Dissertations / Theses on the topic 'CpG Islands'
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1
Thomson, John Paterson. "Defining the protein complement of CpG islands." Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/4885.
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In higher eukaryotes, the DNA base Cytosine can exist in a variety of modified forms when in the dinucleotide CpG. Although a methylated form tends to dominate within the genome, approximately 1% of all CpG dinucleotides are found unmodified at high densities spanning around 1Kb and tend to co-localise to the 5’ ends of around 60% of annotated gene promoters. These unique DNA sequences are known as CpG islands (CGIs) and their role within the genome to date is largely unknown. Methylation of CGIs in cancers however has been linked to silencing of associated genes implying a role in gene regulation. Furthermore these sites are also interesting as they remain specifically nonmodified within a genome rich in methylated CpG. We set out to better understand the roles for CGIs through the characterisation of any specific CGI binding proteins. Digestion of nuclei with methyl sensitive restriction enzymes facilitates the purification of CGI fragments. Subsequent immunohistochemistry on the CGI chromatin fragments along with ChIP-PCR over several CGIs revealed an enrichment of the “active” histone modifications including H3K4me3, a depletion of the “silencing” marks such as H3K27me3, as well as a group of CGI specific binding factors. These latter proteins contained a domain previously shown to bind to non-methylated CpG dinucleotides (the CXXC domain) and as such were ideal candidates for CGI specific factors, in particular a protein called Cfp1. Genome wide sequencing revealed a striking correlation between Cfp1 and H3K4me3 which were both seen at around 80% of islands. Furthermore, the presence of Cfp1/H3K4me3 at islands tended to have a negative correlation with the presence of chromatin rich in the silencing histone modification H3K27me3. Closer investigation of the Cfp1 protein reveals it to be a true non-methyl CGI binding factor in vivo and shRNA reduction of Cfp1 levels to around 10% of wild type resulted in a precipitous drop in H3K4me3 levels over CGIs without a dramatic reduction in global H3K4me3 levels. As Cfp1 has been shown to be part of the Set1 histone H3K4 methyltransferase complex responsible for this modification, this CXXC protein may be attracting this histone modifying complex and as such represents a method whereby the underlying DNA sequence (CpG) can drive the overlying epigenetic state. This study may go some way to understanding the functional significance of CGIs within the genome.
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Wachter, Elisabeth. "Influence of CpG islands on chromatin structure." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/9369.
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CpG islands (CGIs) are short GC rich sequences with a high frequency of CpGs that are associated with the active chromatin mark H3K4me3. Most occur at gene promoters and are often free of cytosine methylation. Recent work has begun to clarify the functional significance of CGIs with respect to chromatin structure and transcription. In particular, proteins associated with histone-modifying activities, such as Cfp1 and Kdm2a, bind specifically to non-methylated CGIs via their CxxC domains. For example, artificial promoterless CpG-rich sequences integrated at the 3’ UTR of genes recruit Cfp1 and generate novel peaks of H3K4me3 in mouse ES cells without apparent RNA polymerase recruitment. There is also evidence that G+C-rich DNA recruits H3K27me3, a gene silencing mark. In this thesis I am exploring the constraints on DNA sequence and genomic location that are required to impose both H3K4me3 and H3K27me3 at CGI sequences. Showing that the generation of novel peaks of H3K4me3 and H3K27me3 over a promoter-less CpG rich sequence in a gene desert region is independent of it’s location in the genome extends earlier findings. These findings suggest that shared features of the primary DNA sequence at CGIs directly influence chromatin modification. Thus CGIs are not passive footprints of other cellular mechanisms, but play an active role in setting up local chromatin structure. However, the relative contribution of CpG frequency versus G+C content remains unclear. Therefore a sequence was generated that contains low levels of CpGs, comparable to the bulk genome, but has a G+C content similar to that of CGIs (Low CpG / High G+C). When this sequence was inserted into a gene desert neither marks of H3K4me3 or H3K27me3 were formed, indicating the importance of CpGs. Surprisingly, the reverse sequence with a high CpG frequency similar to that of CGIs and a low G+C content similar to that of the bulk genome (High CpG / Low G+C) did not establish H3K4me3 or H3K27me3 either. However, it was found that this sequence becomes heavily methylated in contrast to CGI-like sequences that remained unmethylated when introduced into a gene desert. This finding suggests that a high G+C content is important for keeping CGI-like sequences methylation free. Upon insertion of this High CpG / Low G+C sequence into mouse ES cells that were devoid of the de-novo DNA methyltransferases 3a and 3b (Dnmt3a/3b -/-) both H3K4me3 and H3K27me3 marks were established at the inserted sequence. This discovery confirms the importance of CpGs for setting up local chromatin structure.
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Longman, Dagmar. "Contribution of CpG islands to ubiquitous gene expression." Thesis, University of Edinburgh, 1997. http://hdl.handle.net/1842/15231.
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The effect of CpG islands on transgene expression was first tested in cultured cells. In transient transfection it was demonstrated that CpG islands do not influence the expression of a transgene when not integrated into the genome. Even when integrated into the genome of cultured cells, CpG islands are not able to confer position-independent, copy number-dependent transgene expression, as confirmed by the analysis of individual cell lines. However, the results from bulk analysis of primary clones suggest that CpG islands improve the level of expression in cultured cells, and increase the proportion of highly expressing clones. Transgenic mice were used to study the effect of CpG islands on the level and pattern of transgene expression in vivo. Unexpectedly, from the nine transgenic lines generated, transgene expression was detected in only one line. In the rest of the lines transgene expression was silenced, and in these cases the transgene was densely methylated. In half of the silenced lines transgenes were found to localise in the pericentromeric chromatin. The results suggest that full size CpG islands used as promoters do not necessarily overcome the negative effects of neighbouring chromatin to give ubiquitous transgene expression independent of the integration site. During the study of stable cell lines it was observed that cells within a clone do not have the same level of transgene expression, and this heterogeneity in expression was not reduced by the presence of the full size CpG island. In order to elucidate this phenomenon, the hypothesis that intraclonal heterogeneity is caused by individual cells switching transgene expression on and off over a period of time was tested. Clones of cells expressing Green Fluorescent Protein (GFP) were monitored at regular time intervals, and in some cells rapid extinction of fluorescence was observed. It was concluded from this result that transgene expression varies with time.
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Brown, David. "Understanding the role of CFP1 at CpG islands." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:baf2e91a-4417-407a-8938-bbef1f6c411f.
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Vertebrate genomes are punctuated by CpG islands regions, which have an elevated frequency of CpG dinucleotides. CpG islands are associated with over 70% of mammalian promoters suggesting they may contribute to the regulation of transcription. However, despite being discovered over 30 years ago, the function of CpG islands is still not understood. Unlike the majority of the genome, CpG islands are resistant to DNA methylation. This provides a binding site for CFP1 which binds specifically to non-methylated DNA via its zinc-finger CXXC (zf-CXXC) domain. CFP1 is a subunit of the SET1 methyltransferase complex, and is thought to direct the activating histone modification H3K4me3 to CpG islands. Interestingly, CFP1 also contains a PHD domain which is proposed to bind the H3K4me3 mark, potentially producing a feedback loop between H3K4me3 and the SET1 complex. Although the structural basis for discrimination of non-methylated CpGs is known, it is not clear how zf-CXXC proteins distinguish CpG islands amongst the irregular nucleosomal landscape which exists within the nucleus. This thesis is focused on the role of CFP1 in the relationship between CpG islands, SET1 and H3K4me3. To address these questions, it was important to mechanistically dissect the contribution of the PHD and zf-CXXC domains. The proposal that the PHD domain of CFP1 binds selectively to H3K4me3 was confirmed by in vitro experiments, however this study demonstrates that the PHD domain is insufficient for stable interactions with chromatin. Using complementary genome-wide and live cell imaging approaches, the zf-CXXC domain shown to be required for PHD-dependent interactions. Genome-wide snapshots of binding interactions, together with spatial and temporal details, expose a surprising contribution of the SET1 complex to the nuclear mobility of CFP1, providing a new perspective on the role of CFP1 in H3K4 methylation.
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Sertedaki, Amalia. "Study of hypervariable regions and CpG islands in human genomic DNA." Thesis, University of Newcastle Upon Tyne, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.238783.
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Farcas, Anca Madalina. "KDM2B links recognition of CpG islands to polycomb domain formation in vivo." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:cc773afe-703c-4b43-a792-7ee7ba333bcd.
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Mammalian genomes are characterised by global and pervasive DNA methylation and this modification is generally thought to be inhibitory to transcription. An exception to this widespread DNA modification are genomic elements called CpG islands (CGI), contiguous regions of non-methylated DNA which encompass the transcription start site of two thirds of mammalian genes. Although CGIs represent the most prominent feature of mammalian promoters, the contribution of these elements to promoter function remains unclear. Work in this study shows that the histone lysine demethylase KDM2B (FBXL10/ JHDM1B) is a nuclear protein which binds specifically to non-methylated CpG dinucleotides and associates with CGI elements genome-wide through its zinc-finger CxxC (ZF-CxxC) DNA binding domain. Furthermore, in mouse embryonic stem cells, biochemical investigation revealed that KDM2B associates with Polycomb group E3 ubiquitin ligase RING1B to form a variant Polycomb repressive complex 1 (PRC1) characterized by the PCGF1 subunit. Considering that KDM2B has clear DNA-binding activity and that CGIs were reported to function as nucleation sites for polycomb repressive complexes, a potential role for KDM2B in mediating PRC1 recruitment to target genes was investigated. Stable depletion studies indicated that KDM2B is required for the normal targeting of RING1B to CGIs and the regulation of expression of a subset of Polycomb-occupied genes. By taking advantage of a genetic ablation system in which the DNA binding domain of KDM2B can be conditionally deleted, results in this thesis reveal that the ability of KDM2B to recognize non-methylated DNA is essential for polycomb domain formation and normal embryonic development. Finally, through the use of a de novo targeting assay, an unexpected PRC2 recruitment pathway was discovered which is dependent on PRC1-mediated H2AK119ub1 deposition. Together this work uncovers a novel mechanism linking KDM2B-dependent recognition of non-methylated DNA with recruitment of Polycomb proteins and provides the framework on which to further investigate the contribution of CGIs to formation of polycomb domains.
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Zheng, Hao. "Prediction and analysis of the methylation status of CpG islands in human genome." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/43631.
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DNA methylation serves as a major epigenetic modification crucial to the normal organismal development and the onset and progression of complex diseases such as cancer. Computational predictions for DNA methylation profiling serve multiple purposes. First, accurate predictions can contribute valuable information for speeding up genome-wide DNA methylation profiling so that experimental resources can be focused on a few selected while computational procedures are applied to the bulk of the genome. Second, computational predictions can extract functional features and construct useful models of DNA methylation based on existing data, and can therefore be used as an initial step toward quantitative identification of critical factors or pathways controlling DNA methylation patterns. Third, computational prediction of DNA methylation can provide benchmark data to calibrate DNA methylation profiling equipment and to consolidate profiling results from different equipments or techniques.
This thesis is written based on our study on the computational analysis of the DNA methylation patterns of the human genome. Particularly, we have established computational models (1) to predict the methylation patterns of the CpG islands in normal conditions, and (2) to detect the CpG islands that are unmethylated in normal conditions but aberrantly methylated in cancer conditions. When evaluated using the CD4 lymphocyte data of Human Epigenome Project (HEP) data set based on bisulfite sequencing, our computational models for predicting the methylation status of CpG islands in the normal conditions can achieve a high accuracy of 93-94%, specificity of 94%, and sensitivity of 92-93%. And, when evaluated using the aberrant methylation data from the MethCancerDB database for aberrantly methylated genes in cancer, our models for detecting the CpG islands that are unmethylated in normal conditions but aberrantly methylated in colon or prostate cancer can achieve an accuracy of 92-93%, specificity of 98-99%, and sensitivity of 92-93%.
