Medical Biology - Theses
Now showing items 1-12 of 239
Genome in 3D – regulation of lymphocyte development by genome architecture
The functionally and phenotypically diverse cell populations that make up the immune system arise from the expression of a select part of a genome at a given time. The mechanisms governing such differential transcription are still, surprisingly, not fully understood. Only recently has the three-dimensional organisation of chromatin in the interphase nucleus been acknowledged to play a crucial role in modulating transcription. For instance, distal cis-regulatory elements like enhancers can form long-range chromatin loops with promoters to drive transcription, and these chromatin loops are in turn harboured in topologically associating domains (TADs), shielded from interference by outside elements. These three-dimensional structures can be lineage-specific and their roles during cellular differentiation are beginning to be uncovered. The chromosome conformation, or genome architecture, in B- and T-lymphocytes, captured previously as Hi-C data in the lab, has provided immense information about lineage-specific DNA interactions that might be critical during differentiation. Based on this resource, the work herein aimed to develop an approach to identify, characterise and functionally dissect any novel and critical regulatory elements. Using this strategy, I have identified several putative T- and B-cell specific elements and subsequently adopted the CRISPR/Cas9 platform in generating large deletions as to dissect these elements. The approach has identified and confirmed the enhancers of T cell-specific transcription factors Bcl11b and Gata3. Upon closer inspection an uncharacterised long non-coding RNA (lncRNA) Gm13218 was uncovered to associate with the enhancer of Gata3. Given the recent recognition of lncRNAs as important regulator of the 3D genome, I have retrieved the full-length sequence and characterised its expression pattern. It was found that expression of Gm13218 is highly correlated with that of Gata3 during early T cell development in thymus as well as T helper 2 (TH2) cell differentiation. Knockdown and overexpression of Gm13218 transcripts, CRISPR-mediated silencing, activation, demethylation of the locus as well as interference of transcription elongation suggest that Gm13218 may be involved in the establishment, but not the maintenance of Gata3 expression. By utilising Hi-C, RNA-seq, cell division and cell cycle indicators, the spatiotemporal dynamics of genome architecture during B cell activation and terminal differentiation into antibody-secreting cells was examined. It was revealed that genome organisation exhibit two discrete waves of restructuring – the first occurs just prior to the first cell division, with the resulting genome architecture being inherited through the subsequent rapid clonal expansion for many days until the second wave of restructuring upon differentiation into plasmablast. In addition, the first restructuring event was shown to precede the first DNA replication phase, suggesting that genome reorganisation is independent of, and well partitioned from, DNA synthesis and mitosis. In contrast, transcription underwent very early burst and was altered throughout the entire differentiation process. Further analysis suggests that transcription is intricately intermingled with genome organisation in a reciprocal fashion. Overall, the work in this thesis has revealed a number of important findings regarding how the 3D genome controls the development and function of the immune system.
The role of the MYST lysine acetyltransferase TIP60 in human cells and mice
Histone acetylation affects the way DNA and associated proteins are packaged in the cell nucleus and regulate chromatin organisation and gene expression. Acetylation of core histones has been broadly correlated with initiating and maintaining open chromatin, poised or active gene transcription, DNA damage repair, as well as chromosome decondensation during mitosis and meiosis. The acetylation of lysine residues is catalysed by histone lysine acetyltransferases (KATs) and deacetylases (HDACs), which are tightly regulated. Dysregulation of KATs and aberrant lysine acetylation has been associated with tumorigenesis and negative prognoses in a wide range of cancer, presenting a new area of potential therapeutic targets. Potential acetylation targets of KATs catalysing the acetylation of histones have predominantly been studied in cell-free assays, where the enzymes show little substrate specificity. In contrast, histone acetyltransferases appear to acetylate surprisingly specific residues in whole cells. In this thesis I investigate the effects of acute deletion of the MYST lysine acetyltransferase TIP60 (KAT5) using inducible cre-recombinase and CRISPR/Cas9-mediated deletion in human cells and mouse cells, as well as mouse embryos and its potential role in cancer cells. I found that loss of TIP60 caused complete cell growth arrest in human and mouse cells. In the absence of TIP60 cells displayed