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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
21

Arabidopsis thaliana histone deacetylase 1 (AtHD1) and epigenetic regulation

Tian, Lu 30 September 2004 (has links)
Epigenetic regulation is a mechanism by which heritable changes in gene expression are controlled by chromatin status rather than primary DNA sequence. Changes in chromatin structure affect accessibility of DNA elements to the transcriptional machinery and thus affect transcription activity of the gene. A key event in this process is reversible modification of core histones, which is catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDs, HDAs, or HDACs). In general, histone deacetylation is related to transcriptional gene silencing, whereas acetylation is associated with gene activation.To study the role of histone deacetylase in plant gene regulation and development, we generated constitutive antisense histone deacetylase 1 (CASH) transgenic plants. AtHD1 is a homolog of RPD3 protein, a global transcriptional regulator in yeast. Expression of the antisense AtHD1 caused dramatic reduction in endogenous AtHD1 transcription, resulting in accumulation of acetylated histones. Down-regulation of histone deacetylation caused a variety of growth and developmental abnormalities and ectopic expression of tissue-specific genes. However, changes in genomic DNA methylation were not detected in repetitive DNA sequences in the transgenic plants.We also identified a T-DNA insertion line in exon 2 of AtHD1 gene (athd1-t1), resulting in a null allele at the locus. The complete inhibition of the AtHD1 expression induced growth and developmental defects similar to those of CASH transgenic plants. The phenotypic abnormalities were heritable across the generations in the mutants. When the athd1-t1/athd1-t1 plants were crossed to wild-type plants, the mutant phenotype was corrected in the F1 hybrids, which correlated with the AtHD1 expression and reduction of histone H4 Lys12 acetylation. Microarray analysis was applied to determine genome-wide changes in transcriptional profiles in the athd1-t1 mutant. Approximately 6.7% (1,753) of the genes were differentially expressed in leaves between the wild-type (Ws) and the athd1-t1 mutant, whereas 4.8% (1,263) of the genes were up- or down-regulated in flower buds of the mutant. These affected genes were randomly distributed across five chromosomes of Arabidopsis and represented a wide range of biological functions. Chromatin immunoprecipitation assays indicated that the activation for a subset of genes was directly associated with changes in acetylation profiles.
22

Methyl-CpG-Binding domain proteins and histone deacetylases in the stage-specific differentiation of olfactory receptor neurons

MacDonald, Jessica 05 1900 (has links)
DNA methylation-dependent gene silencing, catalyzed by DNA methyltransferases (DNMTs) and mediated by methyl binding domain proteins (MBDs) and histone deacetylases (HDACs), is essential for mammalian development, with the nervous system demonstrating particular sensitivity to perturbations. Little is known, however, about the role of DNA methylation in the stage-specific differentiation of neurons. In the olfactory epithelium (OE), where neurogenesis is continuous and the cells demonstrate a laminar organization with a developmental hierarchy, we identified sequential, transitional stages of differentiation likely mediated by different DNMT, MBD and HDAC family members. Biochemically, HDAC1 and HDAC2 associate with repressor complexes recruited by both MBD2 and MeCP2. HDAC1 and HDAC2, however, are divergently expressed in the OE, a pattern that is recapitulated in the brain. Rather than simultaneous inclusion in a complex, therefore, the individual association of HDAC1 or HDAC2 may provide specificity to a repressor complex in different cell types. Furthermore, distinct transitional stages of differentiation are perturbed in the absence of MBD2 or MeCP2. MeCP2 is expressed in the most apical immature olfactory receptor neurons (ORNs), and is up-regulated with neuronal maturation. In the MeCP2 null OE there is a transient delay in ORN maturation and an increase in neurons of an intermediate developmental stage. Two protein variants of MBD2 are expressed in the OE, with MBD2b expressed in cycling progenitor cells and MBD2a in the maturing ORNs. MBD2 null ORNs undergo increased apoptotic cell death. There is also a significant increase in proliferating progenitors in the MBD2 null OE, likely due, at least in part, to feedback from the dying ORNs, acting to up-regulate neurogenesis. Increased cell cycling in the MBD2 null is also observed post-lesion, however, in the absence of feedback back from the ORNs, a phenotype that is recapitulated by an acute inhibition of HDACs with valproic acid. Therefore, disruptions at both transitional stages of ORN differentiation are likely in the MBD2 null mouse. Together, these results provide the first evidence for a sequential recruitment of different MBD proteins and repressor complexes at distinct transitional stages of neuronal differentiation.
23

