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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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    The transcriptional regulation of maspin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2004) Hollings, Andrew
    Maspin (mammary serine protease inhibitor) is a tumour suppressing member of the serpin superfamily. Maspin is expressed in normal breast and prostate cells, but reportedly down regulated during progression of cancer in these tissues. Maspin has been shown to inhibit cellular migration and invasion in vitro; while in vivo, maspin has been shown to inhibit tumour growth, metastasis, and angiogenesis. Maspin also plays a role in the sensitisation of cells to induced apoptosis. These functions of maspin are independent of serine protease inhibition; however the cellular mobility function is dependent on an intact reactive site loop. Despite this knowledge, the molecular mechanisms for all reported functions of maspin are currently unknown. Maspin is reported to be transcriptionally regulated: to date Ets, Ap1, and p53 transcription factors have been shown to activate transcription of maspin by binding directly to the promoter. Androgen is reported to be a negative regulator through the binding of the androgen receptor to a hormone response element within the promoter. This hormone response element is also responsible for an increase in maspin expression in response to tamoxifen, an anti-oestrogen drug. Transcriptional regulation of maspin has also been reported to be activated by other molecules, including gamma linolenic acid, manganese containing super-oxide dismutase, and nitric oxide, the mechanisms of regulation by these molecules is unknown. Loss of maspin expression in cancerous cells lines has been attributed to loss of one or more of the activating factors, and aberrant methylation of cytosine residues resulting in chromatin compaction. This study investigated the transcriptional regulation of maspin, with the aim of identifying transcriptional effectors important to the regulation of the gene. Identification of such factors may help identify a pathway in which maspin exerts its tumour suppressor functions. To this end, the maspin promoter was cloned and functional assays carried out. identifying several putative regions of the maspin promoter which may be important for the regulation of the gene. To date, the precise activator/repressor binding sites and the cognate proteins responsible for this regulation are unidentified.
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    Functional characterization of two plant type I MADS-box genes in Arabidopsis thaliana : AGL40 and AGL62 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2008) Kaji, Ryohei
    MADS-box transcription factors (TF) are a family of evolutionary conserved genes found across various eukaryotic species. Characterized by the conserved DNA binding MADS-box domain. MADS-box TF has been shown to play various roles in developmental processes. MADS-box genes can be based on MADS-box structural motifs divided into type I and type II lineages. In plants very limited functional characterization have been achieved with type I genes MADS-box genes. In this project we attempted to functionally characterize 2 closely related members of the type I lineage MADS-box genes AGL40 and AGL62 and give further support to the hypothesis that plant type I MADS-box genes are also crucial to normal plant development. Based on our expression domain characterization assay using AGL62: GUS fusion construct, we have shown expression of AGL62 in various tissues but especially strong in developing seeds, pollen and seedling roots and shoots. The web based microarray data suggesting that AGL62 may have a function in seed, pollen and seedling development backed up this result. Interestingly when we carried out PCR based genotyping with segregating population of heterozygous AGL62 T-DNA insertion lines (agl62/+) to identify the homozygous T-DNA insertion lines we detected no homozygous T-DNA insertion line indicating loss-of-function of AGL62 may be lethal to plant. With reference to the AGL62 expression in pollen, seed and seedling root and shoot, we carried out phenotypic assay on each of these tissues in agl62/+ background to investigate whether there was any phenotypic defect observed. Significant reduction in number of seeds was observed in agl62/+ indicating possible role of AGL62 in seed development. Our microscopic observation of seeds from agl62/+ plants showed defective embryos and confirmed that AGL62 plays a role in seed development. Our data on AGL62 is the first report that confirms AGL62's involvement in plant development and can be a ground work for further works on functional characterization of other members of plant type I MADS-box genes.
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    Characterization of the Arabidopsis MADS-box transcription factor, AGL104 : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2007) Reddy, Arti S
    AGL104 is an Arabidopsis MADS-box transcription factor belonging to the MIKC* clade. The exclusive expression of MIKC* genes in the gametophyte generation of both mosses and angiosperms has fueled questions regarding the function of these genes in both these taxa and the notion that the developmental program of the gametophyte generation in both these taxa may be fundamentally similar even though the structures themselves differ greatly in their phenotype. Since transcription factors control development and changes in the developmental control genes is thought to be a major source of evolutionary changes in morphology, characterization of MIKC* genes is expected to provide clues to the evolutionary changes in land plant body form. In angiosperms. AGL104 is reported to be expressed late and exclusively in the male (pollen) and female (embryo sac) gametophyte. Since late pollen development, such as pollen germination and pollen tube elongation, is thought to occur independently of transcription, the exclusive and high level of expression of a transcription factor is thus intriguing. We report the expression of AGL104 in developing anthers, mature pollen, pollen tubes and the egg apparatus of the embryo sac. Our study is the first report of AGL104 expression in the pollen tubes. Our data showing spatial expression of AGL104 in the different developmental stages of pollen, with weak expression in the uninucleate microspore that increases and culminates in the mature pollen, is also novel since spatial expression of this gene during pollen development had not been previously reported. Functional characterization through gain-of-function and loss-of-function analyses shows that AGL104 promotes pollen germination and an increased pollen tube length when measured 4 hours after pollination. The implication of this data is that, despite popular notions, active gene regulation is taking place during pollen germination and tube elongation. Further functional analysis in the pollen and the embryo sac is required to establish the precise role of AGL104 in the angiosperms. This information will then lay the groundwork for future comparisons of MIKC* activity in the basal and higher plants and determine if changes in MIKC* gene function were responsible for evolutionary changes in land plant body form.