Membrane proteins represent a subset of proteins embedded in or associated with the biological membrane. Despite accounting for 30% of the prokaryotic and eukaryotic proteomes and over 50% of current therapeutic targets, the structural and functional studies of membrane proteins still largely lag behind their soluble counterparts. This is predominantly due to the challenges in the isolation and purification of these proteins from their native mmembrane environment. Traditionally, detergents are employed for the extraction of membrane proteins from the native membrane, with subsequent solubilisation in mixed micelles. However, the surrounding lipids could be sequestered and lost during this process, which is potentially denaturing to the proteins. A novel method exploiting the styrene maleic acid (SMA) copolymer for membrane solubilisation (in the total absence of detergents) results in the generation of SMA/lipid particles (SMALPs) or ‘native nanodiscs’ of polymer-encapsulated membrane proteins together with their surrounding lipid moieties. Besides allowing the direct solubilisation of target membrane proteins from their native environment, analyses of native protein-lipid interactions and the characterisation of membrane lipidomes could be implemented, which may provide pivotal information to underpin the development of the next-generation antimicrobial agents. This detergent-free approach was successfully applied to the two cell division proteins, i.e. ZipA and FtsA and another integral membrane lipid phosphatase - PgpB from Escherichia coli. An analytically robust ‘SMALP lipidomics’ method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to elucidate the co-extracted membrane lipidomes of these proteins, resulting in the first comprehensive report of their respective native phospholipid compositions. Biophysical and biochemical characterisations were also conducted on PgpB in the context of SMALP and a detergent benchmark to facilitate the direct comparison between these two approaches for membrane protein studies.

Multiple isoforms of promyelocytic leukaemia protein (PML) participate in various cellular activities including innate immune responses, gene transcription and cell apoptosis. Recent work in our laboratory demonstrated PML isoform II (PML-II) was required for type I interferon (IFN) and IFN-stimulated gene (ISG) transcription by regulating transcription factor recruitment at gene enhancers/promoters. This thesis examines the mechanistic role of PML-II in this process. Considering that the different C-terminal fragments of PML isoforms play distinct roles in PML function, the function of the PML-II C-terminal domain was investigated. A C-terminal region 615-758, defined by deletions Δ1 and Δ2, was essential for its function in the IFN response, while the N-terminal RBCC structure was dispensable. Removal of residues 615-758 greatly impaired PMLII binding with transcription factors NF-κB and STAT1, coactivator (CBP) and SWI/SNF chromatin remodeling complex component Brg-1. These binding sites were further refined using smaller deletions to show that residues 645-665 were critical for PML-II binding with these transcription-related factors. The effect of PML-II on chromatin remodeling and histone modification at target promoters was investigated. Both PML-II and Brg-1 regulated the recruitment of transcription factor (STAT1) and the enrichment/disposition of H3K9me3 histone marks at ISG promoters but had no effect on H3K4me3 at these promoters. Depletion of PML-II also impaired ISG promoter binding by SWI/SNF core subunits Brg-1 and BAF155. Recruitment of activators, coactivators, and other regulators/mediators to ISG promoters occurred sequentially and was interrupted by PML-II depletion. This suggested that PML-II is required for forming and stabilizing the whole transcriptional complex which contains various factors including STAT1, CBP, Brg-1 and BAF155. Finally, the role of PML-II in IFNα-induced cell apoptosis was studied. Knockdown of PML-II enhanced ERK and AKT signaling, suggesting activation of both pro-survival pathways. Moreover, depletion of PML-II greatly decreased expression of IFNα-induced pro-apoptotic proteins including ISG15/54, OAS1, PUMA and TRAIL. It also impaired IFNα-mediated inhibition of AKT signaling and consequently increased cell resistance to IFNα -induced cell apoptosis. Collectively, in this study we have shown the specific involvement of the PML-II C-terminal domain in IFN responses, and mapped the sequences within this domain that are necessary for its interaction with relevant transcription factors. I have also examined in detail the physical and functional interaction of PML-II with the SWI/SNF complex and shown the points in the transcription activation pathway at which PML-II acts. Finally, we have shown how impaired PML-II activity reduces the pro-apoptotic signaling that normally occurs downstream of an IFN response, suggesting a mechanism by which PML may provide its known tumour-suppressor function.

