Medical Biology - Theses
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ItemCharacterising the molecular regulation of erythroferrone( 2023-10)ERFE encodes the hormone erythroferrone which is secreted from erythroblasts in response to increased erythroid drive. ERFE protein suppresses hepcidin, the master iron regulator, which allows iron to be released from body-iron stores and used for red blood cell production. In erythropoietic disorders such as thalassaemia, ineffective red blood cell production results in reduced tissue-oxygen levels, increased erythroid drive, and chronic hepcidin suppression. However, despite representing a possible drug target, the regulation of ERFE has not been well studied. This work has identified a putative ERFE control locus comprising an enhancer and several key transcription factors using in vitro differentiated Human Umbilical Cord Blood-derived Erythroid Progenitor (HUDEP-2) cells. ERFE transcription and chromatin accessibility were tracked during four stages of terminal erythroblast maturation using quantitative PCR and Assay for Transposase-Accessible Chromatin-sequencing (ATAC-seq). These data demonstrated a dynamic chromatin accessibility landscape with distinct erythroid maturation stages and an expression profile that peaked in intermediate erythroblasts (p<0.001). Capture-C then demonstrated contact between ERFE’s 5’ promoter and a putative enhancer that also aligns with trimethylation of lysine 4 and acetylation of lysine 4 on histone 3 (promoter marks), monomethylation of lysine 4 and aceytylation of lysine 27 on histone 3 (enhancer marks) Cleavage Under Targets & Release Using Nuclease (CUT&RUN). Moreover, when ERFE expression is at its highest, CUT&RUN showed that response elements in the enhancer are bound by master erythroid regulators GATA1, KLF1 and TAL1, and the stress erythroid response factor, STAT5, suggesting a role for multiple signalling pathways in ERFE activation. These pathways, and ERFE’s place within them, were further explored using weighted gene correlation network analysis on RNA sequencing from the four progenitor stages, where gene set enrichment analysis demonstrated that ERFE is co-expressed alongside genes that are highly associated with haem metabolism (p=3.65x10-30). Overall, this data provides new insights into the regulation of erythroferrone and may contribute valuable details for identifying therapeutic targets in iron-loading anaemias.
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ItemScreening for breath: identifying Aurkb as a novel regulator of lung development( 2019)The development of the lung is a highly regulated and complex process that is not fully characterised. Although there have been many studies into the development of the lung many of the mechanisms regulating lung organogenesis are still unclear. In recent years, the importance of epigenetic regulators in embryogenesis has been established but epigenetic control of lung morphogenesis is largely underexplored. I have developed a novel in vitro assay to grow embryonic lung stem cells. These cells, called pneumospheres express the early lung progenitor factor Sox9 and recapitulate the E11.5 lung throughout their time in culture. Pneumospheres can be genetically and chemically manipulated to assess the role of signalling pathways and genes-of-interest on lung progenitor cell self-renewal or differentiation. Using pneumospheres I performed a shRNA knockdown screen, targeting 130 genes involved in epigenetic regulation. Nineteen genes were identified in the screen and validated using in vitro and ex vivo culture systems to determine their role lung stem cells and branching morphogenesis. These experiments identified Aurora kinase B (Aurkb) as an interesting candidate gene. Aurkb exerts a dual role as a regulator of cell cycle and epigenetic control through phosphorylation of histones. Disruption of Aurkb either by short-hairpin RNA or by chemical inhibition in vitro abrogates growth of lung epithelial progenitor cells and causes defects in cell cycle, leading to an accumulation of cells in G2/M of the cell cycle. Conditional deletion of Aurkb in the embryonic lung, leads to a complete lack of lung tissue at birth and severe epithelial growth retardation can be seen as early as midgestation. Understanding the regulation of lung development will provide a better understanding of the lung organogenesis and how disruptions in normal biology can cause early lung diseases such as bronchopulmonary dysplasia or can have an impact on lung disorders later in life such as COPD or lung cancer.
