Browsing by Author "Fitzsimons, Helen"

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    Characterising CG5846 (Peep) in Drosophila melanogaster neural function : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2024) Wilson, Sarah Jean
    Histone deacetylase 4 (HDAC4) is a transcriptional regulator that has been implicated in a number of neurodevelopmental and neurodegenerative diseases that are associated with intellectual disability, cognitive defects, and/or memory loss. Both the accumulation of nuclear HDAC4 and its loss-of-function have been linked to these conditions, therefore exploring HDAC4’s role in neuronal function is essential to understand the molecular mechanisms underlying these diseases. In Drosophila, overexpression of HDAC4 results in defects in morphogenesis of axons in the mushroom body, a structure essential for memory formation, as well as long-term memory defects and disruption to the development of the compound eye. The molecular mechanisms underlying these HDAC4-induced phenotypes are currently unknown. RNA-sequencing on fly heads in which HDAC4 was overexpressed has previously been performed and showed few genes were transcriptionally regulated by HDAC4. In addition, an enhancer/suppressor rough eye phenotype screen has also been performed which identified a number of genes that interact genetically in the same molecular pathway as HDAC4. To further investigate the molecular mechanisms underlying HDAC4 dysfunction, an RNA interference (RNAi) based candidate screen for potential HDAC4-interactors was performed, which involved quantification of developmental defects in the mushroom body and eye following RNAi knockdown of each candidate. It was hypothesised that if a phenotype resulting from RNAi knockdown was similar to that induced by HDAC4 overexpression, that candidate may function in similar molecular pathways. A single candidate-interactor was selected (CG5846, named Peep) for further investigation. On overexpression, Peep and HDAC4 co- distribute in nuclei of mushroom body neurons, however no physical interaction was detected. Furthermore, overexpression of Peep did not rescue the HDAC4-induced mushroom body or eye defects. Due to the uncharacterised nature of Peep, a thorough investigation was performed to assess the importance of Peep in survival, longevity, motor function, brain development, courtship learning and memory, and wing development. Peep was observed to be essential for survival of glial cells and for normal mushroom body development, which warrants further investigation. Reduced expression of Peep also resulted in a unique severe necrotic eye phenotype, and through this, Peep was shown to play a potential role in processes involved in regulating mitochondrial and proteasomal function, apoptosis and oxidative stress. These data provide the first documented characterisation of the functional role of Peep in Drosophila development and provide the basis for further investigation into the underlying molecular mechanisms involved in mushroom body and eye development.
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    Development of a reporter gene assay to identify control elements required for dosage compensation in Drosophila Melanogaster : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University
    (Massey University, 1998) Fitzsimons, Helen
    Dosage compensation (equalisation of X-linked gene products) occurs in Drosophila melanogaster by a two-fold transcriptional increase of X-linked gene expression in the male. This involves the binding of four proteins, MSL-1, MSL-2, MSL-3 and MLE (collectively known as the MSLs), to hundreds of sites along the length of the male X. The MSLs are thought to recruit MOF, a histone acetyl transferase, which facilitates the increase in transcriptional activity of X-linked genes. The DNA sequences required to target the MSL complex to the X chromosome (known as dosage compensation regulatory elements, or DCREs) remain elusive, despite numerous attempts over the last ten years to identify them. DCREs are thought to be present at multiple sites along the length of the X chromosome, as antibodies to the MSLs bind to hundreds of sites along the X, and autosomal genes transduced to the X usually become dosage compensated. The first objective of this study was to develop a reporter gene assay to screen for DCREs that would minimise problems previously encountered. A construct consisting of the constitutive armadillo promoter fused to the lacZ reporter gene (called arm-lacZ) was flanked by insulator elements which block the repressive effects of the autosomal chromatin environment. Fragments of X-linked DNA were inserted upstream of the armadillo promoter with the premise that males carrying one copy of an autosomal insertion of this construct would express twice the level of ß-galactosidase as females. Transgenic flies carrying autosomal insertions of X-linked fragments plus arm-lacZ were generated and one dose males and females were assayed for ß-galactosidase activity using a spectrophotometric assay. In all cases, males and females expressed the same level of lacZ. This suggests that no DCREs that could confer dosage compensation onto arm-lacZ were present in the X-linked fragments. arm-lacZ is capable of being dosage compensated as males and females carrying one copy of an X-linked insertion of arm-lacZ produce a 2:1 male to female ratio. This implies that DCREs of the 'strength' required to dosage compensate arm-lacZ are rarer than previously thought. A second method of dosage compensation that is independent of the MSLs is thought to occur in Drosophila. The X-linked gene runt is dosage compensated in the absence of the MSLs. It is possible that runt is sex specifically regulated by the female specific Sex lethal protein (Sxl). Sxl down-regulates msl-2 in females by binding to (U)8 or A(U)7 sequences in the msl-2 5' and 3' untranslated regions (UTRs) of the mRNA. runt mRNA contains three Sxl binding sites in its 3' UTR, as do 20 other X-linked genes. The second objective of this project was to determine if Sxl could down regulate a gene in females, purely by the addition of three Sxl binding sites to the 3'UTR. Sxl binding sites were inserted into the 3'UTR of arm-lacZ in the form of a 40 bp synthetic linker containing three of the sites, and also as a 170 bp fragment from the runt 3' UTR. ß-galactosidase assays of flies carrying the Sxl binding sites from runt showed that males expressed an average of 1.31 to 1.46 times the level of lacZ than females. This shows that Sxl can down-regulate a gene if there are Sxl binding sites in its 3' UTR, however, to achieve two-fold regulation, additional factors may be required, or topologically, the sites may not have been in the right position in the 3' UTR for optimal activity of Sxl. Flies carrying the synthetic linker expressed the same level of ß-galactosidase in both sexes which suggests that either additional elements within the 3' UTR are required, or that the spacing between the sites is critical for the action of Sxl.