Decidualization denotes the differentiation of endometrial stromal cells (EnSCs) into specialized decidual cells that control embryo implantation. This process can be recapitulated in culture upon treatment of primary EnSCs with cyclic AMP analogue and progesterone. In this work, I subjected undifferentiated and decidualizing human EnSCs to Assay for Transposase Accessible Chromatin with sequencing (ATAC-seq) to map the underlying chromatin changes. ATAC-seq is a newly developed technique that utilizes the highly active transposase Tn5 to interrogate accessibility of the genome and map open chromatin regions. These putative cis-regulatory DNA regions can be further explored for “footprints” of transcription factor (TF) binding. In this study, I optimized ATAC-seq and used this technique first to investigate the regulatory mechanisms underlying decidualization of the human endometrial cells. A total of 185,084 open DNA loci were mapped accurately in EnSCs. Altered chromatin accessibility within 10 kb of transcription start sites upon was strongly associated with differential gene expression in decidualizing EnSCs. Analysis of 1,533 opening as well as closing chromatin regions revealed overrepresentation of DNA binding motifs for known decidual TFs and identified putative new regulators, including RAR related orphan receptor A (RORA), aryl hydrocarbon receptor nuclear translocator like (ARNTL) and Meis homeobox 1 (MEIS1). Conversely, downregulation of runt related transcription factor 1 and 2 (RUNX1/RUNX2), SRY-box 12 (SOX12), transcription factor 3 (TCF3), and ETS Proto-Oncogene 1 (ETS1) upon decidualization corresponded to loss of corresponding high-affinity binding sites differentiating EnSCs. Because of its dynamic nature, cyclic human endometrium is a rich source of adult stem cells that could be exploited for clinical purposes. However, clinical use of eMSCs is hampered by differentiation and loss of proliferative capacity of cells in prolonged cultures. As part of my Monash-Warwick alliance studentship, I joined the laboratory of Professor Gargett in Melbourne, Australia, and applied integrated ATAC-seq and RNA-seq analyses to study the impact of TGF-β receptor inhibition on endometrial mesenchymal stem cells (eMSCs) maintained in prolonged culture. I demonstrated that culturing of eMSCs in the presence A83-01, a small molecule inhibitor the of TGF-β receptor, maintains the proliferative capacity and attenuates the loss of stemness features of eMSCs in extended cultures. Furthermore, integrated ATAC-seq and RNA-seq revealed that A83-01 modifies the chromatin accessibility of 5,967 loci and alters the expression 1,463 genes. Mining and cross-referencing of these data sets revealed that A83-01 not only maintains selected stemness-associated genes but also that it represses multiple genes involved in extracellular matrix (ECM) deposition and metabolism. Furthermore, my analysis indicated that induction nuclear receptor subfamily 4 group A member 1 (NR4A1, also known as NUR77) may be an important TF that mediate the repression of ECM genes in response to A83-01 treatment. In summary, by integrating advanced genome-wide expression and DNA accessibility profiling techniques, my work has advanced our understanding of the dynamic changes in the cis-regulatory DNA landscape underpinning decidualization of EnSCs and in the maintenance of a stem-like phenotype of eMSCs in prolonged cultures. Analyses of these two large data sets revealed novel transcriptional regulators in cycling endometrium and putative new targets that could be exploited to accelerate clinical translation of autologous eMSC therapies for a variety of reproductive disorders. Furthermore, the data sets generated during the course of my investigations constitute an important resource to interrogate fundamental molecular questions pertaining to human endometrial cell biology.

Paleovirology is the study of viruses over evolutionary timescales. Contemporary paleovirological analyses often rely on sequence data, derived from organism genome assemblies. These sequences are the germline inherited remnants of past viral infection, in the form of endogenous viral elements and the host immune genes that are evolving to combat viruses. Their study has found that viruses have exerted profound influences on host evolution, and highlighted the conflicts between viruses and host immunity. As genome sequencing technology cheapens, the accumulation of genome data increases, furthering the potential for paleovirological insights. However, data on ERVs, EVEs and antiviral gene evolution, are often not captured by automated annotation pipelines. As such, there is scope for investigations and tools that investigate the burgeoning bulk of genome data for virus and and antiviral gene sequence data in the search of paleovirological insight.

RSC is a member of the multi-subunit SWI/SNF family of ATPase-dependent chromatin remodellers and it is implicated in transcriptional regulation and DNA repair in Saccharomyces cerevisiae. The central ATPase subunit, Sth1, translocates nucleosomes in vitro and mutations in human RSC sub-unit orthologues are implicated in human disease. RSC is found in two isoforms, defined by the presence of either the Rsc1 or Rsc2 subunits, and these appear to confer distinct remodelling functions in different genomic contexts. At the MAT locus, Rsc1 and Rsc2 appear to mediate different forms of nucleosome positioning which are required for efficient mating type switching. Elsewhere in the genome, it has been suggested that RSC can create partially un-wrapped nucleosomes in order to facilitate transcription factor binding. This thesis uses indirect-end-label analysis and chromatin-sequencing technologies to dissect the chromatin remodelling functions of RSC and to determine the roles of Rsc1, Rsc2 and their subdomains. The work presented here suggests that four chromatin-remodelling outcomes arise from RSC activity. Firstly, RSC alters the positions of a tract of nucleosomes abutting HO endonuclease-induced double-strand DNA breaks both at MAT and non-MAT loci in a Rsc1-dependent manner. This activity can be transferred from Rsc1 to Rsc2 by swapping BAH domains. Secondly, RSC can aggregate nucleosomes into a large nuclease-resistant structure, termed an alphasome, in a Rsc2- and Rsc7-dependent manner. Thirdly, RSC positions nucleosomes at tRNA genes in a manner that requires both Rsc1 and Rsc2. Finally, chromatin particles consistent with previously described un-wound nucleosomes are confirmed to be present in specific promoter regions. Although Rsc1- and Rsc2- dependent subsets of these promoters could be identified, and associations with binding motifs for particular transcriptions factors were discovered, it was ultimately not possible to unambiguously define why some gene promoters depend on one RSC sub-unit rather than the other.