cell cycle arrest in G1 and G2/M phase with increased endoreplication, accompanied by chromosomal segregation defects. Remarkably, the proliferation arrest caused by loss of TIP60 also occurred in the absence of the tumour suppressors p53, INK4A and ARF and therefore was independent of these. In contrast, cell survival was not affected. Growth arrest independent of major tumour suppressors flags TIP60 a potential target for novel cancer therapeutics. TIP60 was found to be essential for of H2AZ acetylation, particularly, lysine 7 acetylation. In contrast, global chromatin bound H2AZ levels were not reduced. H2A and H4 acetylation was reduced slightly in TIP60 depleted cells. Identifying H2AZ lysine 7 acetylation as a biomarker for TIP60 activity is a major step in developing TIP60 as a drug target. The mRNA levels of 6236 human and 8238 mouse genes, including many metabolic genes, were dependent on TIP60, supporting a role for TIP60 as a key transcriptional co-activator. Characterization of key mechanisms causing chromosomal aberrations and identifying approaches that can be used to reduce likelihood of genome instability due histone acetylation defects will pave the way for better detection of early changes in cancer and development of therapeutic applications in the future. This work represents important steps towards the development of histone lysine acetyltransferases as drug targets. A comprehensive analysis of histone acetylation activity of TIP60 shines light on the potential of its many proposed roles.
Identification of novel regulators of B lymphocyte biology
The differentiation of B cells into antibody secreting cells (ASCs) and the production of protective antibodies is a critical part of the adaptive immune response to infection. ASCs are also important for the formation of immunological memory which provides protection against reinfection and the generation of ASCs is the goal for almost all current vaccination strategies. Despite the importance of these cells, we still lack a complete understanding of the factors that control B cell differentiation into ASCs, ASC survival and antibody secretion, all of which must be tightly regulated to ensure an optimal immune response. Here, I have developed a CRISPR/Cas9 mediated arrayed screening approach for the identification of novel positive and negative regulators of primary mouse B cell proliferation, survival, differentiation into ASCs and antibody secretion. By interrogating multiple gene sets I have identified all elements within the ASC gene signature that are essential for the in vitro generation of ASCs. I have also identified several novel negative regulators of the B cell differentiation process (AB124611, Arhgef18, A430078G23Rik, Fam43a, Pold1, Ripk3, Rnf130 and Rps6ka5). This work has also uncovered a novel role for 6 genes, (Cdv3, Hspa5, Sec61a1, Selk, Sumo2, Vcp) in driving the proliferation of B cells. One of these genes, Cdv3, has no previous association with proliferation in any cell type and presents an exciting new candidate for further investigation. I have demonstrated that within the ASC gene signature there are 35 genes which are essential for efficient antibody secretion. Interestingly, many of these genes are components of the ER protein processing pathway, however, not all elements of this pathway appear to be essential for antibody secretion. These results raise the possibility of there being a specific pathway for antibody secretion, or that the genes identified in this thesis may represent weak links in the ER protein processing pathway which could potentially be exploited therapeutically to inhibit antibody secretion in disease settings. Finally, I have used an Irf4 deficient mouse model to uncover a novel role for Irf4 in the development of the peritoneal B-1a population. I have shown that Irf4-/- mice lack peritoneal B-1a cells and by examining multiple stages of B-1a cell development I have demonstrated that in the absence of Irf4, B-1a cell development is blocked at the transitional B-1a stage. By employing RNA sequencing to analyse the transcriptional profiles of the remaining Irf4-/- B-1 cells and analysis of previously published ChIP sequencing data, I have revealed a potential role for Irf4 in directly activating the expression of Bhlhe41, a transcription factor that is required for B-1a cell development and homeostasis. Together, the results from this thesis build upon decades of previous work on the genetic regulation of B cell biology. Integrating the novel regulators of B cell proliferation and differentiation that I have identified in this thesis into the current model of ASC generation will improve our understanding of how the decision between undergoing differentiation or maintaining the B cell fate is made. A detailed understanding of how this fate decision is made has far reaching implications for human health and disease as this information can be used to inform vaccine design, reveal the causes of immunodeficiencies or highlight novel avenues for targeting pathogenic ASCs in autoimmunity and cancer.