Epigenetic Silencing of Novel Tumour Suppressor Genes in Medulloblastoma

Kongkham, Paul 26 March 2012 (has links)
Medulloblastomas (MB) are the most common pediatric nervous system malignancy. Known mutations account for only a subset of MB cases. We hypothesized that CpG island methylation-mediated tumour suppressor gene (TSG) silencing contributes to MB pathogenesis, either alone, or in combination with genetic events such as loss of heterozygosity (LOH). We performed a microarray-based genome-wide screen of MB cell lines treated with 5-aza-2’deoxycytidine, identifying genes exhibiting increased expression following treatment. Using this strategy, we identified inhibitors of WNT signalling (SFRP1, SFRP2, and SFRP3) and an inhibitor of the HGF/MET signalling pathway (SPINT2) as putative TSGs silenced by promoter region methylation in MB. Methylation of the WNT signalling inhibitors SFRP1, SFRP2, and SFRP3 was identified using bisulfite sequencing and methylation-specific PCR (MSP). Stable re-expression of SFRP1, SFRP2, and SFRP3 reduced proliferation, impaired anchorage-independent growth, and limited WNT signalling activity. SFRP1 re-expression reduced tumour growth in vivo in xenograft models. Aberrant WNT signalling plays a role in the pathogenesis of a subset of sporadic human MB, as well as MB in cases of Turcot syndrome with germline mutations of APC. Activating mutations of β-catenin are also implicated in a subset of MB. We have identified for the first time an additional mechanism – loss of normal pathway inhibition by SFRP gene silencing – that contributes to MB pathogenesis. SPINT2 methylation was confirmed with bisulfite sequencing and MSP. Stable re-expression of SPINT2 reduced proliferation, impaired cell migratory ability, and decreased the capacity for anchorage-independent growth. In vivo, re-expression of SPINT2 reduced tumour formation in xenograft models. This study identified for the first time SPINT2 as a putative TSG in human MB, and further implicated aberrant HGF/MET oncogenic signalling in the pathogenesis of this disease. The efficacy of targeting the HGF/MET pathway as a novel therapeutic strategy was tested in vitro using the small molecule MET kinase inhibitor PHA665752. Treatment of MB cell lines with PHA665752 reduced cell proliferation, anchorage-independent growth, migration, and limited downstream signalling via the MAPK and PI3K/AKT pathways.
24

GENOMIC IMPRINTING IN Drosophila melanogaster: EPIGENETIC REGULATION OF THE Dp(1;f)LJ9 IMPRINTED DOMAIN

MacDonald, William 09 August 2010 (has links)
Genomic imprinting is an epigenetic phenomenon whereby the expression of a gene, chromosomal region, or entire chromosome, depends on the sex of the transmitting parent. Imprinting results in an otherwise fully functional gene being transcriptionally silenced when transmitted by one parent, yet the same gene, with identical DNA sequence, is active when transmitted by the other. Thus, the gene retains an imprint or “memory” of its genetic history, which is reversible and reset each successive generation by passage through the germline. Within this thesis, I present my findings that show genomic imprinting in Drosophila is regulated by distinct epigenetic mechanisms at different stages of embryogenesis, suggesting the requirement of a transitional stage to stabilize the imprint between establishment in the germline and maintenance in the soma. I futher show that Drosophila utilize epigenetic mechanisms that are involved in regulating genomic imprinting in mammals and plants, such as DNA methylation, histone modification, antisense RNA, and chromatin insulators. These findings demonstrate convergence of the epigenetic mechanisms that regulate genomic imprinting in diverse organisms.
25

