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    Characterisation of pseudogene-like EP400NL in chromatin remodelling and transcriptional regulation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Li, Zidong
    EP400 is an ATP-dependent chromatin remodelling enzyme that has been implicated in DNA double-strand break repair and transcription regulation including Myc-dependent gene expression. It was previously shown that the ectopic expression of the N-terminal domain of EP400 increases the efficacy of chemotherapeutic drugs against cancer cells. This prompted the question of whether the EP400 N-terminal-Like (EP400NL) gene, which resides next to the EP400 gene locus, also plays a similar role in epigenetic transcriptional regulation to the full-length EP400 protein. To characterize the function of the EP400NL nuclear complex, a stable cell line expressing TAP-tagged EP400NL was established, and the EP400NL complex was affinity purified and analyzed by mass spectrometry. EP400NL was found to form a human NuA4-like chromatin remodelling complex that lacks both the TIP60 histone acetyltransferase and EP400 ATPase. However, despite no histone acetyltransferase activity being detected, the EP400NL complex displayed H2A.Z deposition activity on a chromatin template comparable to the human NuA4 complex, suggesting another associated ATPase such as BRG1 or RuvBL1/RuvBL2 catalyses the reaction. In addition to a role in H2A.Z deposition, it was also determined that the transcriptional coactivator function of EP400NL is required for serum and IFNγ- mediated transcriptional activation of the immune checkpoint gene PD-L1. EP400NL, cMyc and multiple identified ATPases such as BRG1, RuvBL1/RuvBL2 were shown to be recruited to the promoter region of PD-L1. To further demonstrate the importance of EP400NL in regulating Myc and IFNγ-mediated PD-L1 expression, CRISPR/Cas9 mediated EP400NL indels were introduced in H1299, a human non-small cell lung carcinoma cell line. These EP400NL indel cell lines show compromised gene induction profiles with significantly decreased PD-L1 expression from both Myc and IFNγ stimulation experiments. In contrast to full-length EP400NL, two deletion mutants (Δ246- 260 and Δ360-419) lacked the ability to enhance the expression level of PD-L1 mRNA or protein, indicating that these regions are important for coactivator activity. Collectively, these data show that EP400NL plays a role as a transcription coactivator for cMyc-mediated gene expression and provides a potential target to modulate PD-L1 expression in cancer immunotherapy.
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    Role of N-terminal domains of p400 ATPase in the ATM interaction and DNA damage response : a thesis presented in partial fulfillment of the requirements for a the degree of Master of Science (MSc) in Genetics at Massey University, Manawatū, New Zealand
    (Massey University, 2016) Weber, Lauren Elizabeth
    Efficient repair of damaged DNA and preservation of genomic integrity is integral in the maintenance of proper cellular function and prevention of unrestricted cell proliferation. One critical threat to the stability of the genome is the double strand break (DSB), arguably one of the most cytotoxic lesions to DNA. Interference with the DSB repair mechanism can lead to dysregulation of cellular systems and the prospective development of malignancies. Two critical proteins in DBS repair are the Ataxia Telangiectasia Mutated (ATM) kinase, a serine/threonine kinase from the Phosphatidylinositol 3-Kinase-related Kinase (PIKK) family, and p400, an ATPase chromatin remodeler. ATM is one of the first responders to DSBs and is responsible for the phosphorylation of a multitude of protein substrates including the histone variant H2AX. Beyond its phosphorylation ability, ATM has been proposed as a potential shuttle for other repair machinery, aiding in the early and efficient recruitment of proteins to the DNA damage foci. One such proposed protein is p400. The exact role of p400 in DSB repair is unknown but previous studies show that there is a decrease in repair efficiency in its absence. A prospective interaction is supported by previous studies in which p400 and p400 N-terminal derivatives co-immunoprecipitate with ATM in vivo in HEK293T cells. This study aimed to confirm the interaction of ATM and p400 N-terminal derivatives in vitro and explore the functional implications of the association in vivo in U2OS cells. It was not possible to isolate full-length p400 derivatives in vitro and thus no conclusive results were obtained. Functional assays revealed the ability of one p400 fragment, F1, to inhibit DNA repair and cell proliferation after DNA double-strand break induction with bleomycin. Ectopic expression of the other two p400 N-terminal fragments, F2 and F3, induced an inhibition of cell proliferation under standard growth conditions. Although no conclusive results were acquired, a trend emerged suggesting that N-terminal fragment F1 is able to interfere with ATM protein-protein interactions resulting in a decrease in the efficiency of the DNA damage response and repair. These results implicate F1 as a potential target for further research in both DNA repair and cancer therapy.
