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  • ItemEmbargo
    Optimisation of TrAEL-seq to study DNA damage and replication in complex and dynamic mammalian cell systems
    Kara, Neesha
    Maintenance of genome stability is critical for cell survival, and consequently cells have evolved a complex set of mechanisms to ensure DNA is repaired and correctly replicated before cell division. Deficiencies in these repair pathways are heavily associated with the development of cancer and ageing, and it is therefore of interest to be able to detect and monitor the distribution of DNA damage and replication events across the genome. This work describes the development and optimisation of TrAEL-seq (Transferase-Activated End Ligation Sequencing), a novel sequencing method for genome-wide detection of DNA double strand breaks, replication fork stalling, and replication fork movement. Our method captures single-stranded 3’ DNA ends, which can be mapped across the genome with base pair resolution. TrAEL-seq requires no labelling, synchronisation or sorting of cells, making it very flexible and simple to implement. In this work I refined the protocol to suit studies of mammalian cell systems; I developed a multiplexing protocol for high-throughput sample processing and comparative quantitation, and optimised themethod’s compatibility with fixed cells. Through collaboration with Artios Pharma, I then used TrAEL-seq to investigate the effect of different DNA damage response inhibitors in cancer cells. Comparison of DNA replication between different therapies and time points provided interesting insights on the mechanism of action of such treatments. Surprisingly, this TrAEL-seq data did not reveal distinct genomic locations vulnerable to replication fork stalling or double-strand breaks, supporting the notion that such events occur in a more random distribution pattern across the genome. I then applied TrAEL-seq to study an alternative model of DNA replication stress and damage; oncogene-induced senescence (OIS) resulting from *HRASG12V* overexpression. Analysis of TrAELseq replication fork movement across a time course of OIS revealed global changes in the levels of DNA replication across the genome. Interestingly, the data revealed an accumulation of TrAEL-seq signal around a subset of R-loops in senescent cells, which were localised in subtelomeric regions. Follow-up investigations which characterised the nature and origin of these peak sites offer new perspectives on cellular senescence. Taken together, this work demonstrates TrAEL-seq as a novel and exciting tool to investigate a range of dynamic and complex mammalian cell systems.
  • ItemOpen Access
    Investigating the role of ACSS2-mediated acetyl-CoA metabolism on EGFR-PI3K-PKB signalling
    Cragoe, Bethany
    The epidermal growth factor receptor (EGFR) signalling network regulates growth, survival and differentiation in mammalian cells. There are multiple signalling pathways downstream of the EGFR, including the phosphoinositide-3-kinase (PI3K)-protein kinase B (PKB) pathway. This pathway is not only essential for cellular function, but has also been commonly implicated in the diseased state, including cancer and immunological deficiencies, therefore understanding its regulation is fundamental in revealing its role in both physiological processes, and diseased states. Recently in the field, there has been a large emphasis on how cells utilise metabolites as nutrient sensors, to coordinate nutrient availability with cellular proliferation. Acetyl-CoA is an essential metabolite that regulates key cellular processes, including *de novo* lipid biosynthesis, energy production and protein acetylation. Whilst there are multiple routes of acetyl-CoA synthesis, acetyl-CoA synthetase short chain family member 2 (ACSS2) is responsible for acetate-dependent synthesis of acetyl-CoA. The objective of this study was to investigate the potential impact of ACSS2-mediated acetyl-CoA on EGFR-PI3K signalling, shedding light on the regulatory roles of metabolism in MCF10a cells' response to external growth factors. A combination of phenotypic assays and mechanistic studies, including the use of the ACSS2 inhibitor VY-3-249, initially suggested that ACSS2 activity directly influences PI3K-PKB signalling. However, the generation of CRISPR/Cas9 ACSS2 knockout cells provided the crucial model required to confirm that the effects observed with VY-3-249 were occurring off-target. This finding is significant for the field, given the widespread citation and general acceptance of the inhibitor. With this in mind, I decided to phenotype ACSS2-KO cells independently of PI3K signalling, to further understand the breadth of ACSS2 activity and importance. Very interestingly, through the use of mass spectrometry and bioenergetic analysis, I have preliminary evidence for the novel role of ACSS2 in the regulation of succinyl-CoA levels, and subsequent succinylation and mitochondrial function. Overall, the work in this dissertation has identified an important insight into off-target effects of a widely used ACSS2 inhibitor, VY-3-249, which should be carefully considered when used in the field. Additionally, I have optimised and created useful tools to study other functions of ACSS2. Our work also highlights an exciting, previously unidentified role of ACSS2 on succinyl-CoA levels and mitochondrial capacity, opening up a new avenue of exploration between metabolism and mitochondrial function.
  • ItemEmbargo
    Characterising FAM117 proteins as novel targets of DYRK1B
    Cassidy, Megan Alice
    Compared to other CMGC kinases the dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) represent an understudied family of protein kinases. Whilst DYRK1A has garnered more attention due to its links to Downs’ Syndrome and Alzheimer’s Disease, the other class I DYRK, DYRK1B, remains comparatively unexplored. Prior work in our lab has explored potential new DYRK1B substrates by utilising a phospho-SILAC screen. From this screen we identified two proteins of the same family, FAM117A and FAM117B, which along with the third family member (FAM117C/GLCCI1) have previously been identified as binding partners of class I DYRKs. FAM117 proteins have no clearly established biological function and so understanding interactions between DYRK1B and FAM117 proteins may help to elucidate further functions of DYRK1B and also identify roles of FAM117 proteins. Here I establish that FAM117 proteins represent DYRK1B inducible phosphoproteins that rely on kinase activity of DYRK1B for their phosphorylation and shown that FAM117B represents a direct substrate of DYRK1B. Furthermore, I demonstrate that both class I and class II DYRKs are capable of inducing post-translational modifications, that likely represent phosphorylation, of FAM117 proteins. This is not a characteristic shared by all CMGC kinases, with only the closely related CLK3 driving similar FAM117 phosphorylation. Whilst no 3D structures exist for FAM117 proteins, prediction software suggests they are highly disordered with many predicted phosphorylation sites. This high level of disorder suggests that stable expression of FAM117 proteins may rely on binding partners and/or modifications such as phosphorylation. Indeed, I show that phosphorylation of FAM117 proteins by DYRKs is associated with their increased expression in both short & long term overexpression systems as well as the endogenous proteins. Using FAM117B as a model FAM117 protein, I identify a high number of DYRK1B inducible phosphorylation sites, many of which are located in the PFAM domain which is conserved between FAM117 proteins. Additionally, the scaffold protein DCAF7, which mediates the interactions of class I DYRKs with their substrates, also binds FAM117 proteins and I demonstrate that FAM117 proteins, DYRK1B & DCAF7 form ternary complexes in cells. Understanding FAM117 function and how their interaction with DYRK1B affects the functions of both proteins may help us to better understand their functions. I demonstrate that expression of FAM117 proteins disrupt the normal distribution of DYRK1B, each in different ways, and that all are capable of perturbing a normal DYRK1B function, in this case the promotion of processing body formation. From observing FAM117 localisation I also show that they partially co-localise with cytoskeletal proteins, such as tubulin, and show strong co-localisation with focal adhesion proteins. FAM117 proteins are reported to be capable of binding the microtubule protein DYNLL1 and work from our lab has identified the intermediate filament protein KANK2 as a possible DYRK1B interactor. Both of these have been suggested to link microtubules to focal adhesions. Here I demonstrate that both DYNLL1 and KANK2 immunoprecipitate with FAM117 proteins and DYRK1B, suggesting a role for DYRK1B and FAM117 proteins in the regulation of focal adhesions which in turn may affect cell adhesion and migration. Given the role of microtubules in regulating membrane organelle dynamics this binding to DYNLL1 may also provide an explanation for the dysregulation of processing bodies caused by FAM117 proteins. These new insights into DYRKs, specifically DYRK1B, and FAM117 proteins are discussed further and used to suggest hypotheses for future investigation. Furthermore, the FAM117 proteins may serve as useful biomarkers for DYRK activation or inhibition.
