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Regulatory Roles of Noncoding RNA in Development and DiseasePandey, Gaurav Kumar January 2013 (has links)
Long noncoding RNAs (lncRNAs) are being realized as important players in gene regulation and their misregulation has been considered as one of the underlying causes for tumor initiation and progression in many human pathologies. In the current thesis, I have addressed the functional role of lncRNAs in development and disease model systems. Genomic imprinting is an epigenetic phenomenon by which subset of genes are expressed in a parent of origin-specific manner. The Kcnq1 imprinted locus is epigenetically regulated by Kcnq1ot1 lncRNA. Deletion of an 890bp region at the 5’ end of Kcnq1ot1 in mouse resulted in the loss of silencing of neighboring ubiqui-tously imprinted genes (UIGs). In addition, we observed loss of DNA methylation at the UIG promoters. We have shown that Kcnq1ot1 RNA establishes CpG methylation by interacting with DNMT1. To explore the stability of lncRNA mediated silencing pathways, we have conditionally deleted Kcnq1ot1 in the mouse in a stage and tissue-specific manner. We have shown that Kcnq1ot1 is continuously required for maintaining the silencing of UIGs, whereas the silencing of the placental im-printed genes is maintained in an RNA independent manner. To identify chromatin-associated lncRNA (CARs) on a genome-wide scale, we purified RNA from the sucrose gradient fractionated chromatin and subjected it to RNA sequencing. Our study has identified 141 intronic and 74 long intergenic CARs. Characterization of one of the CARs revealed that it regulates the expression of neighboring genes in cis by modulating the chromatin structure. We have explored the functional role of lncRNA in tumor progression and initiation by using pediatric neuroblastoma. By transcriptional profiling of low- and high-risk tumors, we have identified several lncRNAs differentially expressed between these subtypes. We report an uncharacterized RNA NBAT-1, expressed at lower levels in high-risk tumors relative to low-risk tumors. Using neuroblastoma cell culture system, we demonstrated that NBAT-1 has anti-cell proliferative and anti-invasive properties. In addition, it promotes differentiation of neurons from undifferentiated neuroblastoma cell lines. In summary, by employing mouse genetics, cell culture based model system and expression profiling in tumors, we have uncovered new roles of lncRNA in gene regulation.
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Duchenne muscular dystrophy : RNA-based therapeutics and microRNA biologyRoberts, Thomas C. January 2012 (has links)
Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by absence of functional dystrophin protein. This thesis describes investigations into the role of small non-coding RNAs in both DMD pathology, and as potential therapeutic molecules. MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions. This study has compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx dystrophic mouse model relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as commonalities between tissues, including previously identified dystromirs. miR-1, miR-133a and miR-206 were found to be highly abundant in mdx serum, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalised in response to peptide-PMO mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable for the development of novel DMD therapeutics and for monitoring their efficacy. Myostatin is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as DMD. This study describes a novel myostatin inhibition approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in cultured myotubes. Silencing was sensitive to treatment with the histone deacetylase inhibitor Trichostatin A, and the silent state chromatin mark H3K9me2 was enriched at the myostatin promoter following siRNA transfection, suggesting epigenetic remodelling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for myostatin and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders. The work in this thesis therefore demonstrates the potential of small RNAs as therapeutic agents and as disease biomarkers in the context of DMD.