The ontogeny of effector regulatory T cells
Regulatory T (Treg) cells are critical for the maintenance of immune homeostasis and peripheral tolerance. Different subsets of Treg cells have recently been described with many studies showing the importance of context-specific differentiation of Treg cells, in particular within non-lymphoid organs. These non-lymphoid organ Treg cells have a fully suppressive Treg cell phenotype with an effector function and are termed effector (e)Treg cells. However, the ontogeny of eTreg cells have not yet been fully described. Additionally, molecular determinants of the eTreg cell program remain incompletely understood. My thesis examines the transcriptional events that regulate the generation of eTreg cells during their thymic development, their homeostasis and response to infection. Using different gene targeted mouse models at steady state and in viral infection models, I studied the intrinsic molecular mechanisms that contribute to eTreg cell differentiation. In particular, I focused on follicular Treg (TFR) cells, which constitute the eTreg cell subset of the germinal centre. The molecular control of eTreg cell fate and function converges on the transcription factors IRF4 and Blimp-1. IRF4 is induced by antigen receptor signals and cooperates with AP-1 factors, BATF and JUN, to regulate transcriptional networks involved in lymphocyte differentiation, function and metabolism. For example, these factors regulate genes important for antibody class switch recombination in B cells and functional differentiation of distinct CD4 T helper (Th) subsets, including Th2, Th9, Th17 and T follicular helper cells. IRF4 expression in Treg cells is critical for effector differentiation, yet the precise mechanisms of how IRF4 regulates the transcriptional program of TFR cells remains unknown. Using a novel transgenic IRF4 reporter mouse we found that IRF4 is highly expressed in TFR cells. Using IRF4 knockout mouse models, we demonstrate that IRF4 is necessary for TFR cell generation in a Treg cell-intrinsic manner. IRF4 controls important aspects of the transcriptional program that drives TFR cell differentiation, including genes essential for Treg cell migration. Furthermore, I identified the transcription factor c-Maf to be essential for TFR cell generation and demonstrated its central role in maintaining a follicular program in Treg cells. Subsequently, using ribonucleic acid (RNA)-sequencing, we generated a “follicular signature” of gene expression from the combined analysis of TFH and TFR cells. Integrated transcriptional analyses showed that in the absence of either IRF4 or c-Maf, the majority of the follicular signature genes were downregulated, indicating that these two transcriptional regulators, aside from Bcl6 are indispensable for follicular TFR cell development. Finally, analyses of IRF4 and c-Maf DNA binding sites, identified by chromatin induced precipitation (ChIP)-sequencing, in combination with open chromatin regions in follicular T cell specific loci, we showed that the precise orchestration of distinct sets of genes is required to promote conserved aspects of the follicular T cell fate. In conclusion, my thesis describes how a key transcriptional network orchestrates fundamental steps in TFR cell differentiation and function, which contributes to the understanding of eTreg cell biology.
New protocols and computational tools for scRNAseq analysis
The fast development of single cell RNA sequencing (scRNAseq) presents new challenges in data analysis and opportunities for protocol development. To address challenges in data preprocessing, I developed scPipe, an R/Bioconductor package that integrates barcode demultiplexing, read alignment, UMI-aware gene-level quantification and quality control of raw sequencing data generated by multiple protocols. Results from scPipe can be used as input for downstream analyses and can be easily incorporated into R-based pipelines with other tools. In order to compare different computational methods for scRNAseq data, I generated a realistic benchmark experiment that included single cells and admixtures of cells or RNA to create `pseudo cells' from up to five distinct cancer cell lines. Multiple datasets were generated using both droplet and plate-based scRNAseq protocols and processed by scPipe. We found pipelines suited to different types of data for different tasks. Our data and analysis provide a comprehensive framework for benchmarking most common scRNA-seq analysis steps. Finally, I developed single cell full-length transcript sequencing by sampling (FLT-seq), together with the computational pipeline FLAMES to perform isoform discovery and quantification, splicing analysis and mutation detection in single cells. With FLT-seq and FLAMES, I performed a comprehensive characterization of the full-length isoform landscape in single cells of different types and species and found conserved functional modules that were enriched for alternative transcript usage in different cell populations, including ribosome biogenesis and mRNA splicing. The datasets, protocols and tools that I developed and generated are useful resources for the single cell research community.