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King, Hamish. "Molecular determinants of chromatin accessibility at CpG islands in mouse embryonic stem cells." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:53ceabe6-40ba-402e-a430-7474eac30f93.
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In eukaryotic cells, transcription factors and polymerases must access DNA in the context of nucleosomes and chromatin. The accessibility of DNA sequences to such trans-acting factors is an important feature of gene regulatory elements, including promoters. In vertebrates, the majority of gene promoters coincide with CpG islands (CGIs), which remain free from DNA methylation and exhibit elevated CpG densities. This hypomethylated and CpG-rich state at CGI promoters is associated not only with transcriptional activity, but also with high levels of chromatin accessibility. However, the causes and consequences of such chromatin accessibility remain unclear. To address this, I have profiled chromatin accessibility in mouse embryonic stem cells (ESCs). In addition to confirming that CGI accessibility is independent of transcriptional activity, I was able to demonstrate that the loss of DNA methylation in ESCs resulted in increased chromatin accessibility at a subset of CpG-rich repetitive elements, suggesting that non-methylated CpG-rich sequences may, at least partially, facilitate open chromatin states. This was supported by preliminary work targeting bacterial CpG-rich sequences into the mouse genome, where they were sufficient to establish novel regions of chromatin accessibility. To examine potential mechanisms by which hypomethylated DNA could serve to promote chromatin accessibility, I profiled chromatin accessibility in mouse ESCs lacking various chromatin-modifying proteins which are normally enriched at CGIs, with the histone demethylases KDM2A/B linked to maintaining open chromatin at CGIs. As an alternative approach to understanding the causes of chromatin accessibility in mouse ESCs, I examined the mechanism by which the pioneer transcription factor OCT4 is able to access previously inaccessible chromatin, and reveal that it requires the chromatin remodeller BRG1 to remodel chromatin and facilitate transcription factor binding at distal regulatory elements. Ultimately, this work provides an insight into some of the molecular determinants of chromatin accessibility in mouse ESCs, although many of the consequences of such chromatin states remain unclear.
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Auchincloss, Catherine Anne. "Investigations into mouse trinucleotide repeat arrays and their putative association with CpG islands." Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/23129.
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A mouse CpG island library was screened for all 10 classes of trinucleotide repeat. Sequence analysis of 89 positive clones revealed that only 32% represented CpG islands, compared to 67% of randomly derived clones. These data implied that trinucleotide repeats are under represented in mouse CpG islands. Where possible PCR primers were designed to amplify these repeats from the mouse genome. The variability of 51 repeat arrays was assessed by their PCR amplification from a panel of sixteen mouse strains. Trinucleotide repeats that exhibited length variability between C57BL/6J and Mus spretus (34/51) were mapped using a relevant interspecific backcross panel. These repeats were then screened by PCR as 'candidates' for causing mouse mutant phenotypes mapping to similar genomic regions. Two complex trinucleotide repeat arrays, which mapped to an identical region of chromosome 7, were found to be expanded in frizzy DNA. Further analysis indicated that neither repeat expansion was the underlying mutation responsible for this phenotype. A transgenic study was also carried out to explore the putative relationship between CpG islands and trinucleotide repeat instability. An expanded human Myotonic Dystrophy repeat was cloned into two similar transgenic constructs, one known to retain its native CpG island properties, and the other mutated to confer non island transgene status. Once transgenic mice had been produced, the methylation of the transgene was assessed by PCR and Southern blot. The introduction of the trinucleotide repeat and a small amount of flanking DNA appears to have complicated the predicted methylation of these transgenic constructs. The stability of the trinucleotide repeat arrays was followed through several generations of mice by fluorescent PCR analysis. Moderate trinucleotide repeat instability was observed in the majority of transgenic lines, with a strong bias towards repeat contraction and instability through the female germline. This instability did not appear associated with transgene CpG island status, as defined by methylation.
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Wong, David J. S. "Methylation of the p16 CpG island during neoplastic progression /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/5074.
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Deaton, Aimée M. "Role of CpG island methylation and MBD2 in immune cell gene regulation." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4758.
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The phenomenon of cell type-specific DNA methylation has received much attention in recent years and a number of DNA methylation differences have been described between cells of the immune system. Of particular interest when studying DNA methylation are CpG islands (CGIs) which are distinct from the rest of the genome due to their elevated CpG content, generally unmethylated state and promoter association. In the instances when they become methylated this is associated with gene repression although it is unclear the extent to which differential methylation corresponds to differential gene expression. I have used an immune system model to assess the role of CGI methylation and the role of the methylation reader MBD2 in regulation of gene expression. A relatively small number of DNA methylation differences were seen between immune cell types with the most developmentally related cells showing the fewest methylation differences. Interestingly, the vast majority of CGI-associated cellspecific methylation occurred at intragenic CGIs located, not at transcription start sites, but in the gene body. Increased intragenic CGI methylation tended to associate with gene repression, although the precise reason for this remains unclear. Most differentially methylated CGIs were depleted for the active chromatin mark H3K4me3 regardless of their methylation state but some of these were associated with the silencing mark H3K27me3 when unmethylated. These findings suggest that intragenic CGIs are a distinct class of genomic element particularly susceptible to cell type-specific methylation. I also looked at the effect of removing the methyl- CpG binding domain protein MBD2 from immune system cells. Immune cells from Mbd2-/- mice showed a number of previously uncharacterised phenotypes as well as a number of differences in gene expression compared to wild-type animals. Most of these genes increased their expression in the absence of MBD2 consistent with MBD2’s role as a transcriptional repressor and Mbd2-/- Th1 cells showed increases in histone H3 acetylation compared to wild-type Th1 cells. This work provides an insight into the role played by cell-specific CGI methylation and MBD2 in regulating gene expression.
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Li, Long. "Involvement of DNA methylation and CpG nuclease in environmental carcinogenesis and cancer chemoprevention." Connect to full-text via OhioLINK ETD Center, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1147792824.
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Thesis (Ph.D.)--Medical University of Ohio, 2006."In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Michael A. Pereira. Includes abstract. Document formatted into pages: v, 152 p. Title from title page of PDF document. Title at ETD Web site: Involvement of DNA methylation and CpG endonuclease activity in environmental carcinogenesis and cancer chemoprevention. Bibliography: pages 65-66, 90-92, 123-125, 137-150.
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Guillet-Renard, Claire. "Évolution des îlots CpG chez les primates." Thesis, Lyon 1, 2009. http://www.theses.fr/2009LYO10162.
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Cette thèse a pour l’objet l’étude des pressions de sélection qui s’appliquent sur les îlots CpG, courtes séquences génomiques qui échappent à la méthylation chez les mammifères. Nous avons tout d’abord étudié les caractéristiques génomiques des îlots CpG, notamment leurs liens avec l’initiation de transcription des gènes et les origines de réplication de l’ADN, en utilisant des jeux de données récemment publiés. Nous avons ensuite déterminé si les caractéristiques de séquence des îlots CpG (richesse en dinucléotides CpG et richesse en GC) étaient sous pression de sélection et pouvaient jouer un rôle dans les fonctions des îlots CpG. Nous avons montré que la richesse relative en dinucléotides CpG des îlots CpG résulte uniquement de la faible méthylation de ces séquences. De plus, la richesse en bases GC des îlots CpG n’est pas soumise à pression de sélection mais semble résulter d’un mécanisme neutre, la conversion génique biaisée vers GC. Nous discutons également du devenir des îlots CpG chez les primates, qui et avons montré que si le taux de GC de ces séquences est en train de diminuer, la richesse relative en CpG quant à elle reste stableThis thesis analyses selective pressures applying on CpG islands, short sequences which escape methylation in mammalian genomes. We first studied genomic characteristics of CpG islands. We namely studied their relationships with gene transcription start, and with DNA replication origins, using recently published data. We then determined wether base peculiar composition of CpG islands (high number of CpG dinucleotides, high GC content) may be under (negative or positive) selective pressures, and thus play a role in their function, or not. We showed that the relative CpG-richness of CpG islands is the mere consequence of the low methylation of these genomic regions. Moreover, we showed that the high GC content of CpG islands is not under selective pressures, and seem to result from a neutral mechanism, biased gene conversion toward GC. We also discussed the future of CpG islands and primates. We showed that the GC content of CpG islands is decreasing, while the relative CpG content remains constant
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Edgar, Rachel. "Meta-analysis of human methylomes reveals stably methylated sequences surrounding CpG islands associated with high gene expression." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/50302.
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DNA methylation is thought to play an important role in the regulation of mammalian gene expression. Part of the evidence for this role is the observation that lack of CpG island methylation in gene promoters is associated with high transcriptional activity. However, CpG island methylation level only accounts for a fraction of the variance in gene expression, and methylation in other domains is hypothesized to play a role (e.g., island shores and shelves). We set out to improve understanding of the human methylome through a meta-analysis approach, using 1737 samples from 30 publicly available studies. An initial screen identified 15224 CpGs that are “ultra-stable” in their state, being always fully methylated or unmethylated across diverse tissues, cell types and developmental stages (974 always methylated; 14250 always unmethylated). A further analysis of ultra-stable CpGs led us to identify a novel class of CpG islands, “ravines”, that exhibit a markedly consistent pattern of low methylation with highly methylated flanking shores and shelves. Our findings were validated using independent and heterogeneous datasets assayed on the same and different technologies. Building on additional existing data types such as gene expression microarrays, DNase hypersensitive sites, and histone modifications, we found that ravines are associated with higher gene expression, compared to typical unmethylated CpG islands. This finding suggests a novel role for methylation in promoters, markedly different from the traditional view that active promoters need to be unmethylated. We propose ravines are a new class of CpG islands, established early in development and maintained through differentiation, that mark universally active genes and provide new evidence that methylation beyond the CpG island could play a role in gene expression.Science, Faculty of
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Saadeh, Heba. "The role of DNA sequence signals in the epigenetic reprogramming of CpG islands during oogenesis and early embryogenesis." Thesis, King's College London (University of London), 2014. https://kclpure.kcl.ac.uk/portal/en/theses/the-role-of-dna-sequence-signals-in-the-epigenetic-reprogramming-of-cpg-islands-during-oogenesis-and-early-embryogenesis(76174739-3129-4655-b3f7-a72a9ea9a11a).html.