Methodology for genome-wide epigenetic profiling of the Drosophila male germline

El Sharnouby, Sherif Maher January 2011 (has links)
No description available.
26

Epigenetic Silencing of Novel Tumour Suppressor Genes in Medulloblastoma

Kongkham, Paul 26 March 2012 (has links)
Medulloblastomas (MB) are the most common pediatric nervous system malignancy. Known mutations account for only a subset of MB cases. We hypothesized that CpG island methylation-mediated tumour suppressor gene (TSG) silencing contributes to MB pathogenesis, either alone, or in combination with genetic events such as loss of heterozygosity (LOH). We performed a microarray-based genome-wide screen of MB cell lines treated with 5-aza-2’deoxycytidine, identifying genes exhibiting increased expression following treatment. Using this strategy, we identified inhibitors of WNT signalling (SFRP1, SFRP2, and SFRP3) and an inhibitor of the HGF/MET signalling pathway (SPINT2) as putative TSGs silenced by promoter region methylation in MB. Methylation of the WNT signalling inhibitors SFRP1, SFRP2, and SFRP3 was identified using bisulfite sequencing and methylation-specific PCR (MSP). Stable re-expression of SFRP1, SFRP2, and SFRP3 reduced proliferation, impaired anchorage-independent growth, and limited WNT signalling activity. SFRP1 re-expression reduced tumour growth in vivo in xenograft models. Aberrant WNT signalling plays a role in the pathogenesis of a subset of sporadic human MB, as well as MB in cases of Turcot syndrome with germline mutations of APC. Activating mutations of β-catenin are also implicated in a subset of MB. We have identified for the first time an additional mechanism – loss of normal pathway inhibition by SFRP gene silencing – that contributes to MB pathogenesis. SPINT2 methylation was confirmed with bisulfite sequencing and MSP. Stable re-expression of SPINT2 reduced proliferation, impaired cell migratory ability, and decreased the capacity for anchorage-independent growth. In vivo, re-expression of SPINT2 reduced tumour formation in xenograft models. This study identified for the first time SPINT2 as a putative TSG in human MB, and further implicated aberrant HGF/MET oncogenic signalling in the pathogenesis of this disease. The efficacy of targeting the HGF/MET pathway as a novel therapeutic strategy was tested in vitro using the small molecule MET kinase inhibitor PHA665752. Treatment of MB cell lines with PHA665752 reduced cell proliferation, anchorage-independent growth, migration, and limited downstream signalling via the MAPK and PI3K/AKT pathways.
27