Optimising colorectal cancer therapies using clinical registries
Colorectal cancer (CRC) remains one of the leading causes of cancer deaths worldwide. Advances in therapy have resulted in significant gains in survival, particularly in the metastatic setting. While the discovery of biomarkers, such as RAS mutations, have helped refine treatment selection to some degree, more accurate biomarkers are urgently needed. Comparison of existing treatments, as well as the evaluation of the efficacy of new therapies, are informed by randomised controlled trials (RCTs), which form the evidentiary backbone of clinical practice guidelines and represent the gold standard of assessment. Despite their high internal validity, they can lack generalisability due to their highly selective inclusion criteria. Prospective, registry-based, randomised controlled trials (RRCTs) have the potential to bridge the gap between RCTs and real-world clinical practice in oncology. The objective of this thesis is to explore how clinical registries can help to advance biomarker research. This thesis applies real-world data to examine the clinical utility and validity of emerging CRC biomarkers, and explores the feasibility of RRCTs in the oncology setting. In a cohort of 99 metastatic colorectal cancer (mCRC) patients, the role of the epidermal growth factor receptor (EGFR) and its ligands, amphiregulin and epiregulin, as potential prognostic and predictive biomarkers for mCRC patients is explored (Chapter 5). This study examines protein expression by immunohistochemistry and includes patients who were not treated with EGFR inhibitors, representing the largest such cohort reported to date. The real-world validity of biomarker trials is explored in Chapter 6, where the characteristics of patients enrolled in these studies are compared to real-world patients. Using an established multi-centre CRC registry as the reference real-world cohort, clinical data was analysed for participants in three types of biomarker trials (retrospective, prospective observational and prospective interventional). This study provides novel insights into recruitment to, and potential validity of, biomarker trials. Finally, Chapter 7 examines the feasibility of an Australian-first RRCT in oncology. This ongoing study is exploring chemotherapy sequencing in first-line treatment of mCRC and leverages an established multi-centre registry as the data collection platform. This study demonstrates the potential of RRCTs to accelerate progress in optimising patient treatments and outcomes. This thesis demonstrates the power of high-quality clinical registries to facilitate prospective randomised trials, while providing opportunities to investigate and validate biomarkers in real-world settings.
Characterization of new regulators in TNFR1-mediated death signalling
Tumor necrosis factor (TNF) is a master inflammatory cytokine that can, depending on the circumstances, promote survival and proliferation or induce cell death. Anti-TNF drugs have proven strikingly successful in treating inflammatory diseases such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD) but it is still unclear exactly why. For a long time, it was thought that they work solely by preventing TNF induced transcription of other inflammatory cytokines, but more recently it has been proposed that one of their major anti-inflammatory functions is to prevent TNF induced death. Therefore, understanding the mechanism by which TNF induced death is regulated may enable the conceptualization of newer or improved approaches in treating a variety of inflammation-associated pathologies. Binding of TNF to its receptor TNFR1 leads to the formation of two distinct signalling complexes. While most previous studies have focused on the membrane-bound, transcription-activating complex (complex-1), the composition and post-translational modifications of the cytosolic, caspase-8-containing, death-inducing complex (complex-2) remain far less well defined. To analyse TNFR1 complex-2 composition at endogenous levels, we decided to generate FLAG tagged caspase-8 knock-in mouse strains. The reagents for the FLAG tag enable very efficient and specific purification and identification of a FLAG tagged protein and its