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The reprogramming of epigenetic marks is a genome-wide process and yet CpG islands escape the overall trend. In addition, not all CpG islands behave in the same way. While the majority of CpG islands resist the de novo DNA methylation establishment in the germ lines, ~1600 CpG islands acquire methylation during oogenesis. The majority of these oocyte-methylated CpG islands remain un-methylated during spermatogenesis, that is, they are maternal germ line differentially methylated regions (maternal gDMRs). All but 25 (permanent) maternal gDMRs lose their methylation post fertilisation and pre-implantation. The DNA sequences of CpG islands was investigated - in the context of transcription in the oocyte - to identify targeting signals for either establishing and/or maintaining, or altogether escaping DNA methylation during oogenesis and in the pre-implantation embryo. The methylation of CpG islands in the oocyte was found to be significantly associated with transcription through CpG islands, as previously observed. However, the sequences of oocyte-methylated CpG islands do not contain a characteristic DNA sequence motif, and neither do the upstream promoters from which transcription through oocyte-methylated CpG islands originates. An analogous de novo motif search successfully identifies TGCCGC, the recognition site of the Zfp57/Kap1 imprint maintenance complex, as a DNA sequence motif that is characteristic for PPM-DMRs. Analysis of the incidence of TGCCGC indicates that not just its presence but also multiple occurrences within a sequence may be required for imprint maintenance. Furthermore, the lack of additional characteristic motifs suggests the absence of additional DNA-binding factors that specifically interact with PPM-DMRs. A period of 8-10bp in the spacing of CpGs in PPM-DMRs was previously observed and proposed as a targeting signal for de novo methylation in the germ line. This observation was reproduced, and the property of the average was found to be representative to only less than the half of the PPM-DMRs. Moreover, the pairs of CpGs 8-10bp apart are in fact depleted in oocyte-methylated CpG islands, consistent with the consequence of accidental deamination over time. The absence of a DNA sequence motif or CpG spacing characteristic of oocyte-methylated CpG islands (including PPM-DMRs) supports a sequence-independent model of de novo methylation establishment during oogenesis. However, the CpG islands that escape this mechanism contain a characteristic CGrich motif that is highly similar to the recognition site of E2F1/2, DNA-binding protein involved in chromatin remodelling that is expressed in oocytes. Logistic-linear regression analyses indicate that the presence of the motif independently conveys significant protection from methylation, regardless of, for example, whether the CpG island is an active promoter. Therefore, the following is proposed: E2F1/2 are part of a mechanism for the active protection of specific CpG islands from de novo methylation in the oocyte.
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Westerberg, Ivar. "CpG islands, but not their methylation level, are key in the regulation of meiotic recombination in chicken (Gallus gallus)." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-376254.
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Meiotic recombination plays a fundamental role in many sexually reproducing species. Recombination shuffles the genetic material during the first meiotic cell division resulting in new combinations of alleles within each chromosome. In many organisms, the rate of recombination is not uniform across the genome but consists of so called hotspots where the recombination rate is remarkably higher than the genome average. In mammals, the regulation and location of recombination hotspots is regulated by a gene called PRDM9. Many nonmammalian animals, like birds, lack this gene and the precise mechanism for recombination rate regulation is still unknown. Previous findings in passerine birds have observed an association between recombination rate and a genomic feature known as CpG islands (CGIs). CGIs are often located in promoter regions of genes and depending on their methylation status constitute accessible chromatin regions. It has therefore been suggested that the proteins involved in the regulation of recombination have better access to less condense chromatin regions. In this study, I tested if the association between recombination rate and CGIs found in passerine birds is also true in chicken. I also tested if methylation levels of CGIs play a role in recombination rate regulation in chicken. To this end, I used previously published data for CGI locations and a methylation map in chicken, and unpublished data of recombination rate estimates. I found that the association between recombination rate and CGIs observed in passerine birds extends to chicken, suggesting that this is an ancestral trait in birds. I did not, however, find a negative association between methylation levels and recombination rate as hypothesised based on a relationship between methylation level and chromatin accessibility. This suggests that DNA methylation level at CGIs is not a strong determinant of recombination in chicken, although there may be some workflow artefacts or unknown factors remaining in my analysis obscuring the relationship between these two variables.
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Paci, Giulia. "Statistical methods for the analysis of DNA sequences: application to dinucleotide distribution in the human genome." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2014. http://amslaurea.unibo.it/7615/.
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Questa tesi si inserisce nell'ambito delle analisi statistiche e dei metodi stocastici applicati all'analisi delle sequenze di DNA. Nello specifico il nostro lavoro è incentrato sullo studio del dinucleotide CG (CpG) all'interno del genoma umano, che si trova raggruppato in zone specifiche denominate CpG islands. Queste sono legate alla metilazione del DNA, un processo che riveste un ruolo fondamentale nella regolazione genica. La prima parte dello studio è dedicata a una caratterizzazione globale del contenuto e della distribuzione dei 16 diversi dinucleotidi all'interno del genoma umano: in particolare viene studiata la distribuzione delle distanze tra occorrenze successive dello stesso dinucleotide lungo la sequenza. I risultati vengono confrontati con diversi modelli nulli: sequenze random generate con catene di Markov di ordine zero (basate sulle frequenze relative dei nucleotidi) e uno (basate sulle probabilità di transizione tra diversi nucleotidi) e la distribuzione geometrica per le distanze.
Da questa analisi le proprietà caratteristiche del dinucleotide CpG emergono chiaramente, sia dal confronto con gli altri dinucleotidi che con i modelli random.
A seguito di questa prima parte abbiamo scelto di concentrare le successive analisi in zone di interesse biologico, studiando l’abbondanza e la distribuzione di CpG al loro interno (CpG islands, promotori e Lamina Associated Domains). Nei primi due casi si osserva un forte arricchimento nel contenuto di CpG, e la distribuzione delle distanze è spostata verso valori inferiori, indicando che questo dinucleotide è clusterizzato. All’interno delle LADs si trovano mediamente meno CpG e questi presentano distanze maggiori. Infine abbiamo adottato una rappresentazione a random walk del DNA, costruita in base al posizionamento dei dinucleotidi: il walk ottenuto presenta caratteristiche drasticamente diverse all’interno e all’esterno di zone annotate come CpG island. Riteniamo pertanto che metodi basati su questo approccio potrebbero essere sfruttati per migliorare l’individuazione di queste aree di interesse nel genoma umano e di altri organismi.
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Maldonado, Mariângela Bueno Cordeiro. "Identificação de SNPs em sítios CpG localizados em regiões genômicas relacionadas à produção em bovinos /." Araçatuba, 2017. http://hdl.handle.net/11449/151514.
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Orientador: Flavia Lombardi LopesBanca: Silvia Helena Venturoli Perri
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Banca: Ricardo da Fonseca
Banca: José bento Sterman Ferraz
Resumo: O objetivo desse estudo foi identificar polimorfismos de nucleotídeo único (SNPs) potencialmente sujeitos a controle epigenético exercido por metilação do DNA via seus envolvimentos na criação, remoção ou deslocamento de sítios CpG (meSNPs) e a partir de tal identificação criar um banco de dados para meSNPs, bem como determinar a possível associação desses marcadores com ilhas CpG (CGIs) e com o perfil metilacional de tecidos submetidos ao ensaio de recuperação de ilhas CpG metiladas combinado com plataformas de sequenciamento de nova geração (MIRA-seq) em bovinos. Usando as variantes anotadas para os SNPs identificados no Run5 do projeto 1000 Bull Genomes e a sequência genômica bovina de referência UMD3.1.1, identificamos e anotamos 12.836.763 meSNPs de acordo com o padrão de variação criado por cada SNP em um sítio CpG. Também analisamos a distribuição genômica desses meSNPs, sendo a maioria deles localizados em regiões intergênicas (68,00%) e intrônicas (26,32%). Globalmente, os meSNPs representam 22,53% dos 56.969.697 SNPs descritos na base de dados e 12,35% deles estão localizados em CGIs. Comparando o número observado com o número esperado de meSNPs nas CGIs e nos tecidos submetidos ao MIRA-seq, verificamos um enriquecimento médio (P<0,01) para meSNPs de 2,47 vezes em CGIs relaxadas e 1,90 vezes em CGIs rigorosas. Nos tecidos, o enriquecimento foi de 1,52 vezes em longissimus dorsi e 2,09 vezes em intestino delgado. Dez meSNPs com metilação diferencial, sendo 1 em longi... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The aim of this study was to identify single nucleotide polymorphisms (SNPs) potentially subject to epigenetic control exerted by DNA methylation via their involvement in creating, removing or displacement CpG sites (meSNPs) and from this identification create a database for meSNPs, as well as to determine its possible association with CpG islands (CGIs) and the methylation profile of tissues submitted to the methylated-CpG island recovery assay combined with next generation sequencing platforms (MIRA-seq) in cattle. Using the variant annotations for SNPs identified in Run5 of the 1000 bull genomes project and the UMD3.1.1 bovine reference genome sequence assembly, we identified and classified 12,836,763 meSNPs according to the pattern of variation caused at the CpG site. We have also analyzed the genomic distribution of the meSNPs, with the majority being located in intergenic regions (68.00%) and then in introns (26.32%) and the remainder distributed among proximal promoters (3.93%), coding regions (1.27%), untranslated regions (UTRs) (0.29%), non-coding RNAs (0.11%) and splice regions (0.08%). Overall, meSNPs represent 22.53% of 56,969,697 SNPs described in the database of which 12.35% are located in CGIs. Comparing the observed number with the expected number of meSNPs in the CGIs and tissues submitted to the MIRAseq we found a mean enrichment (P<0.01) for meSNPs of 2.47 times in the relaxed CGIs and 1.90 times in the strict CGIs. In the tissues the enrichment was of 1.52... (Complete abstract click electronic access below)
Doutor
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Li, Xiaomeng. "Human Promoter Recognition Based on Principal Component Analysis." Thesis, The University of Sydney, 2008. http://hdl.handle.net/2123/3656.
Full textAbstract:
This thesis presents an innovative human promoter recognition model HPR-PCA. Principal component analysis (PCA) is applied on context feature selection DNA sequences and the prediction network is built with the artificial neural network (ANN). A thorough literature review of all the relevant topics in the promoter prediction field is also provided. As the main technique of HPR-PCA, the application of PCA on feature selection is firstly developed. In order to find informative and discriminative features for effective classification, PCA is applied on the different n-mer promoter and exon combined frequency matrices, and principal components (PCs) of each matrix are generated to construct the new feature space. ANN built classifiers are used to test the discriminability of each feature space. Finally, the 3 and 5-mer feature matrix is selected as the context feature in this model. Two proposed schemes of HPR-PCA model are discussed and the implementations of sub-modules in each scheme are introduced. The context features selected by PCA are III used to build three promoter and non-promoter classifiers. CpG-island modules are embedded into models in different ways. In the comparison, Scheme I obtains better prediction results on two test sets so it is adopted as the model for HPR-PCA for further evaluation. Three existing promoter prediction systems are used to compare to HPR-PCA on three test sets including the chromosome 22 sequence. The performance of HPR-PCA is outstanding compared to the other four systems.
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Li, Xiaomeng. "Human Promoter Recognition Based on Principal Component Analysis." University of Sydney, 2008. http://hdl.handle.net/2123/3656.
Full textAbstract:
Master of EngineeringThis thesis presents an innovative human promoter recognition model HPR-PCA. Principal component analysis (PCA) is applied on context feature selection DNA sequences and the prediction network is built with the artificial neural network (ANN). A thorough literature review of all the relevant topics in the promoter prediction field is also provided. As the main technique of HPR-PCA, the application of PCA on feature selection is firstly developed. In order to find informative and discriminative features for effective classification, PCA is applied on the different n-mer promoter and exon combined frequency matrices, and principal components (PCs) of each matrix are generated to construct the new feature space. ANN built classifiers are used to test the discriminability of each feature space. Finally, the 3 and 5-mer feature matrix is selected as the context feature in this model. Two proposed schemes of HPR-PCA model are discussed and the implementations of sub-modules in each scheme are introduced. The context features selected by PCA are III used to build three promoter and non-promoter classifiers. CpG-island modules are embedded into models in different ways. In the comparison, Scheme I obtains better prediction results on two test sets so it is adopted as the model for HPR-PCA for further evaluation. Three existing promoter prediction systems are used to compare to HPR-PCA on three test sets including the chromosome 22 sequence. The performance of HPR-PCA is outstanding compared to the other four systems.