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    Investigating HDAC4 aggregation in a Drosophila model of neuronal development : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2024-06-21) Hawley, Hannah Rose
    Histone deacetylase four (HDAC4) is essential in neuronal development and function, and dysregulation of HDAC4 has been observed in a number of neurodevelopmental and neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. In particular, its aberrant nuclear accumulation is a common feature among these diseases, and it has been observed that upon upregulation or accumulation in the nucleus, HDAC4 forms punctate foci in neuronal nuclei. Previous research in a Drosophila model determined that overexpression of HDAC4 disrupted both neuronal development and long-term memory, and this was largely mediated by the nuclear pool of HDAC4. Based on these data, it was hypothesised that aggregates of HDAC4 are responsible for the neurotoxicity that leads to disrupted neurodevelopment and memory. Therefore, this study aimed to determine whether the presence of HDAC4 nuclear aggregates correlated with neurodevelopmental deficits in a Drosophila model of neurodevelopment, and if so, how they mediate their toxic effects. The N-terminus of HDAC4 forms homo-oligomers in solution, and it was hypothesised that full-length HDAC4 similarly oligomerises, and that this is required for its aggregation in neuronal nuclei. Mutations predicted to prevent oligomerisation were introduced into the N- terminus of HDAC4 and were shown to significantly reduce aggregation of HDAC4 in Drosophila neurons. Furthermore, their presence also reduced the severity of HDAC4 overexpression-induced impairments in neurodevelopment. Conversely, stabilisation of oligomerisation increased aggregation and the severity of neurodevelopmental phenotypes, together indicating that aggregation positively correlates with the severity of neurodevelopmental deficits. HDAC4 aggregates have been previously shown to sequester the transcription factor MEF2, and further investigation revealed that the presence of MEF2 stabilised aggregation and increased the severity of defects in neuronal development. Importantly, targeting the interaction between HDAC4 and MEF2 reduced the severity of these defects. Other than MEF2, the composition of HDAC4 aggregates is unknown, and therefore immunoprecipitation-coupled mass spectrometry was performed on nuclear HDAC4 to identify candidate interactors of aggregates. This revealed a number of proteins with roles in neuronal development and function, as well as those involved in splicing and protein homeostasis, suggesting that aggregates may be impairing these processes to mediate toxicity. Together these data indicate that nuclear aggregation of HDAC4 impairs neurodevelopment, and may constitute a novel biomarker of disease or therapeutic target. Given the overlap in aetiology between neurodevelopmental and neurodegenerative diseases, further investigation of whether HDAC4 aggregation contributes to the severity and/or progression of neurodegenerative disorders is warranted.
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    Investigating the role of HDAC4 in Drosophila neuronal function : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand
    (Massey University, 2022) Tan, Wei Jun
    HDAC4 plays an essential role in brain functions including neurodevelopment and memory formation, and increased levels of HDAC4 have also been associated with neurodegenerative disorders including Alzheimer’s disease. Histone deacetylases are enzymes that are traditionally known to regulate gene expression in the nucleus, however in neurons, HDAC4 shuttles between the nucleus and cytoplasm with a predominant distribution in the cytoplasm. Although studies have identified potential differences in subcellular function in which accumulation of nuclear HDAC4 has been shown to promote neurodegeneration, while cytoplasmic HDAC4 is neuroprotective, the mechanistic pathways through which it acts are still unknown. Therefore, this project aimed to determine the importance of nuclear and cytoplasmic pools of HDAC4 to the neurological functions of Drosophila melanogaster, as well as to determine the domains within the protein that are required for its function(s). This was carried out by expressing HDAC4 with mutations that resulted in altered subcellular distribution or carrying mutations in binding domain/motifs that have previously been shown to be important for HDAC4 function. Increased expression of wild-type HDAC4 disrupted development of the retina and the mushroom body (MB, a brain structure derived from Kenyon cells which are crucial for learning and memory), and expression of each mutant revealed the importance of specific domains/motifs to HDAC4 function in these tissues. Of interest, impairments to MB formation were exacerbated by mutation of the ankyrin-binding site and by mutation of serine residues that promote nuclear exit when phosphorylated (i.e. resulting in restriction to the nucleus). Mutation of the MEF2-binding site ameliorated these phenotypes, suggesting that HDAC4 acts through MEF2 to regulate MB development. However, while deacetylase activity was found to be dispensable in the MB, an active deacetylase domain was required in order for the phenotype to manifest in the retina, and mutation of the MEF2-binding site had no impact on the deficits caused by nuclear restriction of HDAC4 and mutation of the ankyrin-binding domain. Together these data indicate that HDAC4 acts through varying mechanism(s) depending on the cell type. Transcriptional changes in the Drosophila brain resulting from the expression of HDAC4 or its mutant variants was also explored using RNA-Seq. However only wild-type HDAC4 resulted in a large number of differentially expressed genes and the low level of differential gene expression in HDAC4 variants suggests that non-transcriptional processes may be involved in the induction of phenotypes caused by expression of these mutants. Additionally, further analysis of genes that were differentially regulated revealed a number of processes related to mitochondrial energy production. These findings have provided new insights into the role of HDAC4 in Drosophila neurodevelopment which opens up additional research avenues to focus on in the future.