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ItemMechanistic insights into how the epigenetic regulator Smchd1 interacts with and alters the chromatin( 2018)Structural Maintenance of Chromosomes, Hinge Domain containing 1 (Smchd1) is critical for the maintenance of X Chromosome Inactivation (XCI), and transcriptional repression at a subset of autosomal loci (Blewitt et al., 2008; Mould et al., 2013; Gendrel et al., 2013). Gain and loss of function mutations in SMCHD1 have been found to underlie Bosma arhinia micropthalmia syndrome (BAMS) and Facioscapulohumoral muscular dystrophy 2 (FSHD2), respectively - two distinct developmental disorders (Lemmers et al., 2012; Gordon et al., 2017; Shaw et al., 2017). Currently little is known about molecular mechanisms underlying the involvement of Smchd1 in transcriptional repression or disease. This project aimed to better understand how Smchd1 associates with and influences the chromatin. There has been growing evidence in the literature to suggest that Smchd1 and the non-coding RNA Xist might interact directly (Nozawa et al., 2013; Kelsey et al., 2015; Minajigi et al., 2015). We have previously shown that the hinge domain of Smchd1 binds synthetic DNA and RNA oligonucleotides in vitro (Chen et al., 2015). I was therefore interested in whether Smchd1 directly associates with endogenous nucleic acids, and whether such interactions could be important for Smchd1's localisation to the chromatin. To this end, I performed PAR-CLIP to determine whether Smchd1 binds endogenous RNAs genome-wide. I find Smchd1-RNA interactions to be non-specific, and are therefore unlikely to act as a targeting mechanism. I also find that while Smchd1 is dependent on Xist for its localisation to the Xi, this is not due to a direct protein-RNA interaction, but rather due to a dependency on the downstream HnrnpK-polycomb pathway. Evidence from our lab has suggested that Smchd1 may be involved in regulating higher order chromatin organisation (Chen et al., 2015). To investigate changes to the chromatin architecture in the absence of Smchd1, I have performed in-situ Hi-C and ATAC-seq in Smchd1 wild-type and deleted neural stem cells. For the first time my data have demonstrated a role for Smchd1 in chromatin organisation of the Hox cluster, but also the inactive X chromosome. Furthermore, I have identified that in the absence of Smchd1, Hox genes are dysregulated, implicating Smchd1 in Hox gene silencing via a role in chromatin conformation. Taken together the results from the body of work I present here allow me to put forward a model, in which Smchd1 is recruited to target loci by the recognition of a PRC1-mediated chromatin structure. At these sites, I propose that Smchd1 is involved in the maintenance of long-range repressive chromatin structures, which limit promoter-enhancer interactions that are permissive for transcription, potentially by preventing binding of Ctcf, and therefore Ctcf-mediated looping events.
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ItemMechanistic and structure-function characterisation of the epigenetic regulator Smchd1( 2015)Epigenetic regulation of gene expression is fundamental in controlling biological processes in multicellular organisms. Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor that plays critical roles in X chromosome inactivation, genomic imprinting and monoallelic expression of clustered protocadherin genes. In addition, SMCHD1 is crucial for suppressing the D4Z4 repeat implicated in pathogenesis of facioscapulohumeral muscular dystrophy (FSHD). However, the exact molecular mechanism by which Smchd1 regulates gene expression is unclear. Likewise, little is known about the structure and function of Smchd1 protein apart from that it contains two predicted domains, an N-terminal GHKL-ATPase domain and a C-terminal Smc hinge domain. To understand how Smchd1 mediates epigenetic regulation at the molecular level, I determined genome-wide Smchd1 binding sites in male murine neural stem cells by performing chromatin immunoprecipitation coupled with next-generation sequencing. Together with profiling gene expression and epigenetic marks in wild type and Smchd1-deficient cells, I found that Smchd1 binding at several of its known target genes is correlated with differential gene expression, concomitant with changes in epigenetic modifications. Unexpectedly, a significant proportion of Smchd1 occupancy overlaps with that of CCCTC-binding factor (Ctcf) at distal cis-regulatory elements, indicative of a functional relationship between Smchd1 and Ctcf. Indeed, I demonstrated that Smchd1 and Ctcf could evoke opposing effects on the expression of many protocadherin genes. As Ctcf is implicated in mediating chromatin interactions, these results indicate that Smchd1 may impart epigenetic regulation via physical association with chromatin and maintaining a repressive chromatin state that antagonises Ctcf facilitated chromatin interactions. In order to gain further mechanistic insights into how Smchd1 functions, I conducted structural-functional characterisation of the Smchd1 protein. I determined the domain boundaries and generated recombinant Smc hinge domain with its flanking coiled-coil and recombinant protein corresponding to the N-terminal region of Smchd1 encompassing the putative GHKL-ATPase domain. By performing small-angle X-ray scattering (SAXS) analysis, structural characteristics of those two domains were revealed for the first time. By utilizing a suite of biochemical and biophysical assays, I was able to show that the hinge domain could directly bind to nucleic acids in vitro. Furthermore, I found that a single-amino acid substitution within the hinge domain, equivalent to a modification implicated in FSHD pathogenesis, displayed significantly compromised binding activities. These results support the notion that the Smchd1-chromatin interaction via the hinge domain is critical for its role in epigenetic regulation. In addition, I demonstrated that a putative gain-of-function mutation of Smchd1, could potentially induce a conformational change and acquisition of ATP-binding activity of the N-terminal region of Smchd1. Preliminary results also suggest that this mutation could enhance Smchd1 function in the cellular context, potentially resulting from the altered structural-functional properties. Together, work from this thesis provides unprecedented molecular explanations for Smchd1’s action in epigenetic regulation, which are supported by structural-functional characteristics of the Smchd1 protein uncovered by this study. These results not only offer valuable insights into Smchd1 function, but also may inform future development of much needed therapy for FSHD disease where SMCHD1 is critically involved.