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Structural basis of DNA binding complexesWalavalkar, Ninad 30 May 2013 (has links)
The nucleosome remodeling and deacetylase (NuRD) complex is an abundant deacetylase complex, which couples histone deacetylation and chromatin remodeling ATPase activities, and has a broad cellular and tissue distribution. Although the working model of how this complex forms and functions is not well known, we have demonstrated that the coiled-coil interaction between two proteins (MBD2 and p66α) is critical for DNA methylation dependent gene silencing in vivo. Chapter one: ‘Unique features of the anti-parallel, heterodimeric coiled-coil interaction between methyl-cytosine binding domain 2 (MBD2) homologues and p66α dictate high affinity binding’ describes this unique coiled coil interaction. Coiled-coils were studied using a variety of biophysical techniques including analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC) and circular dichroism (CD). Results were compared across homologues and mutation studies were carried out to test our hypotheses. The studies reported in this chapter add to our understanding of coiled-coil interaction and thereby facilitate development of small peptide based drugs which target such interactions in nature.A number of proteins have been identified in humans that specifically bind to methylated CpG via a methyl binding domain (MBD). The human genome encodes at least five MBD proteins: MeCP2 and MBD1 through MBD4, which are homologous in their methyl binding domains but not many similarities are seen outside the MBD. Out of the five MBDs, MBD4 has a c-terminal glycosylase domain through which it recognizes mCpG.TpG mismatch and is important for base excision repair system. Chapter two: ‘Dynamic behavior of MBD4 in methylated DNA recognition’ focuses on MBD4 and its preference for DNA methylation mark. Techniques of surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) spectroscopy are used to study binding affinity for variations of methylated DNA mark. Chemical exchange studies are used to demonstrate how MBD4 scans for methylation mark and these studies have added a new dimension to our understanding of how MBD proteins ‘read’ DNA methylation marks. Chapter three: ‘Solving the solution structure of MBD domain of MBD4 on methylated DNA by NMR’ describes a process of structure determination using NMR spectroscopy. The focus of this chapter is not on developing a new technique but rather on using current resources to solve a protein structure, which can be used to further understand our biological system. Here, I have discussed the workflow used to determine a final three-dimensional structure starting from sample preparation, data collection, data analysis to structure calculation.
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The Effect of Isocitrate Dehydrogenase on the Epigenetics of Human Mitochondrial DNAStrang, John 25 April 2014 (has links)
Aberrant metabolism has become an increasingly interesting area of cancer biology. In many cancers including lower grade glioma, glioblastomas and some leukemias, a mutation in the metabolic enzyme Isocitrate Dehydrogenase (IDH), has been found in more than 70% of cases and has been shown to lead to a distinct hypermethylator phenotype. IDH commonly converts isocitrate to alpha-ketoglutarate in normal cell metabolism. Three isoforms of this enzyme are found in humans: IDH1, IDH2 and IDH3. Studies on IDH1, the cytosolic isoform, have revealed that mutations in the enzyme’s binding site lead to a novel gain of function: the synthesis of an oncogenic metabolite, 2-hydroxyglutarate (2HG). 2HG competitively inhibits alpha-ketoglutarate dependent enzymes such as the TET 5-methylcytosine (5mC) oxygenases and histone demethylases. These oxygenases are responsible for the hydroxymethylation (5hmC) of cytosine residues, ultimately leading to demethylation and gene re-expression. Thus, mutant IDH may lead to an elevation in 5mC levels producing the hypermethylator phenotype described. A similar gain-of-function mutation in IDH2, the mitochondrial isoform of IDH1, has been associated with leukemias and gliomas lacking IDH1 mutations. Mutations in IDH1, IDH2 and TET2 are mutually exclusive, and each yields a similar hypermethylator phenotype. IDH2, along with IDH3, is primarily involved in the TCA cycle and energy production for the cell. Recently, the Taylor lab has uncovered evidence of 5mC and 5hmC residues in mitochondrial DNA, established and maintained by mtDNMT1 and TET2. Changing levels of mtDNMT1 appears to alter the patterns and levels of mtDNA transcription from the mitochondrial genome. We hypothesized that mutant IDH would produce a similar effect on the mitochondrial genome as that found in the nuclear genome and result in a decrease in the level of 5-hydroxymethylcytosine, as well as a subsequent increase in the level of 5-methylcytosine caused by the competitive inhibition of the TET enzymes by 2-hydroxyglutarate accumulation. Using molecular biology techniques such as Western blots and MeDIP (methylated DNA immunoprecipitation) I aim to uncover the role of IDH mutation on mitochondrial DNA methylation and its effect on energy production in mammalian cells.