  • ItemOpen Access
    Investigating the immunosuppressive functions of the GPCR adapter protein Norbin
    Chetwynd, Stephen
    This project investigated the roles of Norbin, an adaptor protein which regulates trafficking and signalling of G protein-coupled receptors (GPCRs). Norbin was originally expected to be exclusively neuronal. However, previous PhD students in our laboratory found that Norbin is also expressed in myeloid cells and made mice with myeloid Norbin deficiency (NcdnΔmye) to investigate its role in these cells. They showed that NcdnΔmye mice have increased immunity during pulmonary *S. pneumoniae* infection, and that neutrophils from these mice kill bacteria better, in a reactive oxygen species (ROS)-dependent manner, and produce more ROS in response to a wide range of stimuli.
    I followed up on their work using immune cell depletion to demonstrate that the elevated immunity of NcdnΔmye mice is neutrophil-derived rather than depending on macrophages. I showed that neutrophils arrive more rapidly in the airways of NcdnΔmye mice during infection with *S. pneumoniae*, and they degranulate in the lungs to a greater extent, although the systemic levels of inflammatory cytokines are normal. I also found ten-fold elevated immunity of NcdnΔmye mice during septic peritonitis. I showed that NcdnΔmye neutrophils degranulate more and produce more neutrophils extracellular traps (NETs) than control neutrophils, and their elevated killing of *S. aureus* is dependent on NETs, whereas their production of inflammatory cytokines is normal. I showed that Norbin controls the Rac-GEFs Vav, which is constitutively active in NcdnΔmye neutrophils, and Tiam1 which is upregulated to cell surface upon C5a-stimulation. I also showed that the elevated ROS production in these cells can be reversed by titrating inhibitors of Rac and Erk.
    Arguably my most important contribution was to investigate the role of Norbin in neutrophil GPCR trafficking. Norbin is known to interact directly with numerous GPCRs, influencing their trafficking and signalling. However, apart from Norbin needing to interact directly with a GPCR, little is known about the mechanisms. I identified novel direct interactions between Norbin and the C-terminal tails of the GPCRs CXCR4 and C5aR1. I showed that the cell surface levels of CXCR4 and C5aR1 are elevated in NcdnΔmye neutrophils whereas the total cellular levels of these receptors are normal. I used various pulse-chase experiments to show that constitutive trafficking of these GPCRs is unaffected, but their agonist-induced internalisation is altered in NcdnΔmye neutrophils, and their recycling back to the plasma membrane faster. Altered binding kinetics of β-arrestin with stimulated C5aR1 may underlie the mechanisms behind these trafficking differences, and the resulting elevated GPCR cell surface levels can explain some of the increased responsiveness of NcdnΔmye neutrophils.
  • ItemEmbargo
    The Role of Autotaxin in Cancer
    Maggs, Lauren
    Autotaxin (ATX/ENPP2) is a secreted phospholipase D enzyme that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA is the agonist for at least six G protein coupled receptors whose activation stimulates cell proliferation, survival and migration. Overexpression of ATX and increased levels of LPA have been linked to fibrotic and inflammatory diseases and to several cancers. ATX is a therapeutic target and the ATX inhibitor IOA-289 is currently in Phase 1B clinical trials for the treatment of patients with metastatic pancreatic cancer, in combination with gemcitabine. ENPP2 is overexpressed in both pancreatic adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC). In HCC, ATX is produced by both the tumour microenvironment (TME) and tumour cells and our data show that ATX signalling can drive tumour cell growth in an autocrine manner. In contrast, in PDAC the TME primarily expresses ATX. The cell lines, PANC-1 and MIA PaCa-2 and the cancer associated fibroblast (CAF) derived cell line, 0082T, were used as a model to explore ATX signalling in PDAC. Consistent with publicly available mRNA expression data, 0082T cells, but not the PDAC cancer cells, secrete ATX and its substrate LPC. Conditioned media from 0082T cells increased the growth of both PANC-1 and MIA PaCa-2 cells. Treatment of 0082T CAF cells with IOA-289 during media conditioning reduced PDAC cell growth, without impacting CAF cell numbers. Lipidomic analysis showed IOA-289 and PF8380, another ATX inhibitor, reduce the generation of LPA in 0082T CAF conditioned media. 22:6 LPA was shown to be generated by an ATX independent pathway, and production of 22:6 LPA was upregulated upon ATX inhibition. Additionally, 22:6 LPA accumulates in conditioned media when 0082T, PANC-1 and MIA PaCa-2 cells are present, but species of LPA generated by ATX are specifically degraded (16:0,16:1 18:0,18:1). Cell surface lipid phosphate phosphatases (LPPs) dephosphorylate LPA to produce monoacylglycerol. This presents the first evidence for LPA species substrate specificity of LPPs and suggests LPPs specifically regulate ATX generated species of LPA. RNA-seq analysis of ATX inhibitor and DMSO treated CAF cells revealed significantly altered expression of genes encoding extracellular proteins, such as CTGF. A combination of modulating the mitogenic CAF secretome and a reduction in LPA is proposed as the mechanism of ATX inhibition in regulating CAF-driven PDAC growth. ATX has non-catalytic functions that could be mediated by specific protein-protein interactions. An HRP-based proximity labelling method was optimised for investigating the extracellular protein interactors of ATX. Several potential interactors are identified, including fatty acid binding protein 5 (FABP5). Modelling with AlphaFold-Multimer indicates FABP5 may interact with ATX at the opening to ATX’s lipid binding active site. Further investigation will be required to validate this idea.