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ItemIdentification of epigenetic modifiers involved in neural stem cell function( 2015)It has become clear that epigenetic modifications of chromatin play a crucial role in regulating gene expression and cell fate decisions in stem cells; however, epigenetic pathways in neural stem cells (NSCs) have not been well characterised. In order to identify new epigenetic factors involved in NSC function, I conducted a pooled competitive RNAi screen in cultured primary embryonic NSCs using a bespoke, high coverage shRNA library that specifically targets epigenetic factors. With this approach, I discovered a novel NSC epigenetic modifier, Prdm8. When Prdm8 expression was reduced in NSCs they displayed a competitive advantage over control cells. Further studies determined that this advantage was mediated by increased cellular proliferation. In a separate targeted individual competition screen of polycomb group complex factors, I also discovered that reduced expression of the Ying Yang 1 gene, Yy1, decreased NSC competitiveness. Further investigation revealed that Yy1 knockdown resulted in a cell cycle defect; specifically, NSCs with reduced Yy1 expression accumulated in the G1 phase of the cell cycle and exhibited increased expression of the cyclin-dependent kinase inhibitor, p21, without co-induction of p53. Correspondingly, Yy1 overexpression led to decreased levels of p21. RNA sequencing of NSCs with reduced Yy1 expression revealed other differentially expressed cell cycle regulatory genes, including Cend1 and Ccna1. Cend1 has a previously published role in driving neural progenitors out of the cell cycle, and in promoting neuronal differentiation and thus the mechanism behind increased Cend1 expression in Yy1 knockdown NSCs is of particular interest. Chromatin immunoprecipitation followed by next generation sequencing using antibodies targeting H3K4me3 and H3K27me3 in control and Yy1 knockdown NSCs revealed the absence of genome-wide alterations or individual peak alterations indicating that the Yy1 phenotype may be correlated with alterations in another histone mark(s). In summary, I have shown that Yy1 and Prdm8 are novel epigenetic factors critical to NSC function. Future experiments will further elucidate their functions in vivo and in human brain cancer as NSCs share many characteristics with cancer stem cells.
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ItemScreening for epigenetic modifiers of X chromosome inactivation( 2013)Epigenetic regulation describes heritable changes in gene expression, but not the genome sequence. X chromosome inactivation is a model epigenetic process that involves the silencing of one of the two X chromosomes in female mammals. Much remains unknown about the mechanisms involved in X inactivation, as about epigenetic control in general, and there is scope for the identification of novel genes or the ascription of new functions to known genes. This project involved the development of different RNA interference screening approaches to identify epigenetic regulators required at different stages of X inactivation. A custom short hairpin RNA library targeting epigenetic regulators was used to perform screens in mouse embryonic fibroblasts for genes involved in the maintenance of X inactivation, by examining reactivation of a green fluorescent protein (GFP) transgene reporter present on the inactive X chromosome. From these screens, knockdown of the H3K9 methyltransferase Setdb1 resulted in reactivation of the silent GFP, implicating it in the maintenance of X inactivation. H3K9 methylation is a modification made to the chromatin of the inactive X, but the enzyme responsible for its presence is currently unknown. Targeting of other H3K9 methyltransferase genes with short hairpin RNAs did not lead to GFP reactivation. Setdb1-null female embryos were found to fail X inactivation as they showed aberrant expression of the transgenic GFP reporter. Some endogenous X-linked genes also showed low levels of reactivation in Setdb1-depleted mouse embryonic fibroblasts by RNA sequencing. An alternative system for simultaneously monitoring the activity of both X chromosomes by flow cytometry was also established, using knock-in alleles of GFP and a red fluorescent protein (mCherry) at the endogenous X-linked housekeeping gene Hprt. Female embryonic stem cells were derived with the knock-in alleles; these cells have two active X chromosomes and undergo X inactivation when differentiated. A screen was commenced to examine the initiation and establishment of X inactivation by monitoring the expression of the two fluorochromes during differentiation. Setdb1 depletion in these cells did not alter the early stages of X inactivation, suggesting that Setdb1 is more important for the maintenance of X chromosome silencing.