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Investigation into the Specification of NURF Recruitment to the GenomeMack, Marissa 01 January 2015 (has links)
The nucleosome remodeling factor (NURF) is a mutli-protein complex that plays a role in the regulation of gene expression through its ability to remodel nucleosomes. The largest subunit of this complex, Bptf (Bromodomain PHD Finger Transcription Factor) is important for many cellular processes as a transcriptional regulator and improper function results in disease or malignancy. To further understand the genome-wide recruitment of the NURF complex, the interaction partner for the N-terminal PHD finger domain of Bptf was investigated through pull down assays followed by mass spectrometry. It was determined that this domain does not recognize histones; instead it recognizes a nonhistone protein, Thoc4 or Hmgb1. The expression of a cDNA corresponding to Bptf was also tested for expression in mouse ES cells after the addition of two exons found to be missing in the original cDNA. Addition of this sequence did not allow for exogenous Bptf expression in ES cells.
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Regulators of DNA methylation in mammalian cellsTermanis, Ausma January 2013 (has links)
Although the many cells within a mammal share the same DNA sequence, their gene expression programmes are highly heterogeneous, and their functions correspondingly diverse. This heterogeneity within an isogenic population of cells arises in part from the ability of each cell to respond to its immediate surroundings via a network of signalling pathways. However, this is not sufficient to explain many of the transcriptional and functional differences between cells, particularly those that are more stable, or, indeed, differences in expression between parental alleles within the same cell. This conundrum lead to the emergence of the field of epigenetics - the study of heritable changes in gene expression independent of DNA sequence. Such changes are dependent on “epigenetic modifications”, of which DNA methylation is one of the best characterised, and is associated with gene silencing. The establishment of correct DNA methylation patterns is particularly important during early development, leading to cell type specific and parental allele specific gene regulation. Besides DNA methyltransferases, various other proteins have recently been implicated in DNA methylation. The absence of these proteins leads to defects in DNA methylation and development that can be even more severe than those in DNA methyltransferase knockouts themselves. In this study I focus on three such accessory proteins: LSH (a putative chromatin remodelling ATPase), G9a (a histone lysine methyltransferase) and SmcHD1 (a structural maintenance of chromosomes protein). To compare DNA methylation between WT cells and cells knocked out for each of these proteins, I used whole genome methylated DNA affinity purification and subsequent hybridization to promoter microarrays. This enabled me to compare the requirement for each protein in DNA methylation at specific genomic regions. The absence of LSH in mouse embryonic fibroblasts (MEFs) resulted in the loss of DNA methylation at 20% of usually methylated promoters, and the misregulation of associated protein coding genes. This revealed a requirement for LSH in the establishment of DNA methylation at promoters normally methylated during pre-implantation as well as post-implantation development. Secondly, I identified hypomethylation at 26% of normally methylated promoters in G9a-/- compared to WT ES cells. Strikingly, this revealed that G9a is required for maintenance of DNA methylation at maternal as well as paternal imprinting control regions (ICRs). This is accompanied by expression defects of imprinted genes regulated by these ICRs. Finally, in collaboration with the Brockdorff lab at the University of Oxford I identified a role for SmcHD1 in establishing DNA methylation at promoters on the X chromosome normally methylated slowly during X chromosome inactivation. Interestingly, SmcHD1 was also required for DNA methylation at autosomal gene promoters, contrary to previous reports that it is mainly involved in X chromosome methylation. I conclude that different accessory proteins are required to facilitate correct DNA methylation and gene repression at distinct regions of the genome, as well as at different times during development. This function of accessory proteins may be in part dependent on the prior establishment of specific chromatin signatures and developmental signals, together comprising a precisely regulated system to establish and maintain appropriate DNA methylation throughout development.