The Arabidopsis HapMap project, and follow-on work carried out by the Bergelson and Nordborg groups, established in broad outline the demographic history and population structure of wild Arabidopsis thaliana. Genome‐wide association studies are likewise making considerable advances in identifying genes associated with ecologically significant traits, and thus in identifying candidate genes likely to be under the action of natural selection. The aim of this project has been to further expand and combine these lines of investigation, by using genomic data to test ecological hypotheses and to grant more complete insight into the rangeof selection pressures acting upon wildpopulations. A method to measure and elucidate the genetic similarity of genomic regions between sampled accessions was therefore developed to facilitate this. 250K SNP data from RegMap accessions was then examined for evidence of patterns of migration and gene flow across Europe. Those observations formed the basis of a simple model of the history of the UK population relative to that of Europe. Comparisons of observed genotypes against expectations derived from the modelallowed the identification of genomic regions under the influence of selection. Loci corresponding to signatures of selection indicated positive selection acting upon phenotypes of disease resistance, flowering time, and seed size.

The bHLH transcriptional repressor HES1 plays a pivotal role in progenitor cell maintenance and cell fate decisions. Hes1 is cyclically expressed during somitogenesis and in a variety of cell types. In order to better understand the mechanism of Hes1 expression, we used comparative genomics to identify additional potential Hes1 cis-regulatory modules (CRMs). This revealed seven phylogenetically conserved sequence blocks within 57 kb upstream of the Hes1 transcription start site in mouse. In vitro reporter assays revealed that these regions have a transcriptional regulatory function as they modulate the activity of both a heterologous and the endogenous Hes1 promoter. Notch signalling is believed to play a role in Hes1 regulation. Consistent with this, sequence analysis revealed that all of the newly identified CRMs contain consensus motifs for the RBP-Jĸ Notch effector. ChIP assays confirmed that RBP-Jĸ binds to all the identified regions in proliferating C2C12 cells, with the exception of CRM7. Further CRM7 sequence analysis identified a conserved M-CAT motif which we showed is specifically bound by TEAD2 and its co-activator YAP using ChIP. The loss of RBP-Jκ or TEAD2 binding correlated with impaired CRM function. Furthermore, mapping of the chromatin architecture of the endogenous Hes1 locus showed that several of the identified CRMs loop onto the Hes1 promoter, consistent with their regulatory function. This CRM-promoter communication is preserved throughout oscillatory expression of the gene. These data suggest a molecular mechanism for Notch and Hippo signalling in Hes1 regulation in proliferating C2C12 cells. Additionally, we have engineered destabilised Venus fluorescent reporters for live cell imaging of CRM function. Venus was modified using protein degradation motifs, including the PEST motif from c-myc and the CL1 yeast sequence, resulting in proteins with half-lives of approximately 76.5 and 28.7 minutes, respectively. These reporters were subsequently multimerised using a 2A peptide to enhance their brightness. The destabilised Venus reporters represent an excellent tool for real-time live cell imaging.

Cis,regulatory modules (CRMs) are the functional DNA elements that encode the spatial and temporal expression patterns of a gene. Each gene is regulated by multiple CRMs, which may be hundreds of kilobases distant from the promoter. Many vertebrate CRMs have been characterised in isolation, but how CRMs act together to regulate complex patterns of expression is relatively unknown. Two CRMs that regulate expression of the muscle, specifying master gene myoD1 have previously been identified. Three additional potential CRMs were identified using a comparative genomics approach.The regulatory roles of these CRMs were investigated alone and in combination. Reporter plasmids containing all thirty,two possible combinations of these CRMs were made,and their expression was assayed in an immunologically,defined subpopulation of transfected mouse myoblast cells by flow cytometry. A statistical mechanics,based model used this exhaustive expression dataset to parameterise the interactions between the CRMs. This identified the ability of particular regulatory modules to have both enhancing and repressive effects upon transcription, dependent upon their surrounding CRM ‘context’. The physical proximity of the regulatory modules and myoD1 promoter in native chromatin during gene expression was confirmed by chromosome conformation capture analysis. To characterise the molecular basis of these properties further, a phylogenetic sequence comparison method was used to identify conserved transcription factor binding sites (TFBSs)within each CRM. A combination of experimental, bioinformatic and literature,derived evidence was used to prioritise binding sites fora large scale mutagenesis study to disentangle the molecular interactions between a prominent CRM pair. This allowed the mechanistic underpinnings of context sensitivity in this particular system to be identified.