partners. After some preliminary tests and trials, I decided to use a 3x FLAG tag which has been reported to be 20–200 times more sensitive than other FLAG tags in immunoprecipitation and detection assays. Before generating the mouse strains, in Chapter 3 I performed extensive in vitro comparison of N-terminally or C-terminally 3x FLAG-tagged caspase-8 using a doxycycline (Dox)-inducible stably integrated lentiviral system. The results suggested that when expressed above endogenous levels, the expression and killing activity of caspase-8 was unaffected by a 3x FLAG tag. Interestingly, when expressed at physiological levels, C-terminally 3x FLAG tagged caspase-8 appeared to be equivalent to untagged caspase-8 and marginally more efficient in mediating TNF-induced death and complex-2 formation compared to N-terminally 3x FLAG tagged caspase-8. In addition, I immunoprecipitated TNFR1 complex-2 from cells expressing endogenous levels of 3x FLAG tagged caspase-8 and performed a mass spectrometry (MS) analysis. According to this analysis, Tankyrase-1 (TNKS1/PARP5a/ ARTD5), a member of the poly ADP-ribose polymerase (PARP) superfamily, appears to be a novel interactor of complex-2. Based on our in vitro data, we generated N-terminally or C-terminally 3x FLAG tagged caspase-8 knock-in mice using CRIPSR/Cas9 technology and these mice were characterized in Chapter 4. Homozygous N-terminally or C-terminally 3x FLAG tagged caspase-8 knock-in mice were viable, fertile and developed normally, indicating that N-terminally or C-terminally 3x FLAG tagged caspase-8 were expressed and active in vivo, at least to heterozygous caspase-8 levels. As expected, the expression of N-terminal or C-terminal 3x FLAG tagged caspase-8 was detectable in tissue and cells from knock-in mice by Western blot and immunofluorescence stain using an anti-FLAG M2 antibody. The 3x FLAG tagged caspase-8 displayed similar tissue distribution and comparable expression levels as endogenous caspase-8. The cell death assay suggested that the primary cells and transformed cells from 3x FLAG tagged caspase-8 knock-in mice responded similarly as wild-type cells to apoptotic and necroptotic stimulations. Moreover, by performing anti-FLAG immunoprecipitation, I successfully purified endogenous TNFR1 complex-2 from knock-in mice derived cells. These data indicated that 3x FLAG tagged caspase-8 knock-in mouse strains are useful tools to study caspase-8 and caspase-8-containing protein complexes at physiological levels. In Chapter 5, I characterized tankyrases-mediated poly(ADP-ribosyl)ation (PARsylation) as a novel checkpoint that limits TNF-induced cytotoxicity. Using primary cells from the 3x FLAG tagged caspase-8 knock-in mice described in Chapter 4, I found that the enzyme tankyrase-1 (TNKS1/TNKS/PARP5a/ARTD5), which was identified by mass spectrometry in Chapter 3, is recruited to the endogenous TNFR1 complex-2. Western blot data indicates that tankyrase-2 (TNKS2/PARP5b/ARTD6) may also be recruited. Tankyrases are poly ADP-ribose polymerases and belong to an ancient group of enzymes that post-translationally modify proteins with ADP-ribose. I found that during TNF signalling, complex-2 becomes poly(ADP-ribosyl)ated (PARsylated) in a tankyrases-dependent manner. Furthermore, tankyrases-specific inhibitors sensitized cells to TNF-induced cell death, which correlated with increased levels of complex-2. This suggested that normally tankyrases help limit TNF induced death. Mechanistically, I showed that tankyrases may modulate the stability of complex-2 by recruiting the E3 ubiquitin ligase RNF146, that in turn promotes ubiquitylation and degradation of complex-2. Moreover, inactivation of tankyrases dramatically increased the killing of the clinical Smac-mimetic (SM) birinapant in a primary acute myeloid leukemia (AML) model. Taken together, this thesis describes 3x FLAG tagged caspase-8 knock-in mice as new tools to study caspase-8 and caspase-8-containing protein complexes at physiological levels. Furthermore, this study identifies tankyrases-mediated PARsylation as a novel checkpoint in TNF signalling that expands our understanding of how TNF induced death is regulated and provides a rationale to use tankyrases inhibitors for cancer therapy.