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Maldonado, Mariângela Bueno Cordeiro [UNESP]. "Identificação de SNPs em sítios CpG localizados em regiões genômicas relacionadas à produção em bovinos." Universidade Estadual Paulista (UNESP), 2017. http://hdl.handle.net/11449/151514.
Full textAbstract:
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O objetivo desse estudo foi identificar polimorfismos de nucleotídeo único (SNPs) potencialmente sujeitos a controle epigenético exercido por metilação do DNA via seus envolvimentos na criação, remoção ou deslocamento de sítios CpG (meSNPs) e a partir de tal identificação criar um banco de dados para meSNPs, bem como determinar a possível associação desses marcadores com ilhas CpG (CGIs) e com o perfil metilacional de tecidos submetidos ao ensaio de recuperação de ilhas CpG metiladas combinado com plataformas de sequenciamento de nova geração (MIRA-seq) em bovinos. Usando as variantes anotadas para os SNPs identificados no Run5 do projeto 1000 Bull Genomes e a sequência genômica bovina de referência UMD3.1.1, identificamos e anotamos 12.836.763 meSNPs de acordo com o padrão de variação criado por cada SNP em um sítio CpG. Também analisamos a distribuição genômica desses meSNPs, sendo a maioria deles localizados em regiões intergênicas (68,00%) e intrônicas (26,32%). Globalmente, os meSNPs representam 22,53% dos 56.969.697 SNPs descritos na base de dados e 12,35% deles estão localizados em CGIs. Comparando o número observado com o número esperado de meSNPs nas CGIs e nos tecidos submetidos ao MIRA-seq, verificamos um enriquecimento médio (P<0,01) para meSNPs de 2,47 vezes em CGIs relaxadas e 1,90 vezes em CGIs rigorosas. Nos tecidos, o enriquecimento foi de 1,52 vezes em longissimus dorsi e 2,09 vezes em intestino delgado. Dez meSNPs com metilação diferencial, sendo 1 em longissimus dorsi e 9 em intestino delgado, causaram uma alteração na sequência genômica, a qual está associada ao fenótipo de eficiência alimentar em bovinos.
The aim of this study was to identify single nucleotide polymorphisms (SNPs) potentially subject to epigenetic control exerted by DNA methylation via their involvement in creating, removing or displacement CpG sites (meSNPs) and from this identification create a database for meSNPs, as well as to determine its possible association with CpG islands (CGIs) and the methylation profile of tissues submitted to the methylated-CpG island recovery assay combined with next generation sequencing platforms (MIRA-seq) in cattle. Using the variant annotations for SNPs identified in Run5 of the 1000 bull genomes project and the UMD3.1.1 bovine reference genome sequence assembly, we identified and classified 12,836,763 meSNPs according to the pattern of variation caused at the CpG site. We have also analyzed the genomic distribution of the meSNPs, with the majority being located in intergenic regions (68.00%) and then in introns (26.32%) and the remainder distributed among proximal promoters (3.93%), coding regions (1.27%), untranslated regions (UTRs) (0.29%), non-coding RNAs (0.11%) and splice regions (0.08%). Overall, meSNPs represent 22.53% of 56,969,697 SNPs described in the database of which 12.35% are located in CGIs. Comparing the observed number with the expected number of meSNPs in the CGIs and tissues submitted to the MIRAseq we found a mean enrichment (P<0.01) for meSNPs of 2.47 times in the relaxed CGIs and 1.90 times in the strict CGIs. In the tissues the enrichment was of 1.52 times in ribeye and 2.09 times in small intestine. Ten meSNPs, differing in methylation status, 1 in ribeye and 9 in small intestine, caused an alteration in the genomic sequence which is associated with a feed efficiency phenotype in cattle.
FAPESP: 2015/20557-5
FAPESP: 2016/07584-6
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Zeng, Jia. "The evolutionary significance of DNA methylation in human genome." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/50308.
Full textAbstract:
In eukaryotic genomes ranging from plants to mammals, DNA methylation is a major epigenetic modification of DNA by adding a methyl group exclusively to cytosine residuals. In mammalian genomes such as humans, these cytosine bases are usually followed by guanine. Although it does not change the primary DNA sequence, this covalent modification plays critical roles in several regulatory processes and can impact gene activity in a heritable fashion. What is more important, DNA methylation is essential for mammalian embryonic development and aberrant DNA methylation is implicated in several human diseases, in particular in neuro-developmental syndromes (such as the fragile X and Rett syndromes) and cancer. These biological significances disclose the importance of understanding genomic patterns and function role of DNA methylation in human, as a initial step to get to know the epigenotype and its manner in connecting the phenotype and genotype.
Two key papers back in 1975 independently suggested that methylation of CpG dinucleotides in vertebrates could be established de novo and inherited through somatic cell divisions by protein machineries of DNA methyltransferases that recognizes hemi-methylated CpG palindromes. They also indicated that the methyl group could be recognized by DNA-binding proteins and that DNA methylation directly silences gene expression. After almost four decades, several key points in these foundation papers are proved to be true. Take the mammalian genome for example, there are several findings indicating the epigenetic repression of gene expression by DNA methylation. These include X-chromosome inactivation, gene imprinting and suppressing the proliferation of transposable elements and repeat elements of viral or retroviral origin. In addition to these, many novel roles of DNA methylation have also been revealed. For example, DNA methylation can regulate alternative splicing by preventing CTCF, an evolutionarily conserved zinc-finger protein, binding to DNA. By using the technique of fluorescence resonance energy transfer (FRET) and fluorescence polarization, DNA methylation has also been shown to increase nucleosome compaction through DNA-histone contacts. What is more important, DNA methylation is essential for mammalian embryonic development and aberrant change of DNA methylation has been related to disease such as cancer. However, it is also notable there are several lines of evidence contradicting the relationship between DNA methylation and gene silencing. For example, comparison of DNA methylation levels in human genome on the active and inactive X chromosomes showed reduced methylation specifically over gene bodies on inactive X chromosomes. Not only in human, DNA methylation is found to be usually targeted to the transcription units of actively transcribed genes in invertebrate species. These results prove that the function of DNA methylation is challenging to be unravel. Besides, due to the development of sequencing technique, whole genome DNA methylation profiles have been detected in diverse species. Comparing genomic patterns of DNA methylation shows considerable variation among taxa, especially between vertebrates and invertebrates. However, even though extensive studies reveal the patterns and functions of DNA methylation in different species, in the mean time, they also highlight the limits to our understanding of this complex epigenetic system.
During my Ph.D., in order to perform in-depth studies of DNA methylation in diverse animals as a way to understand the complexity of DNA methylation and its functions, I dedicated my efforts in investigating and analyzing the DNA methylation profiles in diverse species, ranging from insects to primates, including both model and non-model organisms. This dissertation, which constitutes an important part of my research, mainly focuses on the DNA methylation profile in primates including human and chimpanzee. In general, I will use three chapters to elucidate my work in generating and interpreting the whole genome DNA methylation data. Firstly, we generated nucleotide-resolution whole-genome methylation maps of the prefrontal cortex of multiple humans and chimpanzees, then comprehensive comparative studies for these DNA methylation maps have been performed, by integrating data on gene expression as well. This work demonstrates that differential DNA methylation might be an important molecular mechanism driving gene-expression divergence between human and chimpanzee brains and also potentially contribute to the human-specific traits, such as evolution of disease vulnerabilities. Secondly , we performed global analyses of CpG islands (CGIs) methylation across multiple methylomes of distinctive cellular origins in human. The results from this work show that the human CpG islands can be distinctly classified into different clusters solely based upon the DNA methylation profiles, and these CpG islands clusters reflect their distinctive nature at many biological levels, including both genomic characteristics and evolutionary features. Moreover, these CpG islands clusters are non-randomly associated with several important biological phenomena and processes such as diseases, aging, and gene imprinting. These new findings shed lights in deciphering the regulatory mechanisms of CpG islands in human health and diseases. At last, by utilizing the DNA methylome from human sperm and genetic map generated from the International HapMap Consortium project, we investigated the hypothesis suggesting a potential role of germ line DNA methylation in affecting meiotic recombination, which is essential for successful meiosis and various evolutionary processes. Even thought the results imply that DNA methylation is a important factor affecting regional recombination rate, the strength of correlation between these two is not as strong as the previous report. Besides, high-throughput analyses indicate that other epigenetic modifications, tri-methylation of histone 3 lysine 4 and histone 3 lysine 27 are also global features at the recombination hotspots, and may interact with methylation to affect the recombination pattern simultaneously. This work suggests epigenetic mechanisms as additional factors affecting recombination, which cannot be fully explained by the DNA sequence itself. In summary, I hope the results from these work can expand our knowledge regarding the common and variable patterns of DNA methylation in different taxa, and shed light about the function role and its major change during animal evolution.
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Antunes, João André Rodrigues. "Distâncias inter-simbólicas no ADN." Master's thesis, Universidade de Aveiro, 2013. http://hdl.handle.net/10773/12777.
Full textAbstract:
Mestrado em Engenharia Eletrónica e TelecomunicaçõesO presente trabalho visou estudar o contributo das distâncias-inter simbólicas na segmentação do ADN. Para esse efeito, foi estudada a segmentação das sequências genómicas em código e não código e em ilhas e não ilhas CpG. Desenvolveu-se um estudo das distâncias inter-trinucleótidas no contexto da identificação de regiões codificantes e das distâncias inter-dinucleótidas para a identificação de ilhas CpG. Com base nestas distâncias foi analisado o desempenho de um algoritmo para discriminação de regiões de código e não código, tendo os resultados evidenciado haver ainda margem para aperfeiçoamento e foi desenvolvido um algoritmo para identificação de ilhas CpG tendo as taxas de boa classificação atingido valores elevados.
The present work aimed to study the contribution of the inter-symbolic distances in DNA segmentation. To this end, the segmentation of genomic sequences into coding and non coding regions and CpG islands and non CpG islands was studied. A study of the inter-trinculeotide distances in the context of identifying coding regions and of the inter-dinucleotide distances for identifying CpG islands was developed. Based on these distances the performance of an algorithm to discriminate coding and non coding regions was analyzed, with the results showing there is still room for improvement and an algorithm for identification of CpG islands was designed, resulting in high values of good classification rates.
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Millán, Ariño Lluís 1984. "Genomic distribution and functional specificity of human histone H1 subtypes." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/292370.