  • ItemOpen Access
    Investigating PLEKHS1 adaptor function in PI3K signalling and human cancer biology
    Anwar, Arqum
    The Class-IA PI3K signalling pathway regulates key anabolic cellular processes including proliferation, survival, growth and metabolism. Oncogenic hyperactivation of the pathway occurs across tumour types, commonly via genetic alterations in key pathway genes (e.g., *PIK3CA*, *PTEN*). Despite extensive research, the primary drivers of healthy or oncogenic PI3K signalling *in vivo* are largely unknown and PI3K pathway inhibitors show limited clinical benefit in solid tumours. Identifying upstream regulators of oncogenic PI3K activation can reveal novel therapeutic targets and/or biomarkers. Previously generated *in vivo* interactomics data identified the RTK/IRS network as the primary driver of homeostatic PI3K activation in healthy mouse prostate, and PLEKHS1 as the primary driver of oncogenic PI3K activation in cancerous, PTEN-null mouse prostate. PTEN phosphatase is a tumour-suppressor and inactivated commonly in prostate cancers. PLEKHS1 is otherwise a poorly characterised protein. This thesis presents research investigating the relevance and mechanisms of PLEKHS1 adaptor protein function to oncogenic PI3K pathway remodelling in human cancers, with a focus on prostate cancer. TCGA pan-cancer analyses report *IRS1* downregulation and *PLEKHS1* upregulation in several primary carcinomas, compared to healthy tissue. In prostate, endometrial and breast cancers, *PLEKHS1* expression correlates with PI3K pathway activation and alterations in key pathway genes (*PTEN*, *PIK3CA*). In castration-resistant prostate cancer, *PLEKHS1* is under-expressed in neuroendocrine cancers, compared to adenocarcinomas. ChIP-Seq datasets report binding of key prostate cancer transcriptional regulators to the *PLEKHS1* locus (e.g., AR, FOXA1). To investigate PLEKHS1 adaptor protein function *in vitro*, LNCaP prostate cancer cell line is used due to its PTEN-null status and PI3K pathway hyperactivation. Transient over-expression of PLEKHS1 in LNCaP cells generates a robust PIP3 response via YxxM-dependent interactions with endogenous PI3K regulatory subunits (p85, p55γ). PLEKHS1 phosphorylation and PI3K interaction are driven by SFK activity and independent of serum growth factors. In this context and alike *in vivo* findings, the PLEKHS1 PH domain which specifically binds PIP3 and PI(3,4)P2 mediates positive feedback PI3K activation. PLEKHS1 adaptor function is mostly conserved across mouse and human protein isoforms but altered for human isoform 2 which has a truncated PH domain. Endogenous PLEKHS1 function is also investigated in LNCaP cells. CRISPR/Cas9 mediated PLEKHS1 knockout does not significantly affect PIP3 levels, Akt phosphorylation or cell viability in LNCaP cells cultured as monolayers or spheroids. Consistently, no endogenous PLEKHS1-PI3K interaction is detected using co-immunoprecipitation in LNCaP cells. However, other *in vitro* disease models did provide evidence of endogenous SFK-PLEKHS1-PI3K signalling axis. In the PTEN-null breast cancer cell line HCC70, endogenous PLEKHS1 is highly expressed and SFKs mediate YxxM phosphorylation and interaction with PI3K regulatory subunits (p85, p55γ). However, siRNA mediated PLEKHS1 knockdown does not affect PIP3 levels, Akt phosphorylation or cell viability. Mouse dorsolateral prostate derived organoids are used as an alternative *in vitro* model. PTEN-KO organoids show elevated PLEKHS1 protein expression and PIP3 levels compared to WT. PTEN/PLEKHS1-DKO organoids phenocopy *in vivo* data with reduced PIP3 levels and growth compared to PTEN-KO organoids. Collectively, this work substantially advances prior *in vivo* findings from mouse models by (1) demonstrating the relevance of PLEKHS1 adaptor function to PI3K signalling in human cancers using *in vitro* and clinical data, (2) elucidating the molecular mechanisms of the SFK-PLEKHS1-PI3K signalling axis, and (3) revealing highly context-specific utilisation of PLEKHS1 as a primary driver of oncogenic PI3K signalling.
  • ItemOpen Access
    Understanding the Effects of Ageing on B cells Response to Vaccination
    Lee, Jia Le
    Ageing is often associated with a reduction in antibody-secreting cell formation and vaccine-induced antibody titres, resulting in poor vaccine responses among the older population. Understanding the mechanisms underlying this age-related defect is imperative for guiding strategies to improve vaccine responses in older people. Here, I tested the hypothesis that B-cell intrinsic changes with age contribute to the impaired antibody response to vaccination during ageing. I show that B cells from older people do not have defects in their proliferation and differentiation into antibody-secreting cells *in vitro* compared to those from younger donors. Adoptive transfer of B cells from aged SWHEL mice into young adult recipient mice showed that differentiation into extrafollicular plasma cells was favoured at the expense of B cells entering the germinal centre (GC) during the early stages of GC formation, compared to those from young donors. Nevertheless, by the peak of the GC response, GC B cells derived from B cells of aged mice had expanded to the same extent as those from the younger donors. This indicates that age-related intrinsic B cell changes might delay the GC response but are not responsible for the impaired antibody-secreting response or smaller peak GC response in ageing. Using the B1-8i adoptive transfer system, I replicated the data showing that B cells from aged B1-8i mice were equally able to differentiate into extrafollicular plasma cells and mount a peak GC response, compared to their younger counterparts. Furthermore, GC B cells from aged donor mice had no defects in receiving positive selection signals and undergoing affinity maturation to produce high-affinity clones. Conversely, the transfer of B cells from young adult mice into aged recipient mice resulted in smaller numbers of antigen-specific GC B cells and impaired affinity maturation, compared to transfers into young recipient mice. Together, this work shows that B cells from older bodies are not intrinsically defective in responding to stimulation and becoming high-affinity clones. Rather, B cell-extrinsic factors contribute to the age-associated impairment in humoral immunity. To have an unbiased approach in identifying mechanisms that drive defects in antibody response among older people, I employed a systems immunology approach to investigate the molecular pathways and cellular populations associated with antibody responses to seasonal flu vaccination in individuals aged above 64 years old. Although bulk RNA sequencing analysis of pre-vaccination and day 1 post-vaccination blood samples did not yield differentially expressed genes between vaccine responders and nonresponders, responders exhibited a trend of enhanced upregulation of type I interferon (IFN-I) response genes at day 1 post-vaccination. Furthermore, flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) collected 6-10 days post-vaccination revealed that long-lasting antibody responses to H1N1 antigen positively correlated with higher proportions of HA-binding plasmablasts and circulating T follicular helper (cTfh) cells. These findings suggest that an effective response to seasonal flu vaccine among older individuals relies on a robust antiviral response via IFN-I signalling, coupled with the generation of plasmablasts and cTfh cells. Together, the results from this thesis contribute to the understanding of mechanisms underlying age-related defects in humoral response to vaccines, which can be used to guide the design of more effective vaccine strategies to protect vulnerable members of our population.