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Epigenetic regulation of immune tolerance in intestinal epithelial cellsThorpe, A. J. January 2016 (has links)
Objectives: Tolerance is a hyporesponsive state caused by repeated exposure to a stimulus. In the intestine, dysregulation of tolerance to luminal stimuli may lead to chronic and deleterious inflammation, such as characterizes Inflammatory Bowel Disease. The role of T-cells in immune tolerance is well known, but that of the epithelium requires investigation. Epithelial tolerance is gene-specific and differentially regulated, but the role of and involvement of epigenetics in tolerance regulation is unknown. We hypothesized that prior stimulation may cause epithelial cells to become hyporesponsive (tolerized) and that modification of histone methylation may alter the response to pro-inflammatory stimulation. The aim of this work was to examine if known inhibitors of histone methylation modifying enzymes affected the expression of CXCL8 in response to IL-1β. Methods: CXCL8 production of intestinal epithelial cells was measured by ELISA after stimulation with the pro-inflammatory stimuli P3CK and IL-1β and small molecule epigenetic inhibitors. The CXCL8 production of cells stimulated with a pro-inflammatory stimulus was compared to pre-stimulated cells after a second stimulus. CXCL8 production of IL-1β-pre-stimulated cells was also compared to CXCL8 production when these cells were incubated with epigenetic inhibitors. The effects of these inhibitors on histone methylation levels were examined by Western blotting for the global effect and by ChIP-qPCR for specific effects at the CXCL8 locus. Results: Intestinal epithelial cells stimulated with pro-inflammatory stimuli produced a large CXCL8 response. Pre-stimulation significantly decreased CXCL8 production after a second stimulus. The time-course of CXCL8 expression was measured to ensure that CXCL8 expression due to pre-stimulation was over before the second IL-1β-stimulation. In the presence of specific epigenetic inhibitors, pre-stimulation by IL-1β did not reduce CXCL8 production after a second IL-1β- stimulation. The specific effect of these inhibitors on the epigenetic signature at the CXCL8 locus was confirmed by ChIP. Thus, histone methylation modification disrupted tolerization of intestinal epithelial cells to a pro-inflammatory stimulus. Conclusion: The inflammatory response of the intestinal epithelium can be tolerized by prior stimulation with pro-inflammatory cytokines. Tolerization is lost after incubation with inhibitors known to modify histone methylation status, indicating for the first time, the involvement of histone methylation in this phenomenon.
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The role and regulation of imprinting in Arabidopsis thalianaTopham, Alexander January 2013 (has links)
The ‘epigenome’ refers to a difference in the transcriptional behaviour of a given allele that cannot be explained by differences in the genetic code, or ‘mutation’ at that locus. The epigenome is associated with certain biochemical marks, and generally exerts a silencing effect upon the transcription of genes under its influence. In Angiosperms such as Arabidopsis thaliana, fertilisation of both the egg and central cell– referred to as ‘double fertilisation’ – giving rise to the embryo and endosperm respectively, the latter of which is one of the most important tissues in the human food chain. Upon double fertilisation the gametes of each parent are known to contribute differing epigenetic ‘imprints’, where one gamete contributes a copy of a given allele in a transcriptionally unavailable state, while the other parent’s copy is in an available state. When a gene resides in such a region, the result is that only one parent’s copy is hence transcribed; such a gene is said to be ‘imprinted.’ Imprinting is known to affect the development of the placenta as well as some of the adult tissues in mammalian models, and in plants is most extensively found in the endosperm, where without the imprint of both parents the resulting seed exhibits reduced viability and defective endosperm development. A relative dearth in the number of known imprinted loci in the model angiosperm, A. thaliana makes is difficult to make reliable assessments of its role and regulation. This thesis initially aimed to extend the count of known imprinted genes using a model that proved insufficient to identify novel imprinted genes, and presents a meta-analysis showing that the reliable attribution of imprinted status to a gene is difficult using high-throughput methods as well. In addition, the further characterisation of a novel imprinted gene identified previously by this lab, MPC, with a view to acquiring a more detailed understanding of its role using mutants carrying point mutations in the MPC protein showed only a subtle phenotype to discern them from wild-type plants. There has also been recent speculation of a role for repeat elements in imprinting. This thesis presents findings suggesting that the apparent association of repeat elements with imprinted genes is an artefact rather of an association of endosperm-expressed genes with transposable elements, rather than genes that are specifically imprinted.