SWI/SNF COMPLEXES COORDINATE WITH HISTONE MODIFICATIONS TO REGULATE CHROMATIN REMODELING

Chatterjee, Nilanjana 01 December 2011 (has links)
SWI/SNF, the founding member of ATP dependent chromatin remodelers and its paralog RSC in yeast perform similar yet distinct functions inside the cell. In vitro these complexes use ATP dependent DNA translocation to either mobilize or disassemble nucleosomes. However, how these complexes interact with nucleosomes and the mechanism by which chromatin remodeling is achieved is not fully understood. Further, it is not understood how they perform disparate roles in vivo despite their similar biochemical activities. To understand the fundamental differences between these complexes the substrate specificity of RSC and SWI/SNF and their interaction with different parts of the nucleosome were investigated. SWI/SNF and RSC exhibited almost identical nucleosome binding affinities (~7 nm) with a minimal requirement of 20 bp of extranucleosomal DNA for efficient binding. Hydroxical-radical footprinting of RSC-nucleosome complex showed that RSC, unlike SWI/SNF, interacts extensively with approximately 50 bp of extranucleosomal DNA near the nucleosome entry site. RSC also interacts, but not as strongly as SWI/SNF, with almost one gyre of nucleosomal DNA (SHL-2 to SHL-6) on the same side of the extranucleosomal DNA. Analogous to the previously observed SWI/SNF-footprint the second gyre of nucleosomal DNA had no protection and in fact enhanced cleavage was seen starting from 3-4 helical turns from the dyad axis up to the exit site where DNA leaves the nucleosome. The asymmetry of the DNA footprint pattern confirmed binding of RSC in one preferred orientation guided by the extranucleosomal DNA at one end of the nucleosome like ISW2 and also like SWI/SNF but only when recruited by transcription factors. DNA crosslinking revealed that most of the SWI/SNF contacts are with a small region spanning the DNA translocation start site near SHL2 and does not extend to the rest of the footprint. Further, the SWI/SNF contacts are primarily through its catalytic subunit Snf2 which is found to intercalate between the DNA gyre and the histone octamer at SHL2. Consistent with its DNA footprint, RSC however makes extensive contacts with both nucleosomal and extranucleosomal DNA through five major subunits Sth1, Rsc2, Rsc3, Rsc30 and Rsc4. Excepting the catalytic subunit Sth1 which is highly homologous to Snf2, the remaining four are unique to RSC. Sth1 contacts a much broader region in the nucleosomal DNA than Snf2 with the primary contact being at SHL2 where it wedges between the DNA and the histone octamer surface. The accessory subunits Rsc2, Rsc3 and Rsc30 mostly contribute to the extranucleosomal DNA contacts of RSC. These subunits also make a second major contact near the dyad, with those made by Rsc3 and Rsc30 being the strongest. The histone N-terminal tails that emanate out of the nucleosome structure are implicated in the regulation of chromatin remodeling, in general, and in the activation of several SWI/SNF dependent genes, in particular. Remodeling kinetics studies with tailless nucleosomes revealed that the histone H4 tail is required for nucleosome mobilization, H2A/H2B dimer displacement and nucleosome disassembly by both RSC and SWI/SNF. Further, the H4 tail modulates RSC and SWI/SNF remodeling without affecting ATP hydrolysis or nucleosome binding. These data suggest a similarity between SWI/SNF and ISWI class of chromatin remodelers based on their dependence on the H4 tail. Owing to the presence of acetyl-lysine binding bromodomains in these complexes and to a greater extent in RSC the differences in their remodeling activities, if any, were expected to be accentuated by histone acetylation. Studies with H3 and H4 tail acetylated nucleosomes provided evidence for two pathways that work synergistically to recruit SWI/SNF and RSC to chromatin. While one of the pathways involves transcription activators, the other pathway of SWI/SNF recruitment is dependent on covalent acetylation of histone H3 tail. Bromodomain mediated recognition of these acetyl marks not only facilitates SWI/SNF recruitment but also stimulates their catalytic activity to mobilize nucleosomes. Importantly, extensive conformational changes occur in SWI/SNF in response to H3 tail acetylation. Chromatin remodeling by SWI/SNF and RSC is also regulated to different degrees by H3 tail acetylation depending on the number of bromodomains. The higher responsiveness of RSC to H3 tail acetylation than SWI/SNF can provide additional regulatory mechanisms for RSC which might ultimately account for their different functional roles inside the cell. When these same acetyl marks are within the H3 globular core and reside near the dyad axis of symmetry they are found to act in synergy with RSC and SWI/SNF to facilitate nucleosome movement as well as nucleosome disassembly. Unlike H3 tail acetylation, the remodeling enhancement by H3 core acetylation occurs via an acetyl lysine-bromodomain recognition independent mechanism. Further, supporting this recognition-independent mechanism H3 core acetylation does not affect the recruitment of these complexes. These data illustrate how histone acetylation modulates RSC and SWI/SNF function, and provide a mechanistic insight into their collaborative efforts to remodel chromatin.
28

Characterization of the Global and Locus-Specific Regulation of Gene Expression During Early Myogenic Differentiation

Dixon, Katherine January 2016 (has links)
During cellular differentiation, gene expression is globally regulated through changes in the epigenome. How a single genome can give rise to a diversity of cell and tissue types remains a complex area of investigation, and here we sought to explore the molecular regulation of gene expression during the differentiation of skeletal muscle cells from committed myogenic progenitors. Using a systematic and integrated analysis of global transcriptional and epigenetic data, we characterized the regulation of gene expression in differentiating myoblasts and found that muscle-specific gene expression is regulated through differential activation of tissue-specific regulatory DNA elements by the myogenic transcription factor MyoD. In addition, the genome-wide localization of MyoD, and the mechanisms underlying its function in transcriptional regulation, varies between myogenic progenitors and differentiating myoblasts. Our study explores the recruitment and function of MyoD at regulatory elements of target genes and additionally describes a novel role for ligand-inducible signaling in the regulation of MyoD function and ultimately in myogenic differentiation.
29