  • ItemOpen Access
    Long-read transcriptome profiling of germinal centre B cells
    Gizlenci, Özge
    Alternative splicing (AS) is a major regulatory process underpinning the development and function of the immune system. 29% of alternatively spliced genes are specific to immune cells, yet the regulation and function of AS during B cell activation remain largely unknown. The activation of naive B cells and the subsequent germinal centre (GC) reaction drive the cascade of reactions resulting in short- and long-term antibody responses, making the GC crucial for adaptive immunity. However, abnormal function of GC B cells contributes to autoimmune disease and the development of lymphomas. Hence, GC reaction needs to be tightly regulated. Previous studies have linked individual AS events in GC B cells to B cell malignancies using short-read sequencing; however, this methodology is limited in defining the complete sequence of transcript variants generated by AS. Therefore, many transcript variants remain undefined. During my PhD, I have developed a long-read sequencing methodology Oxford Nanopore Technologies (ONT) workflow to understand post-transcriptional regulation at both gene and isoform levels in human and mouse GC B cells. Because one of the challenges of ONT is the accurate computational analysis of isoforms, we developed the ‘Nexons’ pipeline to identify the differentially spliced transcript variants. Transcriptome characterisation using ONT allowed us to detect differentially expressed transcripts during antigen-mediated activation of GC B cells in human and mouse. Moreover, identification of individual isoforms with Nexons revealed differential splicing of transcripts, including potentially novel splice variants, as well as changes which were undetectable in short-read sequencing data. An in-depth analysis revealed the differential regulation of poison exons (PE) in serine/arginine-rich splicing factors (SRSF) (e.g., SRSF3 and SRSF7). Naive B cells preferentially expressed isoforms carrying PE, leading to nonsense-mediated mRNA decay, whilst the PE were preferentially removed in activated and GC B cells. Notably, we found this regulation of PE in splicing factors is conserved between human and mouse. We validate an ONT/Nexons workflow as a suitable method for the identification and quantification of transcript isoforms and highlight the SRSF family as important candidates for regulating the GC reaction.
  • ItemEmbargo
    Defining the essential regulators of naive human pluripotent stem cell reprogramming
    Bendall, Adam; Bendall, Adam [0000-0002-6865-2625]
    Human pluripotent stem cells (PSCs) exist in two distinct states – naïve and primed, which are interconvertible. Naïve PSCs uniquely harbour several desirable properties that primed PSCs do not, including their ability to model human pre-implantation biology and developmentally-regulated epigenetic phenomena in vitro. Naïve PSCs also have an expanded differentiation potential to produce cell types with possible therapeutic applications. Naïve PSCs are predominantly generated by reprogramming primed PSCs. This project aimed to address a significant gap in our knowledge about the molecular events that underpin reprogramming, which could assist in unlocking the full potential of naïve PSCs. A genome- wide screen identified novel regulators of naïve cell reprogramming, unexpectedly highlighting the Polycomb Repressive Complex 1 (PRC1) subtype PRC1.3. In this project, I sought to understand the role of PRC1.3 in human pluripotency and reprogramming, and determine the PRC1.3-dependent mechanisms that modulate the acquisition of naïve pluripotency. I used CRISPR-Cas9 to delete the core PRC1.3 complex component PCGF3 in primed PSCs and ascertained that PRC1.3 is dispensable for human primed pluripotency but critical for naïve cell reprogramming. I used ChIP-sequencing to identify genes that gain PRC1.3 occupancy during reprogramming and these genes become transcriptionally repressed as cells successfully reprogramme, suggesting a key gene silencing role for PRC1.3. Deletion of PRC1.3 reduces the global abundance of the PRC1-deposited H2AK119ub1 modification, and levels of PRC2-deposited H3K27me3 also, in primed PSCs. Using a proteomics approach, I identified a novel, naïve-specific interaction between PRC1.3 and PRDM14, which cooperate in transcriptional repression of PRC1.3 target genes, I also discovered a developmentally-regulated composition shift in the PRC1.3 complex between human naïve and primed PSCs, which was corroborated in early human embryo datasets. This change in PRC1.3 composition does not appear to alter the enzymatic activity of PRC1.3 in vitro. Overall, I identified that the PRC1.3 complex plays a critical role in human naïve cell reprogramming. The PRC1.3 complex influences the human primed PSC epigenome and is associated with transcriptional repression, with the complex undergoing both intrinsic and exogenous regulation across the cell state transition. These findings advance our knowledge of molecular events that underpin successful naïve cell reprogramming, and this progress could optimise our ability to produce human naïve PSCs, as an in vitro developmental model and a cell type with considerable therapeutic potential.
  • ItemOpen Access
    NANOGP1 as a Model to Study the Consequences of Gene Duplications on Human Pluripotency and Development
    Maskalenka, Katsiaryna
    Gene duplication events play an important role in genome evolution; they can also create developmental strategies that differ between species. However, the functional contribution of duplicated genes in early human development and pluripotency is poorly understood. To address this knowledge gap, I investigated NANOGP1, which is a duplicated pseudogene of a key pluripotency factor called NANOG. NANOGP1 was chosen as a model for studying gene duplication in human pluripotency for several reasons. Firstly, NANOGP1 is an evolutionarily conserved duplicate in Hominidae that appears to have an intact coding sequence. The pseudogene is currently annotated as non-protein-coding, although no functional assays have been performed to test this. Secondly, upon investigating the expression of pseudogenes in human naïve pluripotent stem cells (PSCs), I found that NANOGP1 is among the top 1% of the highest expressed pseudogenes. Because high expression levels of NANOG are crucial for maintaining human pluripotency, I hypothesised that a duplicated copy of this important developmental regulator could have similar properties and might contribute to the regulation of human pluripotency. Gene expression profiling revealed that NANOG and NANOGP1 have overlapping but distinct expression patterns, both in human embryos and in PSC states. NANOGP1 is highly expressed in naïve pluripotent cells but is significantly downregulated in primed pluripotent cells, while NANOG expression levels do not differ to the same extent between the two pluripotent states. RNA splicing analysis predicted that NANOGP1 encodes a protein with an intact homeodomain and transactivation domain, but lacking part of the N-terminus. The divergent N-terminus is the main structural difference between NANOG and NANOGP1 and was therefore used in this study to distinguish between the two genes. Using CRISPR/Cas12a-mediated gene editing in naïve PSCs, I introduced an epitope tag at the start of the predicted protein sequence, and this enabled me to demonstrate for the first time that endogenous NANOGP1 encodes an expressed protein. The ability to be translated into the stable protein raised the possibility that NANOGP1 could have a functional role. To test this, I performed a series of assays and established that at least two key functional properties are conserved between NANOG and NANOGP1: gene autorepression, and the ability to promote primed-to-naïve PSC reprogramming. Alongside this, however, downregulating NANOGP1 expression using inducible CRISPRi in naïve PSCs did not lead to a differentiation phenotype, 4 which is in contrast to NANOG loss of function. Finally, using ChIP-seq, I showed that NANOGP1 shared a subset of chromatin binding sites with NANOG, and also, surprisingly, had a small number of unique, NANOG-independent sites particularly at the promoters of neural-associated genes. Overall, I conclude that NANOGP1, a previously overlooked duplicated copy of NANOG, is an expressed, protein-coding transcription factor in human naïve PSCs. Most of the CDS, and several of the functional properties, are conserved, implying that NANOGP1 could be supporting or cooperating with its ancestral gene copy in stabilising pluripotency. At the same time, differences in the N-terminal of the CDS, binding occupancy, and distinct expression patterns, could potentially contribute to functional diversification. These differences could have significant evolutionary consequences for creating species-specific developmental strategies, such as novel cell type-specific activity, expanded protein interaction networks and interplay with signalling pathways. Collectively, these potential new properties might extend functional potential and, hence, could encourage diversification of developmental mechanisms. Taken together, my work has demonstrated that NANOG/NANOGP1 duplication serves as a paradigm for exploring how pseudogenes could support their ancestral copies, as well as expand the evolutionary potential of conserved developmental programmes.