Full textAbstract:
Seven linker histone H1 variants exist in human somatic cells with distinct prevalence among
cell types and during differentiation. Despite being key chromatin structural components, it
remains elusive how they participate in the regulation of nuclear processes. Moreover, it is
not well understood whether the different variants have specific roles or are differentially
distributed along the genome. By taking advantage of specific antibodies for H1 variants and
HA-tagged recombinant H1s expressed in breast cancer cells, the distribution of somatic
variants H1.2 to H1.5, H1.0 and H1X has been investigated by combining ChIP-qPCR, ChIP-chip,
and ChIP-seq analysis. All H1 variants bind gene promoters and are depleted from the TSS in
active genes, and also from regulatory sites. The extension of H1 depletion at promoters is
dependent on the transcriptional status of the gene and differs between variants. Analyses
show that histone H1 is not uniformly distributed along the genome and differences among
variants exist, being H1.2 the variant showing a more specific pattern and a strongest
correlation with gene repression in breast cancer cells. Results suggest that different variants
may be present at different chromatin types, and this may depend on the cell type,
differentiation state, and whether cells are originated from a neoplastic process. In a second
part of the thesis, it is shown that a previously reported H1.4 knock-down cell line presents
and off-target effect against lamin B2. Therefore, it has been developed a new inducible
knock-down cell line specifically inhibiting H1.4, which resembles previously characterized
H1.2 knock-down. Finally, combined depletion of H1.4/lamin B2 and H1.2/H1.4 causes similar
effects in T47D breast cancer cell line.Fins a set variants de la histona H1 s han identificat en mamífers, les quals mostres una prevalença diferent entre tipus cel lulars i durant el procés de diferenciació. Tot i que la histona H1 juga un paper clau en l estructuració de la cromatina, no s acaba d entendre encara com participa exactament en els diferent processos cel lulars. A més a més, encara no està clar si les diferents variants tenen funcions específiques ni si es distribueixen igual al llarg del genoma. Mitjançant anticossos específics per algunes variants d H1 i de línies cel lulars de càncer de mama que expressen H1s recombinant fusionades a un pèptid HA, s ha estudiat la distribució genòmica de H1.2 a H1.5, H1.0 i H1X, combinant ChIP-qPCR, ChIP-chip i ChIP-seq. Totes les H1s es troben a promotors gènics i empobrides a l inici de transcripció dels gens actius, i també a les regions reguladores. El grau de disminució d H1 al promotor depèn de l estat transcripcional del gen i presenta diferències entre variants. Els anàlisis mostren que la histona H1 no es distribueix uniformement al genoma i que hi ha diferències entre variants, essent H1.2 la variant que presenta un patró més específic i una correlació més forta amb repressió gènica a cèl lules de càncer de mama. Aquests resultats suggereixen que variants d H1 diferents es troben presents als diversos tipus de cromatina, i aquest fet podria dependre de la línia cel lular, l estat de diferenciació, o de si les cèl lules s han originat durant un procés neoplàsic. En una segona part de la tesi, es mostra que una línia cel lular anteriorment descrita que inhibeix H1.4 presenta un efecte inespecífic contra lamina B2. Així, s ha desenvolupat una altra línia que inhibeix H1.4 específicament, la qual s assembla a un mutant anteriorment caracteritzat que inhibeix H1.2. Finalment, la inhibició combinada de H1.4/lamina B2 i H1.2/H1.4 provoca efectes fenotípics semblants a cèl lules de càncer de mama T47D.
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Cluny, Vasco Silva Oliveira. "Exploratory study of age related epigenomic patterns." Master's thesis, Universidade de Aveiro, 2016. http://hdl.handle.net/10773/17887.
Full textAbstract:
Mestrado em Biotecnologia MolecularSabe-se hoje que o genoma humano, para além da sua sequencia nucleotídica, revela várias alterações químicas no DNA, nomeadamente metilações das citosinas. Estas modificações estabelecem padrões específicos que podem ser transmitidos de uma geração para a seguinte e exercem controlo sobre os genes que são expressos a cada momento nas células, tecidos ou orgãos. Esta tese teve como objectivos: explorar as principais bases de dados que contêm dados epigenómicos relevantes; obter ficheiros fastq de bibliotecas bisulfite-seq aplicando métodos de data mining a dados reais de bases de dados públicas de sequenciação de segunda geração; alinhar e mapear estes ficheiros usando software adequado (Methy-Pipe); fazer uma análise comparative por forma obter características associadas ao envelhecimento saudável de indivíduos e á evolução do epigenoma ao longo da vida; finalmente é esperado que, após atingidos os objectivos anteriores, se perceba o contributo do epienoma no envelhecimento saudável das populações .
It is already known today, that the human genome, in addition to its nucleotide sequence, shows multiple chemical modifications at the DNA level, namely cytosine methylations. These modifications changes establish specific patterns that can be transmitted from generation to generation and exercise control over the genes that are expressed at every moment in the life of the cells / tissues / organs. This thesis aimed to: understand the contribution of the epigenome to a healthy lifestyle; to explore the main databases containing relevant epigenomic data; to obtain fastq files of bisulfite-seq libraries by applying data mining methods to real data from next generation public databases; to align and map these files using adequate software (Methy Pipe); to do a comparative analysis in order to identify features associable to a healthy aging of individuals and the evolution of the epigenome in humans throughout life.In doing so, it is expected that this work will contribute to the understanding of the contribution of the epigenome to a healthy lifestyle.
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Illingworth, Robert S. "Comprehensive analysis of human CpG island methylation." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/10972.
Full textAbstract:
We devised a preparative chromatographic tool for the enrichment of CGI sequences based on the empirical criterion of clustered nonmethylated CpGs. Characterisation of a novel somatic CGI set derived from human blood DNA identified a population of islands which were missed by commonly applied bioinformatics criterion. The identified CGIs were enriched in gene rich portions of the genome, and preferentially associated with chromosome ends proximal to the telomeres. Despite association with gene promoters, approximately fifty percent of CGIs were found to be either intra- or intergenic. An arrayed set, comprising ~17,000 unique sequences, allowed the investigation of the methylated CGI component of human blood, brain, muscle and spleen. This identified 6-8% of normally methylated CGIs across the 22 human autosomes. These hypermethylated islands were enriched in regions distal to annotated promoters and preferentially associated with developmental gene loci. The reagents and observations discussed in this thesis allow detailed analysis of the abundance and distribution of methylated CGIs in ‘normal’ human somatic cells.
27
Ali, Isse. "Analysing and predicting differences between methylated and unmethylated DNA sequence features." Thesis, De Montfort University, 2015. http://hdl.handle.net/2086/12616.
Full textAbstract:
DNA methylation is involved in various biological phenomena, and its dysregulation has been demonstrated as being correlated with a number of human disease processes, including cancers, autism, and autoimmune, mental health and neuro-degenerative ones. It has become important and useful in characterising and modelling these biological phenomena in or-der to understand the mechanism of such occurrences, in relation to both health and disease. An attempt has previously been made to map DNA methylation across human tissues, however, the means of distinguishing between methylated, unmethylated and differentially-methylated groups using DNA sequence features remains unclear. The aim of this study is therefore to: firstly, investigate DNA methylation classes and predict these based on DNA sequence features; secondly, to further identify methylation-associated DNA sequence features, and distinguish methylation differences between males and females in relation to both healthy and diseased, sta-tuses. This research is conducted in relation to three samples within nine biological feature sub-sets extracted from DNA sequence patterns (Human genome database). Two samples contain classes (methylated, unmethy-lated and differentially-methylated) within a total of 642 samples with 3,809 attributes driven from four human chromosomes, i.e. chromosomes 6, 20, 21 and 22, and the third sample contains all human chromosomes, which encompasses 1628 individuals, and then 1,505 CpG loci (features) were extracted by using Hierarchical clustering (a process Heatmap), along with pair correlation distance and then applied feature selection methods. From this analysis, author extract 47 features associated with gender and age, with 17 revealing significant methylation differences between males and females. Methylation classes prediction were applied a K-nearest Neighbour classifier, combined with a ten-fold cross- validation, since to some data were severely imbalanced (i.e., existed in sub-classes), and it has been established that direct analysis in machine-learning is biased towards the majority class. Hence, author propose a Modified- Leave-One-Out (MLOO) cross-validation and AdaBoost methods to tackle these issues, with the aim of compositing a balanced outcome and limiting the bias in-terference from inter-differences of the classes involved, which has provided potential predictive accuracies between 75% and 100%, based on the DNA sequence context.
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Nerušil, Václav. "Vyhledávání CpG ostrůvků z DNA sekvencí." Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2015. http://www.nusl.cz/ntk/nusl-221308.
Full textAbstract:
This thesis focuses on searching for CpG islands of DNA sequences based on analysis of DNA spectrograms. The first part is theoretical and deals with the significance CpG island, and a description of the algorithms that are used or have been proposed for their search. The theoretical basis were implemented two algorithms based on the analysis of DNA spectrogram. One is based on the assumption that the region CpG islands has a higher content of guanine and cytosine than the region outside the CpG island and the other on the assumption of a higher frequency of occurrence of CG dinucleotides in the CpG island. The algorithms are implemented through MATLAB programming interface. For evaluation usefulness and effectiveness of solutions, results achieved on the selected DNA sequences implemented algorithms are compared with the results achieved by search engines CpG islands, which are freely available on the internet.
29
Long, Hannah Katherine. "Evolutionary usage and developmental roles of vertebrate non-methylated DNA." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:78b14c1d-1fa3-46f1-815f-a8ba55579c43.
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Vertebrate genomes exhibit global methylation of cytosine residues where they occur in a cytosine-guanine dinucleotide (CpG) context and this epigenetic mark is generally thought to be repressive to transcription. Punctuating this pervasive DNA methylation landscape are short, contiguous regions of non-methylated DNA which are found at two thirds of mammalian gene promoters. These non-methylated regions exhibit CpG content close to expected levels as they escape the depletion of CpGs observed across the methylated fraction of the genome. The unique nucleotide properties of these CpG island (CGI) regions enable their identification by computational prediction in mammalian genomes. Owing to a lack of high-resolution genome-wide DNA methylation profiles in non-mammalian species, these CGI predictions have often been used as a proxy for non-methylated DNA in these organisms. In contrast to mammals, CGI predictions in cold-blooded vertebrates rarely coincide with gene promoters, leading to the belief that CGls are significantly divergent between vertebrate species, and that unique promoter-associated features may have been acquired during warmblooded vertebrate evolution. This thesis is primarily concerned with the location, establishment and biological function of non-methylated islands of DNA in vertebrate genomes. To experimentally determine genome-wide profiles of non-methylated DNA, a novel biochemical technique was established called biotinylated ZF-CxxC affinity purification (Bio-CAP), and development of this method is discussed in Chapter 3. Experimental analysis of non-methylated DNA profiles in this thesis initially addresses two main questions: (1) 'How does the non-methylated DNA landscape compare genome-wide for seven vertebrates considering distinct tissue types and developmental stages?' (2) 'How are vertebrate non-methylated regions of DNA defined and interpreted in the nuclear environment?' To address the first question, non-methylated DNA was profiled by Bio-CAP sequencing across the genomes of seven diverse vertebrate species, representing all major branch points of vertebrate evolution, and the results are discussed in Chapters 4 and S. Contrary to previously held dogma, experimentally determined nonmethylated islands of DNA (NMls) constitute an ancient epigenetic feature of vertebrate gene regulatory elements. However, despite having numerous high-resolution maps of vertebrate non-methylated DNA, the means by which NMls are identified and maintained in the nuclear environment remains poorly understood. To address the second question and identify features which determine the methylation state of DNA, exogenous DNA sequences were introduced into mouse embryonic stem (ES) c~.II~. Non-methylated DNA was profiled by Bio-CAP sequencing to investigate how different features, such as sequence-specific binding motifs, chromatin architecture and nucleotide composition of a given DNA sequence impact local DNA methylation patterns. Interestingly, the majority of exogenous promoters were appropriately non-methylated in mouse ES cells, germline and somatic cells suggesting that gene promoters have retained strong signals for the nonmethylated state across millions of years of evolution (discussed in Chapter 6). During mouse embryogenesis, genome-scale DNA demethylation and remethylation events occur to remodel the epigenetic landscape and loss of DNA methylation during this time leads to embryonic lethality. To investigate the biological function of non-methylated DNA, the third question addressed in this thesis is (3) 'What is the developmental importance of non-methylated islands of DNA during vertebrate embryogenesis?' To investigate this, members of the ZF-CxxC domain-containing family of chromatin modifiers were ablated in zebrafish embryos to perturb the chromatin landscape at NMls, and therefore interfere with their function during early development (Chapter 7). Early embryonic development and patterning was disrupted in knockdown embryos, suggesting that interpretation of non-methylated DNA and placement of chromatin modifications at NMls is essential for normal zebrafish embryogenesis. Together this work sheds light on the evolutionary origins of NMls, the mechanisms involved in the recognition and establishment of nonmethylated loci and provides an insight into the function of non-methylated DNA during early embryonic development.