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ItemThe role of HBO1 during embryonic development( 2012)This thesis presents the first in vivo characterisation of HBO1 (histone acetyltransferase bound to ORC1; MYST2/KAT7) function at the molecular and cellular level. Specifically, HBO1 function was investigated during (a) early embryonic development, by analysing Hbo1 null mutant mice and (b) in brain development during late gestation, by analysing conditional transgenic mice displaying HBO1 deficiency in the central nervous system. HBO1 is essential for H3K14 acetylation (H3K14ac) and the activation of key developmental regulatory genes in both mouse models. In the first study, this process is crucial for the patterning of rapidly differentiating embryonic tissues such as the mesenchyme, vasculature, heart and neural tube during post-gastrulation development. In the second study, HBO1 mediated H3K14ac is indispensible for the expression of genes required for neuronal lineage commitment and differentiation. During embryonic brain development, HBO1 is critical for dentate gyrus patterning, neuronal migration, cortical lamination and layer specific gene expression. Unexpectedly, in contrast to numerous earlier reports implicating HBO1 in DNA replication, this thesis demonstrates that HBO1 is not essential for DNA replication. Together, in vivo and in vitro data presented in this thesis illustrate the dependency on HBO1 for H3K14ac, transcriptional activation and cellular differentiation.
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ItemThe role of Smchd1 in cancer( 2011)Cancer is one of the leading causes of death in Australia, with more than 43,000 people estimated to have died from cancer, and an estimated 114,000 new cases diagnosed in 2010. Cancer is traditionally viewed as a genetic disease, arising from cells that have acquired multiple mutations, some of which may be inherited. However, in recent years it has become increasingly apparent that many epigenetic regulators and processes go awry in cancer, providing evidence for a critical role for epigenetics in the pathology of this disease. Epigenetic regulation encompasses many biological processes including DNA methylation, histone modifications, non-coding RNAs and chromatin remodeling. Deregulated expression, somatic mutations and chromosomal alterations in many epigenetic modifiers involved in all of these processes have been identified in a plethora of human cancers. Much of the ongoing work in this field is dedicated to identifying novel genes or biomarkers, delineating the molecular basis of epigenetic mechanisms in cancer, molecular profiling and stratification of human cancers to better predict response to treatment, development of pre-clinical models for drug testing, and clinical trials for inhibitors of epigenetic modifiers. This thesis describes the role of a novel gene involved in epigenetic silencing of transgenes and X-linked genes in the development of cancer in two mouse models. Structural maintenance of chromosomes hinge domain containing 1 (Smchd1) is ubiquitously expressed in all tissues and cell types examined. Loss of Smchd1 in transformed mouse embryonic fibroblasts (MEFs) enhanced tumour growth in immunodeficient nude mice, providing the first indication for a tumour suppressor activity for Smchd1. Transformed Smchd1 null MEFs out-competed wildtype cells in an in vitro competition assay, suggesting that deficiency for Smchd1 may lead to increased proliferation or enhanced survival. Abrogation of Smchd1 in Eμ-Myc transgenic mice also led to marked acceleration of lymphomagenesis, with a median latency half that of their wildtype counterparts. Pre-malignant Smchd1 null Eμ-Myc transgenic mice exhibited a significant expansion in precursor B cells in the spleen, peripheral blood and bone marrow, which may account for the earlier lymphoma onset in these mice. Collectively, these results suggest that Smchd1 behaved as a tumour suppressor in both model systems. Gene expression profiling in MEFs and Eμ-Myc cells raised an interesting possibility of an association between Smchd1 and Myc and/or Polycomb repressive complex 2 (PRC2). Smchd1 may functionally interact with PRC2 to silence a subset of genes in tumour suppression. The elucidation of the key oncogenic pathways that facilitate the function of Smchd1 as a tumour suppressor would deepen our understanding of epigenetic misregulation in cancer.