Development of an Editable Approach to the Study Parasite-Erythroid Interactions
Malaria remains responsible for an enormous health burden worldwide; considerable research effort is being devoted to finding ways to combat the disease and its transmission. Malaria is caused by Plasmodium species, and P. falciparum causes the most serious disease. The blood stage of the P. falciparum remains critically important to understand for development of treatments and vaccines. To initiate invasion, the P. falciparum merozoite recognises specific proteins on the host red cell membrane, known as invasion receptors. In order to study parasite–host interactions, laboratory adapted P. falciparum strains that invade mature human red cells have been used. Gene modification methods are well established for P. falciparum; however, genetic manipulation of the red cell has not been extensively applied because erythrocytes are not nucleated. The in vitro cultivation of erythroid cell lines facilitates both the scalable production of host cells to support P. falciparum invasion and editing of nucleated precursors that can be genetically modified in a precise manner. In this project, two erythroid cell lines – the Human Umbilical cord blood Derived Erythroid Progenitors (HUDEP-2) and the Bristol Erythroid Line- Adult (BEL-A), both of which can differentiate to more mature forms in vitro – were studied as possible host models. A FACS antibody panel, based on the stage-specific profile of HUDEP-2 and BEL-A cells, provided the means to analyse host invasion receptors as well as erythroid maturation markers. Band 3 is a red cell membrane protein with an uncertain role in merozoite invasion. A gene knockout was constructed in expansion stage BEL-A cells using the lentiviral CRISPR/Cas9 system, targeting band 3 which may be involved in merozoite invasion of human erythrocytes. Single-cell-derived clones were isolated and preliminary validation using PCR and flow cytometry was performed to verify disruption of band 3. Completion of work to validate and functionally characterise the band 3 knockout, and experiments to assess effects on invasion, were curtailed by COVID-19 stay-at-home orders issued to Melbourne between March and July 2020. In summary, a genetically editable in vitro erythroid model was defined to study the function of host invasion receptors for P. falciparum merozoite invasion. Clonal band 3- deficient BEL-A cells were generated, thus paving the way for studying their role as invasion receptors.
Low birthweight and infant growth among children in Papua New Guinea - effect of malaria and other infectious diseases during childhood
Globally, young children continue to die or fail to thrive from treatable and preventable causes including low birthweight (LBW), childhood undernutrition (wasting, stunting, underweight) and infectious diseases. Reducing the burden of these are an essential pillar of global child health and survival targets. These global trends are reflected in Papua New Guinea (PNG), a resource constrained setting that continues to observe high rates of illness and death in young children with LBW, childhood undernutrition and infectious diseases (especially malaria, pneumonia and diarrhoea) continuing to be the leading causes. Improving child health and survival in PNG requires evidence about the risk factors for LBW, sub-optimal growth and infectious diseases, as well as the inter-relatedness of risk factors, that can inform policies and identify appropriate interventions and strategies. This thesis aims to address critical knowledge gaps in this area and provide insights to inform interventions and strategies aimed at reducing low birthweight, childhood undernutrition and malaria in young children in PNG and globally. LBW is caused by a multitude of factors which are often inter-related and with that, it is often difficult to distinguish between independently direct and indirect effects. Moreover, current interventions targeted at preventing LBW generally assume a single dominant cause overlooking the inter-relatedness of risk factors and the possibility of factors exerting joint effects on LBW. These are difficult to establish with widely used standard statistical methods and have therefore been rarely investigated. Using structural equation modelling, we showed intermittent preventive treatment of malaria during pregnancy with sulphadoxine-pyrimethamine (SP) plus azithromycin (AZ) to be independently directly associated with reduced probability of LBW. Unexpectedly, anaemia at enrolment was also directly associated with reduced probability of LBW. Maternal undernutrition at enrolment was independently directly associated with increased probability of LBW. No significant indirect associations between risk factors and LBW were established. After birth, children in lowlands PNG experience high rates of malaria, pneumonia and diarrhoea alongside undernutrition. Infections and undernutrition are believed to have a bi-directional relationship and whilst the effect of undernutrition on risks of illness and death is well known, little is known about the effects of malaria, pneumonia and diarrhoea on growth faltering, particularly among PNG children. By using multivariable regression and distributed lag models for data analysis, we observed malaria pneumonia and diarrhoea to have a differential impact on child growth. The effect of acute malaria on child growth was observed to be long-term while pneumonia and diarrhoea had short-term effects lasting up to 3 months. Of these three diseases, malaria was once ranked the top leading infectious cause of childhood morbidities and mortality in PNG, especially in lowlands PNG. The nationwide scale-up of malaria control interventions significantly reduced overall malaria transmission between 2008 and 2014 but a detailed understanding of the impact of this changing transmission on the epidemiology and risk profile of malaria infections and disease due to the two main species in young children was lacking. By analysing three consecutive longitudinal child cohorts (1-5-year-old children) conducted over the period of improved control (2013), we observed a differential impact of improved control on P. falciparum and P. vivax. Additionally, we showed that with declining malaria transmission, burden of malaria infections and illness were highly spatially localised to areas that had the highest burden prior to scale-up, highlighting potential hotspots of transmission. Collectively, the findings from this thesis provide important insights for improving child health in PNG and globally.