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    ATM and p400 : characterisation of a novel interaction between a DNA repair enzyme and a chromatin remodeler : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry
    (Massey University, 2014) Smith, Rebecca Jane
    The ability to maintain genomic integrity prevents unrestricted cell proliferation and the progression of cancer. DNA repair pathways such as the DNA double-strand break (DSB) response are essential in maintaining this integrity. This system requires activation of the serine/threonine kinase ataxia telangiectasia mutated (ATM) through acetylation by TIP60, a histone acetyl transferase, and subsequent ATM autophosphorylation. During DNA repair, activated ATM phosphorylates the histone variant H2AX several kilobases either side of the break site. This phosphorylation acts a signal for additional repair proteins and chromatin remodeling complexes which repairs DNA. In a previous study, H2AX phosphorylation was induced through the over expression of TIP60 or the SWI3-ADA2-N-CoR-TFIIIB (SANT) domain of p400. It was hypothesised that over expressed TIP60 or SANT domain was able to sequester a putative negative regulator from the ATM-TIP60 complex and artificially induce activation. This study aimed to investigate if a single domain of TIP60 or if a single helix from the three helix SANT domain was responsible for the activation of the ATM-TIP60 complex. Here, the ability of the chromo domain and zinc domain of TIP60 individually and the combined zincHat domain of TIP60 to induce H2AX phosphorylation as well as three helix deletion mutants of the SANT domain of p400 was examined. While all constructs were able to be expressed in human cell lines, the induction of H2AX was variable and non-reproducible. ATM belongs to the phosphatidylinositol 3-kinase-related kinase family (PIKK). Members of the PIKK family show domain homology, where the domain of one protein is replaced with the homologous domain of another member and the function of the protein is not altered. As p400 has been previously shown to interact with TIP60 and also Transformation/transcription domain-associated protein (TRRAP), a member of the PIKK family, it was hypothesised that p400 could interact with ATM (which also interacts with TIP60). This study confirms this novel interaction between ATM and p400 through the use of co-immunoprecipitation and protein localisation using confocal microscopy. This study provides a platform to further investigate the involvement of an ATM-p400 complex during DNA repair.
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    The role of HP1α and HP1β in breast cancer progression : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2012) Campbell, Tahnee Maree
    Breast cancer is the foremost cause of cancer-related deaths in New Zealand women. Metastasis of breast tumours increases the likelihood of fatality of the disease as treatment becomes more difficult and the tumours may interfere with the function of multiple organ systems. Consequently, the identification of biomarkers that may indicate the potential for a tumour to become metastatic are of great importance and may allow for the selection of more targeted treatment regimes. Heterochromatin Protein 1 (HP1) is a chromatin associating protein that facilitates heterochromatic spreading through its interaction with trimethylated H3K9. There are three HP1 isoforms found in mammals, HP1α, HP1β and HP1γ, each with differing functions and chromatin localisation patterns. Previous research has demonstrated that deregulation of either HP1α or HP1β expression occurs in several types of cancers. Both increases and decreases in HP1α expression have been reported in breast tumour samples, with a decrease in HP1α associated with breast metastases. However, what role loss of HP1α may have in promoting a metastatic phenotype is unclear, and any contribution of HP1β to this process is also explored. This thesis examined the roles of HP1α and HP1β in breast cancer progression through the creation of breast cancer cell lines with knock-down of either HP1α or HP1β. These cell lines were characterised for changes in proliferation, cell cycle profile, global chromatin compaction, invasive potential and anchorage independence. Though no changes were observed in the majority of these characteristics, a novel role for HP1β as a potential suppressor of anchorage independence was identified. Additionally, it was found that HP1α may act to enhance anchorage independence. This information could help to further knowledge of how breast cancer cells proceed towards metastasis, and provide new avenues of research into the potential for levels of HP1α or HP1β to be used as biomarkers for breast cancer progression.