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The histone demethylase KDM1A sustains the oncogenic potential of MLL-AF9 leukaemia stem cellsHarris, William January 2013 (has links)
Rearrangements involving the mixed lineage leukaemia (MLL) gene are found in 5-10% of human leukaemias and are likely propagated by a deregulated self renewing pool of leukaemia stem cells (LSCs). Targeting of the LSC pool represents a key novel strategy for the treatment of AML. In recent years epigenetic dysfunction has been identified as a key driving factor in a range of solid tumours and haematological malignancies. Evidence for this includes identification of mutations in the genes coding for critical epigenetic modifiers, characterisation of localised regions of abnormal chromatin at oncogene or tumour suppressor genes and the efficacious use of epigenetic-targeted therapies already present in the clinic. The data submitted in this thesis identify the histone demethylase KDM1A as a critical regulator of LSC potential in MLL-AF9 acute myeloid leukaemia (AML). Of all the histone demethylases, we found that only Kdm1a expression correlated positively and significantly with LSC frequency in murine models of human MLL fusion AML. Genetic knockdown or Cre-mediated excision of Kdm1a resulted in loss of LSC potential, reduced expression of LSC maintenance transcriptional programs and induction of macrophage differentiation in MLL-AF9 cells. These effects were phenocopied by chemical inhibition of KDM1A using the monoamine oxidase inhibitor tranylcypromine (TCP), as well as novel TCP analogues which inhibit KDM1A with greater potency and selectivity. These results were seen in murine, human cell line and primary patient cells harbouring MLL rearrangements. Global transcriptome and epigenome analyses revealed a key role for KDM1A in maintaining the histone three lysine four (H3K4) methylation status at highly expressed MLL-AF9-bound genes. In vivo transplantation of Kdm1a knockdown MLL-AF9 cells conferred a significant survival advantage compared with control littermates. Similarly, TCP analogue treatment of mice transplanted with MLL-AF9 cells revealed a reduction in LSC potential of the donor-derived AML cells but little impact on normal recipient haematopoietic stem and progenitor cells (HSPCs). Critically the clonogenic and repopulating potential of normal HSPCS, of both murine and human origin, was spared following either knockdown or chemical inhibition of KDM1A. Taken together, the data presented establish KDM1A as a potential therapeutic target in MLL fusion leukaemia.
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Characterisation of the non-canonical zinc finger protein ZFP263 in mouseDelahaye, Celia January 2018 (has links)
Multicellular organisms are composed of a number of different specialised cells that all carry the same genetic material but are highly divergent in their functions and characteristics. This diversity is only allowed because sets of specific genes are expressed in one type of cells and silent in others. A precise control mechanism is required to fine-tune gene regulation and relies on chromatin structure and regulatory proteins. One of the largest families of DNA-binding factors that influence this in human and mouse is the KRAB zinc finger protein (KZFP) family. KZFPs are thought to have rapidly evolved alongside transposable elements and be mediators of transcriptional repression. The few KZFPs that have been characterised so far have been shown to be involved in a wide range of regulatory and biological processes; hence it is hard to make functional generalisations. During my PhD, I studied one member of the KZFP family in mouse, ZFP263, with the aim of understanding its mechanism of action in mouse embryonic stem cells (mESCs) and its role in mice. My work has shown that ZFP263 is an ancient protein highly conserved in mammals and under purifying selection. One of its two functional domains however is divergent from the consensus sequence found in most KZFPs and suggests that ZFP263 might have lost the ability to recruit repressive chromatin states. My research identified the targets of ZFP263 binding in mESCs and showed that it does not bind and silence transposable elements. Indeed it targets unique regions of the genome, mostly within transcribed regions of genes. These genes show a wide range of expression levels and are involved in several key biological processes. Surprisingly, binding sites are not associated with the canonical KZFP co-factor but mostly co-localize with active histone marks. My findings lead me to hypothesise that ZFP263 has evolved to target active epigenetic states to unique regions that are positive regulators of transcription, in contrast to the more canonical model of KZFP function. To test this hypothesis, I have generated targeted mutations at Zfp263 in mice using CRISPR-Cas9 and my preliminary results suggest that Zfp263 mutants have growth defects indicating a role for this protein in mouse development. My findings indicate that ZFP263 is a unique KZFP with non-canonical properties and provide novel insights into the evolution and functions of KZFPs in mammals.
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