  • ItemOpen Access
    Investigating the role of DPPA2 and DPPA4 in the Epigenetic Control of Lineage Programs in Human Embryonic Stem Cells
    Malcolm, Andrew; Malcolm, Andrew [0000-0001-6240-7701]
    The precise co-ordination of cell fate specification during human early development is a vital yet poorly understood process. To navigate the dynamic transcriptional and epigenetic changes associated with germ layer allocation, pluripotent cells maintain developmentally important genes and their regulatory regions in a poised but repressed chromatin state. The poised state has been proposed to allow for precise and coordinated activation or complete repression of gene expression depending on the instructive signals from the external and intrinsic environment. Poised promoters and enhancers adopt multivalent histone modification states, comprised of both active and repressive modifications, such as H3K27me3, H3K4me3 and H3K4me1. Combining opposing modifications may help to maintain robustness of genes to low levels of signal, whilst retaining the capacity to respond upon the appropriate level. Despite their importance in controlling cell fate decisions, our understanding of the mechanisms by which poised states are established and maintained in human cells is currently lacking. Here, I discover a role for the transcription factors DPPA2 and DPPA4 in maintaining poised chromatin in human pluripotent stem cells (hPSCs). I found that DPPA2/4 bind to the majority of CpG islands, poised promoters and a large subset of poised enhancers. CRISPR-Cas9 mediated knockout of DPPA2/4 in primed hPSCs led to changes in the expression of developmentally critical genes, particularly those associated with signalling. Primed hPSCs lacking DPPA2/4 exhibit increased spontaneous differentiation even in self-renewing conditions, display altered cell fate commitment III during differentiation and show axial patterning defects upon human gastruloid formation. Epigenomic profiling following the loss of DPPA2/4 revealed a marked depletion of H3K27me3 at a subset of DPPA2/4-target regions, predominantly near to poised chromatin regions. Regions losing H3K27me3 were typically highly accessible and had high levels of H3K4me3 in both wild-type and DPPA2/4 DKO hPSCs. The depletion of H3K27me3 upon DPPA2/4 loss leaves these regions in a more active conformation, potentially driving changes in the expression of these genes. These data reveal new roles for DPPA2/4 in safeguarding the robustness of poised chromatin states and in regulating genes that are important for cell fate specification. Understanding further how these transcription factors and chromatin states jointly contribute to gene expression dynamics is critical to uncover the principles of developmental gene regulation and to improve generation of specialised cell types from human pluripotent cells.
  • ItemOpen Access
    New roles of Rac-GEFs in Neutrophils and in Glucose Homeostasis
    Machin, Polly
    Rac-GEFs (guanine-nucleotide exchange factors) are proteins that activate Rac GTPases, thereby enabling Rac-dependent cytoskeletal dynamics and cellular processes such as adhesion and migration. I used mice with genetically modified Rac-GEFs to identify new functional roles of these proteins in two distinct biological systems, neutrophil adhesion/migration and glucose homeostasis. In the first part of my thesis, I investigated cytoskeletal dynamics controlled by the Rac-GEF Tiam1 in neutrophil adhesion/migration. We previously found a paradoxical increase in the adhesion of Tiam1–/– neutrophils (unpublished). This was surprising, as deficiencies in other neutrophil Rac-GEFs impair adhesion. I showed deregulated neutrophil polarisation, Filamentous-actin (F-actin) polarity, F-actin dynamics and focal adhesion structures in Tiam1–/– neutrophils adhering to integrin ligands. I demonstrated increased integrin avidity in Tiam1–/– neutrophils stimulated with CXCL1, and increased migration of Tiam1–/– neutrophils under shear stress. These results contribute to our elucidation of the mechanisms underlying the paradoxical increase in the adhesion of Tiam1-deficient neutrophils. In the second part, I investigated spatiotemporal patterns of Rac activity generated during neutrophil adhesion/migration by several major neutrophil Rac-GEFs. The aim was to identify specific roles for these Rac-GEFs which all signal in response to the activation of GPCRs. I used our Rac activity FRET reporter mouse strain (RFC) (Johnsson, Dai et al. 2014), crossed to mice deficient in the Rac-GEFs P-Rex1/Vav1, DOCK2 or Tiam1. I demonstrated that Rac activity is increased in RFC Tiam1–/– neutrophils adhering to integrin ligands, which may explain the increased adhesion. In contrast, RFC DOCK2–/– and RFC P-Rex1–/– Vav1–/– neutrophils had reduced Rac activity, as expected for Rac-GEF deficient cells, confirming a unique and paradoxical role of Tiam1 in limiting Rac activity and Rac-dependent cell responses. The loss of Rac activity was global in RFC DOCK2–/– neutrophils but more localised in RFC P-Rex1–/– Vav1–/– cells. This project has identified specific roles of various Rac-GEFs in neutrophil adhesion and migration. In the third and final part, I investigated adaptor functions of P-Rex family Rac-GEFs P-Rex1 and P-Rex2 in glucose homeostasis. We previously showed that P-Rex1 and P-Rex2 deficient mice have accelerated glucose clearance during glucose challenge, along with low fasting blood glucose levels and altered insulin sensitivity (unpublished). In order to address the underlying mechanisms, I used mice with catalytically inactive P-Rex1 or P-Rex2 (GEF-dead mice) which we recently generated (unpublished). I demonstrated that the increased glucose clearance is an adaptor function of P-Rex Rac-GEFs, whereas fasting blood glucose levels and insulin sensitivity are Rac-GEF activity dependent. I showed increased plasma insulin levels in P-Rex1–/– and P-Rex2–/– mice upon glucose challenge and increased glucose-stimulated insulin secretion from P-Rex1–/– and P-Rex2–/– pancreatic islets. Use of P-Rex1 GEF-dead mice showed that these latter phenotypes were again adaptor functions, suggesting that these responses contribute to the accelerated glucose clearance in P-Rex-deficient mice. Combined, my work has provided a substantial body of data identifying unexpected novel roles for Rac-GEFs in both neutrophil biology and in glucose homeostasis, providing mechanistic insight in addition to new functions in both systems.