SYMBIONT REGULATED HOST DNA METHYLATION IN EUPRYMNA SCOLOPES – VIBRIO FISCHERI SYMBIOSIS

Xiao, Rui 01 May 2018 (has links)
Advancement in the study of host-microbe interactions has shown that microbes can induce and maintain long lasting changes in gene expression in host cells to facilitate beneficial symbiosis through changes in methylation of the host’s genomic DNA. The beneficial symbiosis between Hawaiian Bobtail squid, Euprymna scolopes and Gram negative bioluminescent bacteria Vibrio fischeri provides an excellent system for studying beneficial microbes’ effect on host DNA methylation. The symbiosis is highly specific, in that only V. fischeri colonizes the squid’s symbiotic organ from a background of 106 diverse bacteria per mL of sea water. DNA methylation (DNAm) refers to the covalent addition of methyl (CH3) groups to the nucleotides of organism’s genomic DNA. The most well researched DNA methylation type is 5- methyl cytosine methylation (5mC). Previous publications show DNAm provides an extra tier of regulation for organisms to control their gene expression, without altering their DNA sequences. Two types of DNAm have been discovered in invertebrate systems: gene promoter methylation and gene body methylation. The amount of methylated cytosine on gene bodies is positively correlated with the specific gene’s expression state. We hypothesize that V. fischeri plays an important role in regulating host DNA methylation during both colonization of the animals (juvenile) and maintenance of the symbiosis I (adult). To start to address this hypothesis, our specific aims are (1) validate DNA methylation in E. scolopes; (2) identify DNA methylation machinery genes in squid at the transcript level and quantify the level of their expression based on state of symbiosis; (3) analyze squid DNA methylation at the whole genome level as well as gene specific level.
30

Control of Histone H3 Lysine 27 Trimethylation in Neurospora crassa

Jamieson, Kirsty 14 January 2015 (has links)
Trimethylation of histone H3 lysine 27 (H3K27me3) marks facultative heterochromatin, containing silent genes. My research investigated factors that influence the distribution of H3K27me3 in the filamentous fungus Neurospora crassa. The H3K27 methyltransferase complex, PRC2, is well conserved in eukaryotes and consists of four core members: E(Z), EED, SUZ12 and P55. I showed that three of the PRC2 subunits (SET-7, the homolog of E(Z), EED and SUZ12) are required for H3K27me3 in Neurospora, while NPF, the homolog of P55, is only required for a subset of H3K27me3 domains. H3K27me3 is organized into large, gene-rich domains in Neurospora and normally does not overlap with constitutive heterochromatin, which is marked by both H3K9me3 and DNA methylation and bound by heterochromatin protein 1 (HP1). I discovered that loss of HP1 binding results in a genome-wide relocalization of H3K27me3. Specifically, it is lost from many of its normal domains while it becomes associated with much of the genome that is constitutive heterochromatin. This contrasts plant and mouse studies in which the loss of DNA methylation relocalizes H3K27me3. The DCDC complex is the H3K9-specific methyltransferase consisting of DIM-5, DIM-7, DIM-9, CUL4 and DIM-8. Separate deletions of DCDC subunits, with the exception of dim-7, relocalized H3K27me3 to constitutive heterochromatin, presumably due to the loss of HP1 binding. The deletion of dim-7 resulted in the loss of all H3K27me3, suggesting a novel role for dim-7. To look for a recruitment signal for PRC2, I moved large fragments contained within an H3K27me3 domain to loci devoid of H3K27me3, his-3 and csr-1. None of the fragments induced H3K27me3, demonstrating that a recruitment signal is not present within every fragment of H3K27me3-marked DNA. Large chromosomal rearrangements had profound effects on H3K27me3 domains, resulting in the loss of some H3K27me3 domains and the formation of others. In Drosophila and mammals, a subset of PRC2 complexes contains the histone deacetylase, Rpd3. A close homolog of Rpd3 in Neurospora, HDA-3, did not appear to be a member of PRC2 in Neurospora. This dissertation includes both previously published and unpublished co-authored material.

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