Functional and structural characterisation of the epigenetic regulator, SMCHD1
Structural Maintenance of Chromosomes Hinge Domain-containing protein 1 (SMCHD1) has been established as an epigenetic regulator, with critical roles in X-chromosome inactivation, autosomal gene silencing and genomic imprinting. Recently, variations in SMCHD1 have been associated with two human conditions: facioscapulohumeral muscular dystrophy (FSHD) and Bosma arhinia microphthalmia syndrome (BAMS). There has therefore been a growing interest in unveiling SMCHD1’s atomic structure and the molecular mechanisms underlying its function in both a healthy and diseased state. To provide a better understanding of Smchd1’s molecular structure and function, I successfully expressed and purified the full-length 2007-amino acid mouse Smchd1 protein. Electron microscopy analyses of the Smchd1 dimer revealed an elongated rod-like structure that displays a high conformational flexibility, similar to that of other structural maintenance of chromosomes (SMC) proteins. This flexibility is largely conferred by the intermediate region of the protein that connects Smchd1’s two functional domains: the N-terminal GHKL ATPase and the C-terminal SMC hinge domain. In follow-up studies of the two individual domains, we revealed the first atomic-resolution structure of Smchd1’s hinge domain, providing a novel insight into its DNA-binding and dimerisation modes. Contrary to previously suggested models describing the DNA interaction mode of canonical SMC proteins, I showed that nucleic acids are not threaded through the central pore region of the Smchd1 hinge domain. Subsequent immunofluorescence studies additionally revealed that the hinge domain targets full-length Smchd1 to chromatin, and that a functional hotspot within the hinge is required for chromatin localisation in cells. SMCHD1’s ATPase domain has been of particular interest due to the identification of disease-related variants that are frequently located within this region of the protein. However, the mechanisms by which some of these pathogenic variants affect SMCHD1 function are poorly understood. Using analytical ultracentrifugation, I demonstrated that the wild-type SMCHD1 ATPase undergoes dimerisation, which was reliant on the inclusion of both the UBL domain and the presence of substrate, ATP. Follow-up cellular studies revealed that Smchd1’s catalytic activity, as well as the presence of the newly- identified UBL domain, are both necessary for the localisation of full-length Smchd1 to chromatin. Together, these studies provide an insight into the molecular basis of Smchd1 function and highlight how chromatin binding may be compromised in human disease. Future studies will further investigate the cellular localisation and dimerisation properties of disease-associated SMCHD1 variants, contributing towards our ongoing drug development program aimed at developing therapeutic treatments for FSHD patients.