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Garg, Dharmendra Kumar. "Quantification of CpG island methylation in the human large bowel." Thesis, University of Newcastle upon Tyne, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427274.
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Ladopoulos, Vasileios. "Regulation of CpG island promoters by the histone methyltransferase MLL2." Thesis, University of Birmingham, 2013. http://etheses.bham.ac.uk//id/eprint/4030/.
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MLL2 is a H3K4 specific HMT which vital for normal embryonic development in the mouse. Little is known on how MLL2 is recruited to its target genes and activates transcription. To gain insight into the molecular mechanism underlying MLL2 function, we focused on a known MLL2 target gene: Magoh2. This gene is controlled by a CpG island promoter and is ubiquitously expressed. Our results demonstrate that in the absence of MLL2, the Magoh2 promoter is methylated and Magoh2 is transcriptionally silenced. The Magoh2 promoter adopts the active conformation only in the presence of MLL2. Pol II is lost from the Magoh2 promoter in the absence of MLL2, resulting in Magoh2 down-regulation. We observed loss of H3K4me\(_3\)and H3K9ac and relocation of a nucleosome over the promoter, coinciding with the onset of DNA methylation. Use of ORB and a-amanitin demonstrated that neither transcription nor the presence of Pol II are required for the maintenance of H3K4me\(_3\). Magoh2 silencing can be overcome by re-introducing full-length MLL2. We investigated the role of MLL2 in haemopoiesis and demonstrated that MLL2 is vital for macrophage differentiation from embryoid bodies. MLL2 may be required for correct upregulation of Flk1 and generation of haem angioblast cells. When M//2 was deleted in haemangioblasts, the haemopoietic transcriptional program was perturbed suggesting that MLL2 may also play a role at this later developmental stage.
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Liu, Mengning. "Evolutionary landscape of CpG island methylation in X chromosome inactivation." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611328.
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Rahmatpanah, Farahnaz B. Caldwell Charles W. "Large scale CpG island methylation profiling of small B cell lymphoma." Diss., Columbia, Mo. : University of Missouri-Columbia, 2008. http://hdl.handle.net/10355/6863.
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The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 1, 2010). Vita. Thesis advisor: Charles W. Caldwell. "May 2008" Includes bibliographical references
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Dahlin, Anna. "The CpG island methylator phenotype in colorectal cancer : studies on risk and prognosis." Doctoral thesis, Umeå universitet, Patologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-41270.
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Background Colorectal cancer (CRC) is the second most common malignancy in developed countries. The mortality is high, with nearly half of patients dying from the disease. The primary treatment of CRC is surgery, and decisions about additional treatment with chemotherapy are based mainly on tumor stage. Novel prognostic markers that identify patients at high risk of recurrence and cancer-related death are needed. The development of CRC has been described in terms of two different pathways; the microsatellite instability (MSI) and chromosomal instability (microsatellite stable, MSS) pathway. More recently, the CpG island methylator phenotype (CIMP), characterized by frequent DNA hypermethylation, has been described as an alternative pathway of tumorigenesis. The event of DNA methylation is dependent on one-carbon metabolism, in which folate and vitamin B12 have essential functions. The purpose of this thesis was to study CIMP in CRC. The specific aims were to investigate the potential role of components of one-carbon metabolism as risk factors for this subgroup of tumors, and the prognostic importance of CIMP status, taking into consideration important confounding factors, such as MSI and tumor-infiltrating T cells. Methods CRC cases and referents included in the Northern Sweden Health and Disease Study (NSHDS, 226 cases and 437 referents) and CRC cases in the Colorectal Cancer in Umeå Study (CRUMS, n=490) were studied. Prediagnostic plasma concentrations of folate and vitamin B12 were analyzed in NSHDS. In both study groups, CIMP status was determined in archival tumor tissue by real-time quantitative PCR using an eight-gene panel (CDKN2A, MLH1, CACNA1G, NEUROG1, RUNX3, SOCS1, IGF2 and CRABP1). MSI screening status and the density of tumor-infiltrating T cells were determined by immunohistochemistry. Results An inverse association was found between plasma concentrations of vitamin B12 and rectal, but not colon, cancer risk. We also found a reduced risk of CIMP-high and CIMP-low CRC in study subjects with the lowest levels of plasma folate. We found that patients with CIMP-low tumors in both NSHDS and CRUMS had a poorer prognosis compared with CIMP-negative, regardless of MSI screening status. We also found that MSS CIMP-high patients had a poorer prognosis compared with MSS CIMP-negative. The density of tumor-infiltrating T cells and CIMP status were both found to be independent predictors of CRC patient prognosis. A particularly poor prognosis was found in patients with CIMP-low tumors poorly infiltrated by T cells. In addition, the density of T cells appeared to be more important than MSI screening status for predicting CRC patient prognosis. Conclusion Rather than being one disease, CRC is a heterogeneous set of diseases with respect to clinico-pathological and molecular characteristics. We found that the association between risk and plasma concentration of vitamin B12 and folate depends on tumor site and CIMP status, respectively. Patient prognosis was found to be different depending on CIMP and MSI screening status, and the density of tumor-infiltrating T cells.
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Siqueira, Flavia Ramos de. "Restrição no consumo de sódio durante a gestação é responsável pelo baixo peso ao nascimento e pela resistência à insulina da prole na idade adulta: estudo do mecanismo epigenético por metilação do DNA." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/5/5148/tde-13082014-142638/.
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Sabe-se que algumas alterações nutricionais maternas durante o período perinatal estão associadas com doenças metabólicas na vida adulta das proles, tais como diabetes melito tipo 2, resistência à insulina, obesidade e hipertensão arterial. O período da gestação em que estas alterações nutricionais influenciam a prole na idade adulta ainda não está elucidado. Modificações epigenéticas têm sido propostas como mecanismos responsáveis por estas desordens metabólicas. Ratas Wistar de doze semanas de idade foram alimentadas com dieta com conteúdo baixo (HO - 0,15% NaCl) ou normal (NR - 1,3% NaCl) de sódio desde o primeiro dia de gestação até o nascimento da prole ou HO durante a primeira (HO10) ou segunda (HO20) metade da gestação. O peso corpóreo e a ingestão de água e ração foram avaliados semanalmente durante a gestação. Teste de tolerância à insulina (ITT) e à glicose (GTT) e HOMA-IR foram realizados nas proles adultas. Expressão gênica por qRT-PCR e metilação do DNA na região promotora dos genes foram mapeadas utilizando tratamento com bissulfito de sódio e avaliadas por pirosequenciamento. O ganho de peso materno foi menor no HO e HO20 na terceira semana de gestação em comparação com NR e HO10. O peso ao nascimento da prole foi menor em machos e fêmeas dos grupos HO e HO20 em relação ao NR e HO10. O HOMA-IR foi maior nos machos com 12 semanas de idade do grupo HO em comparação com NR e com 20 semanas de idade do grupo HO10 em comparação com NR e HO20. Nas fêmeas com 12 semanas de idade o HOMA-IR foi maior no HO10 comparado com HO. Os níveis de insulina no soro foram maiores tanto nos machos com 20 semanas de idade do grupo HO10 comparado com NR quanto nas fêmeas com 12 semanas de idade do grupo HO10 comparado com HO. A área sob a curva do GTT indicou intolerância à glicose nos machos do grupo HO. A porcentagem de metilação das ilhas CpG no promotor dos genes de Igf1, Igf1r, Ins1, Ins2 e Insr no fígado de machos e fêmeas neonatais e no fígado, tecido adiposo branco e músculo em machos com 20 semanas de idade foi influenciada pela baixa ingestão de sal durante a gestação. Nenhuma destas alterações foi identificada nas fêmeas com 20 semanas de idade. Em conclusão, a baixa ingestão de sal na segunda metade da gestação é responsável pelo baixo peso ao nascimento em ambos os sexos. A intolerância à glicose observada na prole adulta ocorreu somente se a dieta hipossódica é dada durante a gestação inteira. Por outro lado, a resistência à insulina em resposta ao consumo de dieta hipossódica durante a gestação está relacionada com o momento em que ocorre este insulto e com o envelhecimento da prole. Também foi observado que alterações na metilação do promotor do gene Igf1 está correlacionado com o baixo peso ao nascimento em resposta a ingestão de dieta hipossódica durante a gestaçãoIt is known that some maternal nutritional alterations during pregnancy are associated with metabolic disorders in adult offspring, such as insulin resistance, type 2 diabetes mellitus, obesity and arterial hypertension. The period of pregnancy in which these nutritional alterations influence adult offspring remains uncertain. Epigenetic changes are proposed to underlie these metabolic disorders. Twelve-week-old female Wistar rats were fed a low-salt (LS - 0.15% NaCl) or normal-salt (NS - 1.3% NaCl) diet since the first day of gestation until delivery or LS during the first (LS10) or second (LS20) half of gestation. Body weight, food and water intake were weekly evaluated during gestation. Blood glucose, insulin (ITT) and glucose (GTT) tolerance tests, HOMA-IR were performed in adult offspring. Gene expression and DNA methylation were mapped using bisulfite treatment evaluated by pyrosequencing in the male and female neonates and adult offspring. Weight gain was lower in LS and LS20 dams than in NS and LS10 dams in the third week of pregnancy. Birth weights were lower in male and female LS20 and LS rats compared with NS and LS10 neonates. HOMA-IR was higher in 12-week-old LS males compared with NS and in 20-week-old male LS10 rats compared with NS and LS20 rats. In 12-week-old LS10 females, HOMA-IR was higher than in LS. Serum insulin levels were higher in 20 week-old LS10 male compared with NS rats and in 12-week-old LS10 female compared to LS rats. The area under the curve of GTT indicated glucose intolerance in 12- and 20-week-old LS male. Methylation of CpG islands of the Insr, Igf1, Igf1r, Ins1 and Ins2 genes in liver in neonates male and female offspring and liver, white adipose tissue and muscle in 20-week-old male offspring were influenced by low-salt intake during pregnancy. None of these alterations was identified in 20-week-old females. In conclusion, low-salt diet consumption in the second half of pregnancy can result in low birth weights in the males and females offspring. Glucose intolerance observed in adult offspring occurred only if low salt intake was given throughout pregnancy. However, insulin resistance in response to low salt intake during pregnancy is related to the time at which this insult occurs and to the age of the offspring. Alterations in the DNA methylation of Igf1 were observed to be correlated with low birth weight in response to low salt feeding during pregnancy
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Gautrey, Hannah Elizabeth. "Age-associated changes in promoter CpG island methylation and their potential role in cancer development." Thesis, University of Newcastle Upon Tyne, 2013. http://hdl.handle.net/10443/1824.