The genome of the Trichostrongylid nematode parasite of small ruminants Haemonchus contortus is being sequenced at the Pathogen Sequencing Unit of the Wellcome Trust Sanger Institute, Cambridge, UK. Currently, in excess of 800 Mb of genomic sequence is available for this on-going project (http://www.sanger.ac.uk/Projects/H_contortus/). Once available, the fully sequenced and assembled genome of H. contortus will be an extremely valuable resource for both novel drug discovery and biological research into this important pathogen. H. contortus resides in the same Clade (Clade V) of the phylum Nematoda as the free-living model organism Caenorhabditis elegans. Therefore, it is ideally placed to extrapolate the wealth of genomic and biological data available for C. elegans. The extent to which such data can be applied to parasitic nematode research was a major focus of this project. I have concentrated on two well documented and important gene classes: those comprising the β-tubulin gene family and those of the RNA-interference (RNAi) pathway. Control methods for H. contortus are becoming increasingly restricted due to the rise in resistance to current anthelmintic drugs. Benzimidazoles (BZ) are a class of anthelmintic to which there is widespread resistance. Mutations and deletions in both of the β-tubulin genes previously identified from H. contortus, isotypes-1 and 2, have been shown to correlate with BZ resistance. I have identified an additional two β-tubulin loci within the H. contortus genome, which now gives a total of four genes for this family. Using C. elegans as a surrogate expression system together with antibody immunolocalisation in H. contortus I have investigated the expression pattern of three of these H. contortus β-tubulin genes and encoded proteins, and compared these with those of the C. elegans β-tubulin gene family. In addition, I have characterised the phylogenetic relationships of all available Trichostrongylid β-tubulin polypeptide sequences. This has allowed the determination of the evolution of this gene family, and possible association of isotypes-1 and 2 with BZ resistance, across these nematodes. RNAi is a well established technique used in C. elegans to silence gene expression using double stranded RNA (dsRNA). However, RNAi is far less effective and repeatable in parasitic nematode species. Using gene searching techniques I have examined whether genes required for RNAi in C. elegans are present and conserved in the H. contortus genome. Although I identified putative homologues of Dicer (dcr-1) and several other RNAi genes, no sequence homologous to the C. elegans rde-4 gene could be found. This gene is essential for the generation of small inhibitory RNAs (siRNAs) in the C. elegans RNAi pathway. Furthermore, no H. contortus genomic sequence encoding a homologue of Ce-SID-2 was identified. SID-2 is essential for dsRNA uptake from the environment, and sequence differences between C. elegans and C. briggsae SID-2 are responsible for the lack of environmental RNAi in the latter. I have also searched the available genomic sequence databases of Pristionchus pacificus and Brugia malayi for RNAi pathway genes and concluded that components of the RNAi pathway may not be conserved across the phylum Nematoda, although full genome sequences will be required to confirm these findings.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common form of muscular dystrophy in Caucasians. FSHD is caused by contraction of a 3.3kb repeat array, D4Z4, to below 11 repeat units. Each of these repeat units contains an ORF encoding the DUX4 gene and at the beginning of the work described in this thesis expression of transcripts from the most distal repeat of this D4Z4 array had been reported. However, expression data from the DUX4 gene was new and most research focussed on contraction of the array having a position affect on the expression of neighbouring genes. There is a similar Dux array located in the mouse genome. This array also contains an ORF in each repeat and transcripts from this gene have been detected in a number of different tissues. This array is not present in the same chromosomal location as the D4Z4 array in humans so is unlikely to be a true ortholog, however it is the only Dux array in the mouse genome and may therefore be functionally equivalent. The work described in this thesis provides evidence for transcription from multiple repeats of the D4Z4 array in a human embryonal carcinoma cell line and gives information on the sequence variation within these transcripts. In addition, this work contributes to an understanding of expression from the mouse Dux array. Similar to the human array, expression appears to come from multiple repeat units, and analysis of the sequences amplified by RT-PCR identifies variants which suggest some of these transcripts are non-coding. The aim of this work is to determine whether human DUX4 and mouse Dux genes have equivalent functions with the long-term goal of producing a mouse model for FSHD