Molecular control of haematopoiesis via the JAK-STAT signalling pathway
Maintenance of the haematopoietic system is controlled by intercellular signalling molecules known as cytokines. Cytokines function by binding to receptors at the surface of target cells and activating a number of signalling pathways inside the cell that lead to changes in gene transcription and ultimately a cellular response, whether it be differentiation, division, cell death or other. These processes need to be tightly controlled and regulated, and so there are cellular mechanisms for controlling both the duration and intensity of the signal produced by cytokines. This thesis investigates the structure and function of two regulators of cytokine signalling; PTP1B and LNK. PTP1B is a protein tyrosine phosphatase that interacts with and dephosphorylates the JAK proteins, returning them to their inactive state. Similarly, LNK, a lymphocyte adaptor protein also interacts with JAK2, and negatively regulates a subset of cytokines by an unknown mechanism. The work presented in this thesis details the study of the interactions between PTP1B and LNK with the JAK proteins using structural and biochemical techniques, allowing for the characterisation of the mode of substrate recognition, and substrate preference. The studies in chapter 3 revealed that the PTP1B phosphatase can directly dephosphorylate JAK kinases, inducing their transition to an inactive state. The interaction between PTP1B and the motif on JAK that is its target was studied in detail. While previous studies of PTP1B substrate recognition have defined a second aryl binding site that allows PTP1B to interact with substrates containing two consecutive phosphotyrosine residues, here it is shown that there is a second mode of binding where this second binding site is not used. Given that PTP1B is the prototypical phosphatase in the category I phosphatases, this may warrant redefining this category. Moreover, the accessibility and position of the JAK activation loop to phosphatases was shown to be critical for dephosphorylation rate, and so it can be postulated that some conformations of the JAK proteins may inhibit their dephosphorylation by phosphatases. The results presented in chapter 4 study the interaction between the LNK SH2 domain and various phosphorylated targets, including JAK. It was shown that LNK can interact with several sequence motifs from proteins involved in haematopoiesis but that JAK2 and JAK3 are its favoured targets. Structural studies were performed to uncover the molecular basis for this high-affinity interaction with JAK. In addition, studies of mutations found in the LNK SH2 domain which were identified in patients with myeloproliferative neoplasms revealed how these changes to the SH2 domain reduced the ability of LNK to bind to its targets, which may contribute to disease. In summary, the information presented in this thesis add to our understanding of the regulation of the JAK-STAT pathway, and how changes to essential protein- protein interactions may be involved in disease.
Manipulation of host signalling for the characterisation and control of dengue fever
Dengue fever is a mosquito-transmitted disease of the tropics and sub-tropics that is caused by dengue virus (DENV). There are an estimated 60-100 million clinical cases of dengue fever per year, resulting in at least 10,000 deaths. Most clinical cases of dengue are characterised by flu-like symptoms. However, for unknown reasons, a small proportion (1-2%) of clinical cases progress to a life-threatening form of disease referred to as “severe dengue”. Severe dengue is characterised by cytokine storms, heightened endothelial permeability and associated sequelae such as shock and haemorrhage. During the onset of severe dengue, viraemia and viral antigenaemia are sharply declining or absent. Therefore, it is logical to deduce that dysregulated host signalling is the underlying cause of the cytokine storm phenotype and symptoms of severe dengue. However, although many host factors have been characterised in the context of DENV infection, the root cause of this signalling dysregulation is still poorly understood. Furthermore, there are currently no drug treatments available for the treatment of severe dengue, and although there is a licensed dengue vaccine, it confers only moderate protection, and administration of this vaccine to dengue naive individuals is contraindicated by the World Health Organisation. In the first part of this thesis, I characterised how genetic disruption of key host signalling pathways altered the response of macrophages and mice to DENV infection. I found that infection of cells and mice that had a co-deletion of genes encoding cellular inhibitor of apoptosis proteins (cIAPs) resulted in decreased production of virus, and an exaggerated production of inflammatory cytokines. In the second part of this thesis, I determined whether clinical stage cancer therapeutics could be repurposed as treatments for severe dengue. To investigate this, I established an in vivo mouse model of severe dengue and treated these mice with anti-inflammatory compounds. However, these drug treatments did not reduce clinical manifestations of infection or improve the survival of the infected mice. These studies suggest that cIAPs facilitate the efficient replication of DENV. In addition, I hope that the negative results from my therapeutic experiments can inform future experimental plans, and contribute to reducing the worldwide burden of severe dengue.