b>
Creators: name: Kwizera, Ronard
Publisher: name: Purdue University Graduate School
Year: 2024
Subjects: classificationCode: 320505
subject: Medical biochemistry - nucleic acids
classificationCode: 320501
subject: Medical biochemistry - amino acids and metabolites
classificationCode: 320601
subject: Gene and molecular therapy
classificationCode: 300110
subject: Transgenesis
classificationCode: 321101
subject: Cancer cell biology

description: The eukaryotic genome is organized and packaged into DNA-protein complexes called chromatin. Nucleosomes, the repeating unit of chromatin, are composed of DNA in complex with histone octamers containing two of each of the four core histones, the H2A, H2B, H3, and H4. Accessibility of nucleosomal DNA by other DNA-binding proteins can be regulated, in-part, by the type and level of nucleosome-associated post-translational modifications (PTMs). PTMs, such as acetylation of lysine residues, including H3K9, H3K14, and H4K16, can neutralize their positive charge, thereby reducing the affinity of acetylated histones for the negatively charged DNA sugar phosphate backbone. This, in turn, promotes the formation of relaxed open chromatin which is crucial for the accessibility of DNA by the transcription machinery. Consequently, histone acetylation is associated with active gene expression, while deacetylation, which restores positive charges of lysine residues, is linked with condensed chromatin structure, and the associated decrease in gene expression. Other PTMs such as histone methylation on H3K9, which prevents H3K9 acetylation, promotes gene silencing via serving as a binding site for HP1, a structural component of silenced heterochromatin. Interestingly, this epigenetic control of gene expression, typically used to regulate endogenous genes, also influences ectopic gene expression in host cells. Transgenes delivered into target cells undergo host cell-mediated assembly into chromatin. As a result, transgenes assembled into hypoacetylated condensed chromatin experience a dramatic loss of expression shortly after delivery into target cells. As part of this thesis, we assessed the impact of "priming" plasmid-based transgenes to adopt accessible chromatin states on transgene expression. Nucleosome positioning elements were introduced at promoters of transgenes, or vectors were pre-assembled into nucleosomes containing either unmodified histones or histone mutants mimicking constitutively acetylated states at residues H3K9 and H3K14, or H4K16 prior to their introduction into human cells. Transgene expression was then monitored by epifluorescence microscopy and flow cytometry over time. We found that DNA sequences capable of positioning nucleosomes influenced the expression of adjacent transgenes in a distance-dependent manner, even in the absence of pre-assembly into chromatin. Intriguingly, pre-assembly of plasmids into chromatin facilitated prolonged transgene expression compared to plasmids that were not pre-packaged into chromatin. Interactions between pre-assembled chromatin states and nucleosome positioning effects on reporter gene expression were also assessed. Overall, nucleosome positioning played a more significant role in influencing gene expression than priming with hyperacetylated chromatin states. These findings have direct relevance to ongoing efforts to develop durable plasmid-based gene therapies for genetically-derived disorders such as cancer.In this thesis, we also explored the roles of the tumor suppressor enzyme fumarate hydratase (FH) in cellular responses to DNA replication stress. In humans, biallelic loss of function mutations in FH predisposes individuals to hereditary leiomyomatosis and renal cell carcinoma (HLRCC). However, the role of fumarase in HLRCC is not fully understood. The eukaryotic genome experiences thousands of lesions per cell each day, with DNA double-strand breaks (DSBs) being among the most deleterious. If unrepaired or repaired incorrectly, DSBs can lead to various disorders, including cell death and cancer. Cellular processes such as metabolism, transcription, and DNA replication are among the major endogenous sources of DSBs. Impediments to DNA replication that result in persistent stalling of replication forks can lead to fork collapse, thereby exposing unprotected single-stranded DNA (ssDNA) to endonuclease attack and the formation of double-strand breaks (DSBs). DNA-protein crosslinks that form irreversible complexes, an imbalance or exhaustion of deoxynucleotides (dNTPs), as well as the presence of inherently difficult-to-replicate genomic regions such as common fragile sites (CFSs), are some of the many obstacles that can halt fork progression. Intrinsically, however, all organisms have evolved response mechanisms designed to sense, prevent, detect, and resolve the different sources of DNA replication stress. These response mechanisms are governed by the highly conserved DNA replication checkpoint (DRC), a part of the intra-S phase checkpoint. The main function of the DRC is to halt cell cycle progression and activate DNA damage responses, such as DNA repair and subsequent replication fork restart. The highly conserved tricarboxylic acid (TCA) cycle enzyme fumarase (FH in humans, Fum1p in yeast), which functions to convert fumarate to malate in the mitochondrial TCA cycle, has been implicated in DRC responses in the cell’s nucleus. Upon exposure of yeast model organism to hydroxyurea (HU), an inhibitor of ribonucleotide reductase (RNR) that results in the exhaustion of cellular dNTPs, the expression of Fum1p is upregulated and Fum1p is translocated to the nucleus. Fum1p’s metabolite fumarate suppresses sensitivity to HU in yeast in a manner independent of modulating cellular dNTP levels. Notwithstanding, our understanding of these extra-mitochondrial functions of fumarase remains incomplete. For example, the nature of the impediments to DNA replication that are affected by fumarase is presently unclear. In addition, across all eukaryotes, fumarase lacks a canonical nuclear localization signal, and the means of its nuclear translocation upon DNA replication stress remain a mystery. In this study, our immunofluorescence experiments revealed that, similar to yeast, exposure of human cells to HU promoted the nuclear translocation of fumarase. We also performed Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) using human cells exposed to DNA replication stress by HU to identify replication stress-dependent protein-protein interactions of FH. We observed that FH co-precipitated MUS81, a structure-specific endonuclease and a crucial component of cellular responses to DNA replication stress. MUS81 is localized to stalled replication forks during the S phase of the cell cycle, where it cleaves the three-way junctions created by stalled forks. MUS81 also functions to resolve recombination intermediates and Holliday junctions (HJs) during the S phase. MUS81 also localizes at common fragile sites (CFSs) during the G2/M phase. CFSs are genomic regions that are difficult-to-replicate, late-replicating, and tend to exit the S phase with under-replicated DNA. These CFSs are targeted by MUS81, cleaved, and replicated via Break-Induced Replication (BIR) that is dependent on POLD3 in a process called mitotic DNA synthesis (MiDAS). In our immunofluorescence studies, we observed that HLRCC-derived cancer cells expressing low levels of catalytically inactive fumarase exhibited bulky anaphase bridges. These observations are especially intriguing because the loss of MUS81 or inhibition of POLD3 is known to increase the frequency of bulky anaphase bridges, a phenotype associated with defects in MiDAS. Additionally, we observed that exposure of fumarase-deficient cells to aphidicolin (APH), an inhibitor of POLD3, dramatically increased the frequency of anaphase bridges. Furthermore, we found that upon exposure of human cells to APH, fumarase translocated to the nucleus. Whether fumarase suppresses the MiDAS defects observed in HLRCC cells is still under investigation. Taken together, the work described herein uncovers previously unknown roles of fumarase in DNA damage responses and provides direct links toward understanding how fumarase may function to safeguard genomic integrity.