  • ItemOpen Access
    The roles of DPPA2, DPPA4 and SMARCA5 in mouse zygotic genome activation, epigenetic reprogramming and development
    Kubinyecz, Oana Nicoleta
    The epigenetic remodelling that takes place in order to transform a fertilised oocyte into an embryo and then a whole organism, is one of the most intriguing cellular transformations in biology. The whole process starts with the meiotic resumption of an oocyte when the translational activation of the dormant mRNAs takes place, followed by their gradual elimination in the zygote, and ending with the full transcriptional activation of the newly reprogrammed embryonic genome. All these complex processes constitute the maternal to zygotic transition (MZT), which is accompanied by the global epigenetic reprogramming of the embryo. In this dissertation, I focus predominantly on the epigenetic factors that potentially contribute to the initiation of transcription, a process also called zygotic genome activation (ZGA). In mice, this takes place in 1 cell embryos – minor ZGA, followed by a burst of transcription - major ZGA, at the 2 cell stage. Previous screens in the lab, using mouse embryonic stem cell (mESC) identified the small DNA binding proteins DPPA2, DPPA4 and the chromatin remodeler SMARCA5 as potential inducers of major ZGA. Here I focus on validating these three factors and I assess their roles in vivo, using conditional knock-out mouse models and a targeted protein depletion system, together with the characterisation of the transcriptomic and epigenetic changes. In Chapter 1, I give an overview of the biological context for my study together with the latest findings, and depict some of the technologies used to describe the changes that take place during epigenetic reprogramming in the early embryo. Chapter 2 outlines the materials and methods used to address my questions, and Chapters 3, 4 and 5 contain a detailed description of my results and their significance in the wider context. Chapter 3 is focused on the definition of comprehensive ZGA gene lists and of control genes, building a base for chapters 4 and 5. Using six published transcriptomics datasets from independent studies using different library generation methods, I define minor and major ZGA signatures that give a complex picture of the transcriptional landscape in zygotes and 2 cell stage embryos, by waning the biases introduced with each individual study. In Chapter 4, I assess the role that maternal DPPA2 and DPPA4 play in mouse embryonic development and major ZGA. For this, I used single and double conditional knock-out models to deplete maternal deposits of these proteins, and showed that major ZGA still takes place in their absence. This work was recently published. In Chapter 5, I assess the role that the maternal ISWI ATPase SMARCA5 plays in embryonic development, ZGA and chromatin remodeling in early embryos. For this I use both a conditional knock-out mouse model lacking SMARCA5 in oocytes, but also a targeted protein depletion system in early wild-type zygotes, followed by transcriptomics, methylation and chromatin accessibility characterisation of the embryos. Both systems confirmed that SMARCA5 plays an important role not only in the transcriptional activation but also in global chromatin changes that take place at the 2 cell stage. Chapter 6 contains a summary of my findings, conclusions and their relevance in the wider context of the field, and ends with proposed future directions. My dissertation provides new insights into the mechanisms and factors that regulate mouse ZGA, and highlights the importance of validating in vitro findings in a relevant in vivo system.
  • ItemOpen Access
    RNA binding proteins ZFP36 and ZFP36L1 limit CD8+ T cell differentiation and effector function
    Mitchell, Twm
    Post-transcriptional regulation of gene expression is mediated in part by RNA binding proteins (RBPs). The ZFP36 family of RBPs are key regulators of gene expression in the immune system. The founding member ZFP36 is a well-known regulator of cytokine mRNA stability. Paralogs ZFP36L1 and ZFP36L2 act redundantly during thymopoiesis and in developing B lymphocytes, where they limit DNA damage response and cell cycle progression. Furthermore, ZFP36L2 has been shown to repress translation of preformed Ifng mRNA and maintain quiescence in CD8+ memory T-cells. However, roles for the ZFP36 proteins in the differentiation and effector functions of CD8+ T cells remain to be explored. My project utilized mice featuring the OT-1 transgenic T cell receptor to produce cytotoxic T lymphocytes (CTLs) and memory-like CD8+ T cells in vitro. I made use of knockout mouse models with CD4cre-mediated conditional deletion of Zfp36 and Zfp36l1, to determine the role of these RBPs in limiting CD8+ T cell differentiation and effector functions. Using this approach, I demonstrated that ZFP36L1 acts to limit the cytotoxicity of CTLs and, by employing a CRISPR/Cas9 mediated gene knockout system, I demonstrated that this occurs early after initial T cell stimulation. I also demonstrated that upon adoptive transfer, memory-like CD8+ T cells form part of the central memory niche and respond to Listeria monocytogenes-OVA infection by differentiating and proliferating and that cells that lack ZFP36 and ZFP36L1 show more rapid terminal differentiation and improved effector functions. To begin to assay the molecular mechanisms by which ZFP36L1 limits CD8+ T cell differentiation, I made use of a recently published improved individual nucleotide crosslink immunoprecipitation methodology to identify directly bound target mRNAs. I demonstrated that transcripts encoding for factors in the MAPK, PI3K-AKT, and JAK/STAT signaling pathways are bound by ZFP36L1 and transcription factors IRF8 and Notch-1, and multiple cytokines and chemokines, including IFN-γ, TNF-α, IL-2 and CCL3/4. In addition, I describe a novel reporter mouse model of ZFP36L1 with an N-terminal fusion of a red-fluorescent protein expressed from the endogenous Zfp36l1 locus. Using this model, I analyzed the kinetics of ZFP36L1 expression in response to TCR stimulation. These findings demonstrate a role for ZFP36 and ZFP36L1 in coordinating both the differentiation and effector functions of CD8+ T lymphocytes. In the future, I hope to determine the mechanisms of action for ZFP36 RBPs in limiting CD8+ T cell differentiation.