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Changes in DNA methylation patterns are a hallmark of both cancer and ageing, and may underlie susceptibility to developing age-related diseases such as cancer. To uncover the impact that such variation has on health and ageing, we assessed DNA methylation patterns in the peripheral blood leukocytes (PBL) of 480 participants in the Newcastle 85+ study to determine the levels, and degree of inter-individual variation, of DNA methylation in the promoter region of a panel of genes by Pyrosequencing. We found considerable inter-individual variation in promoter CpG methylation in several genes and a remarkable similarity to leukaemic patterns of aberrant methylation. This included specific methylation of the same sets of genes, strong correlations between methylation of the genes (in a pattern reminiscent of the CpG island methylator phenotype observed in cancer) and the presence of densely methylated alleles in highly methylated individuals, identical to patterns observed in cancer cells. This suggests that ageing and cancer related methylation may be closely linked. Further analysis of PBL DNA methylation levels in Newcastle 85+ study participants with a previous history of cancer (n=113) versus cancer-free individuals (n=113) found significantly higher methylation in those with a cancer history (10.71% vs. 10.21%, p=0.04). Further, a separate group of 72 individuals diagnosed with cancer within the 3 year duration of the study had similarly increased methylation levels (10.96% vs. 10.36%, p=0.03), suggesting that pre-existing methylation in normal cells may increase risk of cancer and may be evident prior to clinically detectable disease. In addition, individuals with increased DNA methylation were more likely to be categorised as frail (as defined by Fried) than those with lower DNA methylation measures, indicating that disrupted methylation patterns are associated with detrimental effects on healthy ageing. Subsequently, a GWAS analysis found that SNPs in two genes, DSCAM and DSCAML1, appeared to be associated with determining DNA methylation levels in the Newcastle 85+ study participants.
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Shi, Gongping. "Pattern of RECK CpG methylation as a potential marker for predicting breast cancer prognosis and drug-sensitivity." Kyoto University, 2016. http://hdl.handle.net/2433/216181.
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http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path[]=8620&pubmed-linkout=1Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第19927号
医博第4147号
新制||医||1017(附属図書館)
33013
京都大学大学院医学研究科医学専攻
(主査)教授 山田 泰広, 教授 妹尾 浩, 教授 武田 俊一
学位規則第4条第1項該当
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Vannini, Andrea <1983>. "Transcriptional responses of the Helicobacter pylori cag pathogenicity island." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2013. http://amsdottorato.unibo.it/5824/.
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The severity of Helicobacter pylori infections largely depends on the genetic diversity of the infecting strain, and particularly on the presence of the cag pathogenicity island (cag-PAI). This virulence locus encodes a type-IV secretion system able to translocate in the host cell at least the cag-encoded toxin CagA and peptidoglycan fragments, that together are responsible for the pathogenic phenotype in the host. Little is known about the bacterial regulators that underlie the coordinated expression of cag gene products, needed to assemble a functional secretion system apparatus. To fill this gap, a comprehensive analysis of the transcriptional regulation of the cag-PAI operons was undertaken.
To pursue this goal, a robust tool for the analysis of gene expression in H. pylori was first implemented. A bioluminescent reporter system based on the P. luminescens luxCDABE operon was constructed and validated by comparisons with transcriptional analyses, then it was systematically used for the comprehensive study and mapping of the cag promoters.
The identification of bona fide cag promoters had permitted to pinpoint the set of cag transcriptional units of the PAI. The responses of these cag transcriptional units to metabolic stress signals were analyzed in detail, and integrated with transcription studies in deletion mutants of important H. pylori virulence regulators and protein-DNA interaction analyses to map the binding sites of the regulators.
Finally, a small regulatory RNA cncR1 encoded by the cag-PAI was identified, and the 5’- and 3’-ends of the molecule were mapped by primer extension analyses, northern blot and studies with lux reporter constructs. To identify regulatory effects exerted by cncR1 on the H. pylori gene expression, the cncR1 knock out strain was derived and compared to the parental wild type strain by a macroarray approach. Results suggest a negative effect exerted by cncR1 on the regulome of the alternative sigma54 factor.
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Allinen, M. (Minna). "DNA damage response genes and chromosome 11q21-q24 candidate tumor suppressor genes in breast cancer." Doctoral thesis, University of Oulu, 2002. http://urn.fi/urn:isbn:9514267141.
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Abstract
As the defects in DNA repair and cell cycle control are known to promote tumorigenesis, a proportion of inherited breast cancers might be attributable to mutations in the genes involved in these functions. In the present study, three such genes, TP53, CHK2 and ATM, which are also associated with known cancer syndromes, were screened for germline mutations in Finnish breast cancer patients.
In combination with our previous results, three TP53 germline mutations, Tyr220Cys, Asn235Ser and Arg248Gln, were detected in 2.6% (3/108) of the breast cancer families. The only observed CHK2 alteration with a putative effect on cancer susceptibility, Ile157Thr, segregated ambiguously with the disease, and was also present in cancer-free controls. The available functional data, however, suggests that the altered CHK2 in some way promote tumorigenesis. Furthermore, compared to the other studied populations, Ile157Thr seems to be markedly enriched in Finland. Thus, the clinical significance of Ile157Thr requires further investigation among Finnish cancer patients.
ATM germline mutations appear to contribute to a small proportion of the hereditary breast cancer risk, as two distinct ATM mutations, Ala2524Pro and 6903insA, were found among three families (1.9%, 3/162) displaying breast cancer. They all originated from the same geographical region as the AT families with the corresponding mutations, possibly referring to a founder effect concerning the distribution of these mutations in the Finnish population.
The genes important for tumorigenesis in sporadic disease might also contribute to familial breast cancer. Therefore, four putative LOH targets genes in chromosome 11q21-q24 were screened for intragenic mutations, and five were analyzed for epigenetic inactivation in sporadic breast tumors. The lack of somatic intragenic mutations in MRE11A, PPP2R1B, CHK1 and TSLC1 led us next to investigate promoter region hypermethylation as a mechanism capable of silencing these genes, as well as the ATM gene. Only TSLC1 demonstrated involvement of CpG island methylation, which was especially prominent in three tumors. This suggests that together with LOH, methylation could result in biallelic inactivation of the TSLC1 gene in breast cancer.
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Zhu, Yiping [Verfasser]. "Epigenetic regulation of the pKi-67 gene promotor. DNA methylation analysis of its CpG island / Yiping Zhu." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2011. http://d-nb.info/1010394193/34.
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Berglund, Jonas. "Meiotic Recombination in Human and Dog : Targets, Consequences and Implications for Genome Evolution." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-233195.
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Understanding the mechanism of recombination has important implications for genome evolution and genomic variability. The work presented in this thesis studies the properties of recombination by investigating the effects it has on genome evolution in humans and dogs. Using alignments of human genes with chimpanzee and macaque orthologues we studied substitution patterns along the human lineage and scanned for evidence of positive selection. The properties mirror the situation in human non-coding sequences with the fixation bias ‘GC-biased gene conversion’ (gBGC) as a driving force in the most rapidly evolving regions. By assigning candidate genes to distinct classes of evolutionary forces we quantified the extent of those genes affected by gBGC to 20%. This suggests that human-specific characters can be prompted by the fixation bias of gBGC, which can be mistaken for selection. The gene PRDM9 controls recombination in most mammals, but is lacking in dogs. Using whole-genome alignments of dog with related species we examined the effects of PRDM9 inactivation. Additionally, we analyzed genomic variation in the genomes of several dog breeds. We identified that non-allelic homologous recombination (NAHR) via sequence identity, often GC-rich, creates structural variants of genomic regions. We show that these regions, which are also found in dog recombination hotspots, are a subset of unmethylated CpG-islands (CGIs). We inferred that CGIs have experienced a drastic increase in biased substitution rates, concurrent with a shift of recombination to target these regions. This enables recurrent episodes of gBGC to shape their distribution. The work presented in this thesis demonstrates the importance of meiotic recombination on patterns of molecular evolution and genomic variability in humans and dogs. Bioinformatic analyses identified mechanisms that regulate genome composition. gBGC is presented as an alternative to positive selection and is revealed as a major factor affecting allele configuration and the emergence of accelerated evolution on the human lineage. Characterization of recombination-induced sequence patterns highlights the potential of non-methylation and establishes unmethylated CGIs as targets of meiotic recombination in dogs. These observations describe recombination as an interesting process in genome evolution and provide further insights into the mechanisms of genomic variability.
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MacKenzie, Douglas James. "A functional dissection of the relationships between BRAF, DNMT3B and the CpG island methylator phenotype in colorectal cancer." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8867/.
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Approximately 10-20% of human colorectal cancers harbour an activating BRAFV600E mutation, which acts as a founder mutation for an alternative, serrated pathway of colorectal carcinogenesis. Conversely, BRAFV600E mutations are detectable in hyperplastic colonic polyps: lesions traditionally considered not to harbour malignant potential. Furthermore, both in vitro and in vivo, activated oncogenic BRAFV600E induces a stable proliferation arrest: oncogene induced senescence, which is a fundamental intrinsic tumour-suppressor mechanism. Thus, for neoplastic transformation to occur, it is clear that additional genetic and epigenetic events are required. BRAFV600E-mutant colorectal cancers are frequently associated with a CpG island methylator phenotype (CIMP), which is proposed to promote neoplastic transformation by bypass of intrinsic tumour-suppressor mechanisms, such as silencing of CDKN2A/INK4A. Consistent with this, neoplastic transformation in the serrated pathway is characterised by the progressive development of a CIMP phenotype. An emerging body of evidence supports a model in which the BRAFV600E mutation directly induces CIMP through the de novo methyltransferase, DNMT3B. Separately, elevated DNMT3B expression has previously been linked to the development of CIMP in both murine and human colorectal cancer. The published data however do not universally support this model, and significant questions over its validity remain. In the present work, the relationships between BRAFV600E mutation, DNMT3B expression and the CpG island methylator phenotype were examined by multiple approaches. A panel of DNMT3B antibodies were first characterised and validated. Significantly, the antibody previously used to link DNMT3B and CIMP in human colon cancer was demonstrated not to react with human DNMT3B. The ability of BRAFV600E to induce CIMP was next tested by whole genome bisulfite sequencing in a primary cell culture model. Surprisingly, activated BRAFV600E repressed expression of DNMT3B and failed to induce a CIMP phenotype. Consistent with this, human colorectal cancer cell lines expressing activated BRAFV600E typically expressed a low level of DNMT3B, and inactivation of DNMT3B in a BRAFV600E-mutant, CIMP-positive cell line did not reverse gene silencing characteristic of CIMP. An in vitro model system was next designed to test functional interactions between BRAFV600E and DNMT3B. Ectopic expression of DNMT3B antagonised BRAFV600E-induced proliferation arrest: a hallmark of senescence. Moreover, ectopic DNMT3B expression was demonstrated to accelerate BrafV600E-induced intestinal carcinogenesis in a mouse model, and conversely, Dnmt3b knockout impaired BrafV600E-induced murine intestinal carcinogenesis. Analysis of human colorectal cancer TCGA data was next undertaken, and confirmed that expression of DNMT3B is frequently elevated in human colorectal cancer and that this is often linked to amplification of the DNMT3B gene. However, more detailed analysis of human TCGA data revealed that BRAFV600E mutation is neither necessary nor sufficient to induce CIMP, and that both BRAFV600E mutations and CIMP are both linked to low expression of DNMT3B. Thus, while both BRAFV600E and DNMT3B both harbour oncogenic potential, they do not appear to cooperate to induce CIMP, and do not appear to cooperate frequently in human colorectal cancer by any mechanism.
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Dodge, Jonathan Eldon. "Selective variegated methylation of the p15/INK4B CpG island is a high frequency event in acute myeloid leukemia (AML)." Diss., The University of Arizona, 2000. http://hdl.handle.net/10150/284143.