  • ItemOpen Access
    The RNA-Binding protein PTBP1 plays a role in the activation of mouse CD8 T cells
    D'Angeli, Vanessa
    CD8 T cells play a pivotal role in immune responses against intracellular pathogens, including viruses and bacteria, and in tumour surveillance. After encountering an antigen, CD8 T cells undergo cell growth, clonal expansion, and acquisition of effector functions. These steps are a result of controlled gene expression changes dependent on transcriptional and post- transcriptional mechanisms. These processes are tightly regulated, but little is known about the mechanisms through which these processes are integrated. RNA-binding proteins play a pivotal role in controlling and regulating these processes. Here we show how the RNA-binding protein PTBP1 is dispensable for T cell development but has an essential role in regulating CD8 T cell activation, proliferation, and production of effector molecules. To investigate the roles of PTBP1 in CD8 T cells we generated a mouse model which conditionally deletes Ptbp1 in mature T cells. We found that PTBP1 has an essential role in regulating early events in CD8T cell activation resulting in the production of the effector molecules IL-2 and TNFa. It is also required for optimal proliferation and survival of clonally expanding CD8 T cells. PTBP1 controls a program of Alternative Splicing of many genes. One of these, the catalytic subunit of Calcineurin Ab and Ag may be linked to translocation of c-Fos, NFATc2 and NFATc3 in the nucleus of T cells. These findings reveal a crucial role for PTBP1 in regulating post-transcriptional regulation of genes involved in CD8 T cell activation and effector functions.
  • ItemOpen Access
    Elucidating the effects of dietary change on replicative ageing in Saccharomyces cerevisiae
    Horkai, Dorottya
    Saccharomyces cerevisiae, baker's yeast, is an established model organism of ageing research. There are many different pathologies associated with the ageing of yeast, ranging from phenotypic to global transcriptome changes. We have followed changes of several ageing markers and alteration of the global transcriptome under different dietary conditions and introduced genetic modifications to be able to uncover cellular and molecular mechanisms that play a role in the process of replicative ageing. We used imaging flow cytometry to monitor parameters and fluorescence levels of different markers of ageing and bulk RNA-sequencing to follow transcriptome changes in yeast cells during ageing. One particular focus of our research was the change in mitochondrial Tom70-GFP intensity, which has recently been found to undergo a dramatic accumulation throughout ageing, especially when the cell enters into senescence. We have tested the effects of different dietary environments on this and other markers of ageing, by growing cells on glucose (GLU), where the fermentation process is preferred, or an alternative carbon source galactose (GAL), where cells utilise both fermentation and respiration. Interestingly, ageing on GAL resulted in significantly lower Tom70-GFP marker accumulation than ageing on GLU, while cells had similar replicative age on both sugar sources. Furthermore, we have shown that the first 24 hours of growth is the primary determinant of the level of Tom70-GFP fluoresce intensity, even if the cells are shifted to a different carbon source. Differences in the global transcriptome revealed an overall more “young-like” transcriptome of GAL aged cells compared to GLU grown cells of the same timepoint. We demonstrated that increased respiration during ageing on GLU is sufficient to lower the Tom70-GFP marker aggregation phenotype. This was achieved by overexpression of the HAP4 gene, a transcriptional activator of mitochondrial biogenesis, and also overexpression of the SAK1 gene, which is a key regulator of the Snf1/AMPK pathway. On the other hand, we found that hindering respiration on GAL with a cox9Δ gal80Δ double mutation results in higher Tom70-GFP marker intensity after 48 hours of ageing. Forced reduction of the Snf1 pathway activity via deletion of the SNF4 gene in a GAL environment also resulted in increased ageing marker. These changes are also reflected in the extent of global transcriptional de-repression. In this thesis work we have shown that dietary change without restriction can also protect against phenotypic and transcriptome changes associated with ageing in yeast cells. Furthermore, we have demonstrated that early life respiration and the Snf1/AMPK pathway activity is important in the control of yeast ageing pathology. We can also conclude that global transcriptome changes are at least partly influenced by the respiration process. Therefore, our work has revealed potential signalling pathway targets for the improvement of yeast ageing process. Due to the conserved nature of these pathways it can also further our understanding of improved ageing health in higher organisms too.
  • ItemOpen Access
    Role of Histone Lysine Demethylase, KDM1B, in Trophoblast Stem Cell Self-Renewal and Differentiation
    Lea, Georgia
    The first cell fate decision in development occurs at the blastocyst stage with the emergence of the trophectoderm (TE) and the inner cell mass (ICM). The TE is the precursor population of all major placental cell types. Reflecting this developmental plasticity, trophoblast stem cells (TSCs) can be derived from the TE of mouse blastocysts. TSCs have proven an invaluable research tool to study processes of early placentation in vitro. Despite the placenta’s central role in reproduction, our understanding of the regulatory networks that orchestrate TSC self-renewal and differentiation remains incomplete. In this project, I characterised an epigenetic modifier, KDM1B, for its role in TSC self-renewal and differentiation. I identified this factor as a putative novel regulator of trophoblast stem cell fate and in vitro differentiation from transcriptomics data as its expression is markedly induced at the onset of differentiation. Furthermore, Kdm1b had been implicated in mouse development and placentation, via directing DNA methylation of maternal imprints in the oocyte. KDM1B is a histone lysine demethylase whose activity is directed to H3K4me1 and H3K4me2, particularly within the gene body of actively transcribed genes. By generating CRISPR-Cas9-mediated knockout TSCs ablated for Kdm1b, I show that Esrrb is consistently down-regulated but Gcm1 is up-regulated in Kdm1b-/- TSC clones as measured by RT-qPCR, indicative of precocious differentiation into the syncytiotrophoblast lineage. By performing a large cohort of integrated genome-wide analyses, notably RNA-seq and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) for the relevant histone modifications H3K4me1, H3K4me2, H3K4me3 and H3K36me3, I show that KDM1B regulates intragenic H3K4me1-marked enhancers, while not impacting H3K4me3. Remarkably, KDM1B null trophoblast cells also show an increased instability of chromosome 13, the same chromosome on which Kdm1b itself is located. This chromosome hosts several gene families that arose from gene duplication events, with vital roles in trophoblast development. The instability of chromosome 13 that gave rise to these gene families is apparently exacerbated by loss of KDM1B, or by CRISPR Cas9-induced cutting of the chromosome. In addition to interrogating the role of KDM1B during differentiation, I utilised the transcriptomic and ChIP-seq data to identify several trophoblast-specific transcripts via location of distal H3K4me3 peaks. Using previously published ChIP-seq data in combination with my own, I link enhancer activity in stem cells to gene expression throughout differentiation. Finally, I identify a striking and novel redistribution of H3K36me3 in 5 day differentiated trophoblast cells, to the promoter regions of expressed genes. In conclusion, this work presents an in-depth analysis of the transcriptional and epigenomic rearrangements that occur both in WT trophoblast and as a consequence of Kdm1b deletion. These data lend important insights into the functions of this epigenetic modifier in the fine-tuning of the transcriptional networks that direct TSC self-renewal and differentiation.