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We attempted to define target genes that were inactivated in acute myeloid leukemia (AML) by DNA methylation. We hypothesized that hypermethylation of 51 CpG islands is associated with transcriptional silencing of the corresponding gene and participates in either the emergence of drug resistance or the conversion of normal cells to cancer cells. To test this hypothesis the DNA methylation status of the 5' CpG islands of dCK containing 49 CpGs, p15 containing 80 CpGs, and p16 containing 53 CpGs was determined by sodium bisulfite sequencing of normal human peripheral blood lymphocytes (PBL) and bone marrow (NBM), human leukemia cell lines, and cytosine-arabinoside (ara-C)-resistant adult acute myeloid leukemia (AML) patients. In PBL and NBM dCK, p15, and p16 were all unmethylated. dCK was unmethylated in the paired ara-C-sensitive (/S) ara-C-resistant (/R) leukemia cell lines HL60/S & /R and K562/S & /R, and in the 8 AML patients analyzed. p16 was unmethylated in KG-l and KG-1a and both had detectable p16 mRNA and protein. None of the 8 AML patients had aberrant methylation of p16. For p15, a variegated pattern of aberrant methylation was found in KG-1, and complete methylation of p15 was found in KG-1a. The variegated pattern of p15 methylation seen in KG-1 and the complete methylation seen in KG-1a were both associated with no detectable p15 mRNA or protein. p15 was aberrantly methylated in 6 of the 8 AML patients, 5 had a variegated pattern of methylation, and 1 showed complete methylation. We next introduced ectopic p15 and p16 into the p15 and p16 negative human T-cell lymphocytic leukemia cell line Jurkat. The p15 positive clones grew at a slower rate than the parent cell or p16 positive clones as measured by growth in liquid culture and MTS assay. cDNA microarray expression analysis differentiated p15 and p16 positive subclones from the parent cell line but not from each other. This suggests that despite the selective methylation of p15 but not p16 in AML, p15 and p16 are functionally similar.
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Zhou, Jin Chuan. "Biochemical characterisation of KDM2A." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:030faa1d-5d3c-4066-9e9f-a44cd13cf85c.
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Mammalian genomes are characterised by unique regions of non-methylated DNA known as CpG islands (CGIs). These genomic elements are characterised by a high density of CpGs and an elevated GC content compared to the surrounding, bulk of the genome. CGIs are prevalently associated with the 5’ end of genes and represent key nucleation sites where specific transcription factors and chromatin modifiers are recruited to impact on gene function. This thesis is focused at understanding the biochemical properties of the recently discovered H3K36-specific histone demethylase, KDM2A. This enzyme is specifically recruited to CGIs but how it interfaces with local chromatin in vivo remains unknown. Using defined chromatin templates in vitro, this study demonstrates that KDM2A binding to DNA relies on a zinc finger CXXC domain that preferentially recognizes non-methylated CpGs. In particular, nucleosomes represent a major barrier to KDM2A binding and chromatin substrates are interpreted by the CXXC domain through specific interaction with CpGs within linker DNAs. Moreover, the adjacent PHD domain does not contribute to KDM2A binding to chromatin. Together these observations suggest that sequence, methylation status and accessibility of DNA define how CGI chromatin is interpreted by CXXC domain proteins. In particular, the precise targeting of KDM2A to CGIs contributes to the creation of a unique chromatin architecture that highlights gene regulatory regions within large and complex mammalian genomes.
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Corless, Samuel. "Role of DNA supercoiling in genome structure and regulation." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/9623.
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A principle challenge of modern biology is to understand how the human genome is organised and regulated within a nucleus. The field of chromatin biology has made significant progress in characterising how protein and DNA modifications reflect transcription and replication state. Recently our lab has shown that the human genome is organised into large domains of altered DNA helical twist, called DNA supercoiling domains, similar to the regulatory domains observed in prokaryotes. In my PhD I have analysed how the maintenance and distribution of DNA supercoiling relates to biological function in human cells. DNA supercoiling domains are set up and maintained by the balanced activity of RNA transcription and topoisomerase enzymes. RNA polymerase twists the DNA, over-winding in front of the polymerase and under-winding behind. In contrast topoisomerases relieve supercoiling from the genome by introducing transient nicks (topoisomerase I) or double strand breaks (topoisomerase II) into the double helix. Topoisomerase activity is critical for cell viability, but the distribution of topoisomerase I, IIα and IIβ in the human genome is not known. Using a chromatin immunoprecipitation (ChIP) approach I have shown that topoisomerases are enriched in large chromosomal domains, with distinct topoisomerase I and topoisomerase II domains. Topoisomerase I is correlated with RNA polymerase II, genes and underwound DNA, whereas topoisomerase IIα and IIβ are associated with each other and over-wound DNA. This indicates that different topoisomerase proteins operate in distinct regions of the genome and can be independently regulated depending on the genomic environment. Transcriptional regulation by DNA supercoiling is believed to occur through changes in gene promoter structure. To investigate DNA supercoiling my lab has developed biotinylated trimethylpsoralen (bTMP) as a DNA structure probe, which preferentially intercalates into under-wound DNA. Using bTMP in conjunction with microarrays my lab identified a transcription and topoisomerase dependent peak of under-wound DNA in a meta-analysis of several hundred genes (Naughton et al. (2013)). In a similar analysis, Kouzine et al. (2013) identified an under-wound promoter structure and proposed a model of topoisomerase distribution for the regulation of promoter DNA supercoiling. To better understand the role of supercoiling and topoisomerases at gene promoters, a much larger-scale analysis of these factors was required. I have analysed the distribution of bTMP at promoters genome wide, confirming a transcription and expression dependent distribution of DNA supercoils. DNA supercoiling is distinct at CpG island and non-CpG island promoters, and I present a model in which over-wound DNA limits transcription from both CpG island promoters and repressed genes. In addition, I have mapped by ChIP topoisomerase I and IIβ at gene promoters on chromosome 11 and identified a different distribution to that proposed by Kouzine et al. (2013), with topoisomerase I maintaining DNA supercoiling at highly expressed genes. This study provides the first comprehensive analysis of DNA supercoiling at promoters and identifies the relationship between supercoiling, topoisomerase distribution and gene expression. In addition to regulating transcription, DNA supercoiling and topoisomerases are important for genome stability. Several studies have suggested a link between DNA supercoiling and instability at common fragile sites (CFSs), which are normal structures in the genome that frequently break under replication stress and cancer. bTMP was used to measure DNA supercoiling across FRA3B and FRA16D CFSs, identifying a transition to a more over-wound DNA structure under conditions that induce chromosome fragility at these regions. Furthermore, topoisomerase I, IIα and IIβ showed a pronounced depletion in the vicinity of the FRA3B and FRA16D CFSs. This provides the first experimental evidence of a role for DNA supercoiling in fragile site formation.
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Trimarchi, Michael Paul Trimarchi. "Identification of endometrial cancer methylation features using a combined methylation analysis method." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1461302615.
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Kouri, Kimberly. "Genetic characterization and molecular evolution of the cag pathogenicity island of Helicobacter pylori." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0029/MQ64384.pdf.
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Bechtel, Jason M. "Characterization of Genomic MidRange Inhomogeneity." University of Toledo Health Science Campus / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=mco1217365784.
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Kgatle, Mankgopo Magdeline. "An investigation of genome-wide promoter region cytosine-phosphate-guanine (CpG) Island methylation profiles in patients with chronic hepatitis B virus infection." Doctoral thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/8800.
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Includes bibliographical references.Hepatitis B virus (HBV) is oncogenic and a major cause of hepatocellular carcinoma (HCC) in the developing world. It integrates parts of its genome such as the HBx gene, core and surface antigens into the human genome. The integrated viral DNA disrupts gene function resulting in physiological changes that cause liver disease. The viral inserts are inactivated through methylation. This is a protective innate response driven by human DNA methyltransferases triggered by the presence of viral DNA inserts. This thesis investigates the hypothesis that during the innate response to methylate integrated HBV DNA, there is unintended methylation of genomic DNA around the intercalated viral DNA that could be adjacent host promoter Cytosine-phosphateGuanine (CpG) islands. This would activate or silence genes including tumour suppressors and result in the clinical disease phenotypes of hepatic inflammation, fibrosis and HCC that characterise chronic HBV infection. Genome-wide microarray analysis was used to investigate for the presence of promoter CpG island methylation in a cohort of patients with liver disease due to HBV infection, HCC, autoimmune hepatitis which is a non-viral liver disease and normal cases with no liver disease. The study identified hypermethylation in promoter regions, transcription start sites, gene exons and introns. Only sites in the promoter region and within 100bp upstream of a transcription start site were analysed for this thesis presentation. Using an extended cohort of patients with chronic HBV infection and normal controls, bisulfite DNA sequencing was used to validate and confirm the presence of DNA methylation in a selection of some of genes identified. HBV infected patients were shown to have hypermethylation in the promoter CpG island regions of several genes that regulate hepatic metabolism, tumour suppression, ribonucleic acid splicing, vitamin D receptor binding, protein ubiquitination and the cell cycle. Many of these genes have transcriptional binding factors that are known to be affected by the transcriptional transactivator HBx protein, suggesting that HBx protein is important in the pathogenesis of liver disease. Amongst the most hypermethylated core promoter regions identified were those for cyclin kinases genes such as Cyclin D3 (CCND3). CCND3 gene is important in liver regeneration and wound healing and its abnormal function has been linked to the development of liver fibrosis and HCC. Increased methylation of CCND3 gene was associated with HBV e antigen positive status and genotype D, supporting the hypothesis that increased methylation is associated with host and viral factors. Methylation induced alteration in the function of the identified gene promoters would affect cellular signalling with effects on cell growth, differentiation, proliferation and apoptosis. These changes would explain the development of hepatic inflammation, apoptosis, fibrosis and malignant transformation seen in chronic HBV infection. Further investigation of these genes will provide new insights on mechanisms of HBV induced liver disease and the development of new molecular diagnostic tools or therapeutic interventions.
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Cruz, Guilherme Marcello Queiroga. "Desvendando as interações entre retrotransposons e genomas vegetais, com ênfase em cana-de-açúcar." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-27012015-084619/.
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Esta tese é estruturada em dois capítulos. O primeiro capítulo explora os retrotransposons com LTR (LTR-RT) em cana-de-açúcar e grande parte de seus resultados foram publicados no artigo \'\'Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns\'\'. Nossos resultados mostraram que as diferentes famílias de LTR-RT em cana-de-açúcar possuem estruturas e regulação distintas. O segundo capítulo desta tese visa responder a perguntas que surgiram durante a primeira metade deste trabalho, mas ao invés de focar no genoma de uma planta optamos por trabalhar com linhagem Del de LTR-RT em dez genomas de angiospermas sequenciados. Os resultados desta parte do trabalho foram submetidos para publicação no artigo intitulado \'\'Virus-like attachment sites and plastic CpG islands: landmarks of diversity in plant Del retrotransposons\'\'. Os resultados mostraram que a LTR é uma região dinâmica e importante para a evolução dos LTR-RTs. Nós especulamos que mudanças nas LTR atuem como gatilhos para a diversificação dos LTR-RTs.This doctoral thesis is structured in two chapters. In the first chapter we explore the LTRretrotransposons (LTR-RT) in sugarcane, these results were published in an article entitled \'\'Analysis of plant LTR-etrotransposons at the fine-scale family level reveals individual molecular patterns\'\'. In this paper we show that different sugarcane LTR-RT families have distinct structure and are differentially regulated. In the second chapter we try to find answers to questions that came up in the first half of this work, but instead of focusing in one plant genome we chose to work with the Del lineage of LTR-RT in tem angiosperm sequenced genomes. These results are submitted to publication as an article entitled \'\'Virus-like attachment sites and plastic CpG islands: landmarks of diversity in plant Del retrotransposons\'\'. Our results indicate that the LTR region is dynamic and important in the evolution of LTR-retrotransposons, we speculate that it is a trigger for retrotransposon diversification.