  • ItemOpen Access
    Investigation of the three dimensional organisation of the genome in B cell development
    Rogers, Carolyn
    In order to detect and eliminate a myriad of different antigens, the adaptive immune systems of jawed vertebrates have evolved to generate diverse repertoires of antigen receptors (AgRs). AgR diversity is primarily generated by a form of somatic recombination known as V(D)J recombination, where different variable (V), diversity (D) and joining (J) gene segments are cut-and-pasted together. V(D)J recombination of the immunoglobulin (Ig) loci, critical to the generation of diverse antibody repertoires, occurs during B lymphocyte development in the bone marrow. The three dimensional conformations of the multimegabase murine immunoglobulin heavy chain (Igh) and immunoglobulin kappa chain (Igκ) loci must not only facilitate recombination between gene segments separated by up to 3Mb, but diverse recombination events. To understand how this is achieved a capture Hi-C (CHi-C) approach was developed to investigate interactions within the Ig loci at high resolution. Hi-C libraries were enriched for the Ig loci and other genes of interest, using a biotinylated RNA bait system. In this thesis, I used the CHi-C assay to generate a high-resolution, unbiased view of murine Igh locus conformation in pro-B cells, resolving conflicting models in the field. I observed that the Igh locus folds into subdomains and two regulatory elements that bind CTCF, the 3’ CTCF Binding Elements (3’CBEs) and the Intergenic Control Region 1 (IGCR1), mediate contacts with the entire VH region. In collaborative studies the CHi-C data was used for polymer modelling to generate possible single conformations of the locus, giving further insights into the mechanisms of recombination. I revealed the first high-resolution, unbiased spatial conformation model of the murine Igκ locus in both pro-B and pre-B cells. I confirmed that the locus folds into discrete subdomains at the pro-B cell stage, but undergoes reorganisation at the pre-B cell stage. I then examined the roles of regulatory elements in the locus, including using a machine learning approach to probe the mechanisms underpinning their interactions with the Vκ region. Furthermore, I have proposed that the conformation of the Igκ locus, combined with published data on the binding of transcription factors and the distribution of active histone modifications and non-coding transcription, suggests the existence of a, so far, undescribed regulatory element in the Igκ locus. Moreover, the CHi-C assay used enabled the interrogation of interchromosomal contacts of baited regions and revealed that the Ig loci mediate numerous interchromosomal interactions during B cell development. I have shown that the interchromosomal interactions of the Ig loci are developmental stage specific. Additionally, I have demonstrated that, predominantly at the pre-B cell stage, the Ig loci and key lymphocyte-specific transcription factors share interaction partners. I characterised these interaction partners, and showed that many are implicated in immune system processes, indicative that these interaction networks have a regulatory function. Together these findings give new insights into the three dimensional organisation of the genome in B cell development.
  • ItemOpen Access
    The Rac-GEF P-Rex1 Regulates Agonist-Induced GPCR Trafficking
    Hampson, Elizabeth
    P-Rex guanine-nucleotide exchange factors (GEFs) activate the small GTPase Rac following stimulation of a variety of cell surface receptors, including G protein-coupled receptors (GPCRs). By activating Rac, P-Rex proteins control gene expression, cell survival and motility, among other responses, and therefore play important roles in regulating physiological processes including innate immunity, glucose homeostasis, thermogenicity, pigmentation and synaptic plasticity. Ligand binding to GPCRs not only induces signalling (within seconds) but also the internalisation of the receptor by clathrin-mediated endocytosis (within minutes) to switch off signalling. Previous data from our lab for the GPCR Sphingosine-1-Phosphate Receptor 1 (S1PR1) suggested a new role of P-Rex in GPCR trafficking. P-Rex overexpression blocks the first step of agonist-induced S1PR1 endocytosis - receptor phosphorylation - independently of catalytic Rac-GEF activity (unpublished). My PhD builds on this previous research providing a comprehensive insight into the role of P-Rex1 in GPCR trafficking. Upon generating a P-Rex1 knock-out PC12-S1PR1-GFP cell line, I used imaging and cell fractionation methods to show that endogenous P-Rex1 limits the agonist-induced internalisation of S1PR1 and quantified this effect. I investigated receptor specificity, finding that the role of P-Rex1 in blocking receptor trafficking extends to all GPCRs tested, regardless of which type of heterotrimeric G protein these receptors couple to. In contrast, receptor tyrosine kinases were unaffected. Mechanistically, an early stage of receptor internalisation, receptor phosphorylation, was inhibited by P-Rex1 in a GEF-activity independent manner. I also showed that endogenous P-Rex1 levels correlate with the SDF1α-induced internalisation of CXCR4 in MDA-MB-231 and MCF7 breast cancer cell lines. Finally, I investigated the role of P-Rex1 in physiological responses of neuronal cells, revealing novel GPCR signalling-dependent roles in the activation of Rac3 and Akt, and in cell spreading, as well as constitutive roles in cAMP production, cell-cycle progression and proliferation.
  • ItemOpen Access
    Investigating the role of the Polycomb Repressive Complex 2 in human in vitro pancreatic differentiation
    Fabian, Charlene
    The potential to use human pluripotent stem cells in regenerative medicine is an idea that continues to excite and captivate much of the science community, decades after the initial discovery of human embryonic stem cells. This interest has grown with the discovery of induced human pluripotent stem cells, combined with the continual improvement of differentiation protocols. These protocols allow the formation of an ever-increasing pool of mature and functional cell types within an in vitro laboratory environment. However, a number of important questions still exist in terms of the production of these cells, including their functionality and how closely these cells resemble the in vivo counterparts. One such area still to be explored is the epigenetic aspect of the in vitro derived cells. Beyond this understanding, the potential to apply epigenetic modulation to improve differentiation programmes, and/or the final cell product, is an important area of research. In this dissertation, I use the in vitro production of human pancreatic endocrine cells as a model to study histone methylation changes, and associated transcription, of differentiating cells produced in culture from a pluripotent stem cell population. I describe the dynamic epigenetic and transcriptional changes that occur in a bulk population of human pluripotent cells developing through a number of intermediary cell types to form pancreatic endocrine cells. This analysis was then used to study the differences between in vitro end-cell populations and in vivo derived cells, highlighting the large variation in gene expression and persistent H3K27me3 modifications present in the in vitro cells. I show that manipulation of Polycomb protein-mediated H3K27me3 levels in differentiation at specific time-points has a variable effect on end-cell population, with an apparently negative effect early in differentiation, but with potentially a more positive effect later in the differentiation. Lastly, I was able to introduce a reversible knockdown system within the human pluripotent cells, which will allow us to study the functional role of Polycomb-proteins in the context of in vitro differentiation. In summary, my work has established a role of aberrant histone modifications in limiting in vitro differentiation capabilities and provides a new framework for manipulating epigenetic processes to improve differentiation outcomes that may be relevant for improving targeted cell production.