1,720,991 research outputs found
Investigating the Direct Role of Promoter DNA Methylation in Gene Expression using a Targeted TET1CD-dCas9 Demethylase Tool
Full text linkDNA hypermethylation of gene promoter regions has long been associated with transcriptional repression. Furthermore, the establishment of dense promoter methylation within tumour- suppressor genes has been widely-characterised as a prominent hallmark of oncogenesis. The presence of dense DNA methylation, concentrated to promoter region CpG islands, is known to recruit methyl-CpG binding domain proteins (MBDs) and transcriptional repressors which manipulate the local chromatin environment to downregulate transcriptional activity.
However, there is an accumulating body of evidence which now suggests, in some specific contexts, that promoter hypermethylation is also associated with high or increased levels of active gene expression. Recent research has uncovered this phenomenon in the context of cutaneous melanoma. Early B-cell factor 3 (EBF3) has been identified as a putative ‘epigenetic driver’ of metastasis in melanoma, associated with an increase in aggressive phenotypic behaviour. Interestingly, EBF3 acquires substantial promoter DNA methylation during the transition to metastasis, which is associated with a corresponding increase in gene expression. Moreover, when treated with a global demethylating agent, EBF3 expression was subsequently reduced.
Unfortunately, investigations using global demethylation inhibitors do not conclusively demonstrate that promoter-specific methylation gain is responsible for a change in gene expression. However, with the recent emergence of Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-based epigenetic editing technologies, there is now scope for more targeted investigation into this relationship.
Here, I have constructed a CRISPR-based targeting system to investigate the EBF3 promoter region in more detail. The CRISPR construct is fused to the SUperNova Tagging (SunTag) protein scaffold, which allows for the recruitment of effector proteins to induce active demethylation at the target locus. In this case, the effector protein is a catalytic domain derived from the human ten-eleven translocation 1 (TET1) dioxygenase.
Directed by locus-specific guide-RNA (gRNA) molecules, this system was used to induce site- specific active demethylation within the target promoter region of EBF3, in human melanoma cell lines. Methylation changes across this target locus have been evaluated using Combined Bisulfite Restriction Assay (CoBRA) and methylation-specific Illumina MiSeq sequencing.
Variable levels of targeted demethylation have been observed, of up to 58.8% absolute methylation difference between edited and control samples.
In generating a modifiable, RNA-guided construct for site-specific DNA methylation editing, this work has laid a strong platform for further investigation into the direct relationship between DNA methylation and gene expression with regards to both EBF3, and a limitless range of additional contexts in future
Investigating the Direct Role of Promoter DNA Methylation in Gene Expression using a Targeted TET1CD-dCas9 Demethylase Tool
No full textDNA hypermethylation of gene promoter regions has long been associated with transcriptional repression. Furthermore, the establishment of dense promoter methylation within tumour- suppressor genes has been widely-characterised as a prominent hallmark of oncogenesis. The presence of dense DNA methylation, concentrated to promoter region CpG islands, is known to recruit methyl-CpG binding domain proteins (MBDs) and transcriptional repressors which manipulate the local chromatin environment to downregulate transcriptional activity.
However, there is an accumulating body of evidence which now suggests, in some specific contexts, that promoter hypermethylation is also associated with high or increased levels of active gene expression. Recent research has uncovered this phenomenon in the context of cutaneous melanoma. Early B-cell factor 3 (EBF3) has been identified as a putative ‘epigenetic driver’ of metastasis in melanoma, associated with an increase in aggressive phenotypic behaviour. Interestingly, EBF3 acquires substantial promoter DNA methylation during the transition to metastasis, which is associated with a corresponding increase in gene expression. Moreover, when treated with a global demethylating agent, EBF3 expression was subsequently reduced.
Unfortunately, investigations using global demethylation inhibitors do not conclusively demonstrate that promoter-specific methylation gain is responsible for a change in gene expression. However, with the recent emergence of Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-based epigenetic editing technologies, there is now scope for more targeted investigation into this relationship.
Here, I have constructed a CRISPR-based targeting system to investigate the EBF3 promoter region in more detail. The CRISPR construct is fused to the SUperNova Tagging (SunTag) protein scaffold, which allows for the recruitment of effector proteins to induce active demethylation at the target locus. In this case, the effector protein is a catalytic domain derived from the human ten-eleven translocation 1 (TET1) dioxygenase.
Directed by locus-specific guide-RNA (gRNA) molecules, this system was used to induce site- specific active demethylation within the target promoter region of EBF3, in human melanoma cell lines. Methylation changes across this target locus have been evaluated using Combined Bisulfite Restriction Assay (CoBRA) and methylation-specific Illumina MiSeq sequencing.
Variable levels of targeted demethylation have been observed, of up to 58.8% absolute methylation difference between edited and control samples.
In generating a modifiable, RNA-guided construct for site-specific DNA methylation editing, this work has laid a strong platform for further investigation into the direct relationship between DNA methylation and gene expression with regards to both EBF3, and a limitless range of additional contexts in future
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Targeted Epigenetic Editing Using a dCas9- SunTag Methylation System
Full text linkDNA methylation is a stable and somatically heritable epigenetic process. It is classically understood as a gene silencing mechanism, whereby hypermethylation of gene promoters is largely associated with transcriptional silencing. However, emerging research in the past decade has found a different association, particularly in the context of cancer and metastasis. These studies have demonstrated a seemingly paradoxical correlation between high levels of promoter methylation and subsequent transcriptional activation. As such, we are yet to fully unravel the complex mechanism underlying this relationship. This warrants further investigations to better understand this dynamic molecular mechanism and thus its implications in cancer pathobiology and metastasis.
Previously established methods of manipulating DNA methylation have largely involved the use of chemical drugs, such as decitabine. These drugs act globally on the epigenome and are thus non-specific. Importantly, as they are inhibitors of DNA methylation, they cannot definitively demonstrate that an increase in promoter methylation is directly responsible for an increase in gene expression. Therefore, by using a CRISPR-based system for targeted epigenetic editing, the precise mechanism of paradoxical gene activation by DNA methylation can now be investigated.
Our group has recently demonstrated this paradoxical correlation in a study characterising epigenetic changes in metastatic melanoma. The Early B-cell Factor 3 (EBF3) gene promoter region was found to be substantially hypermethylated in metastatic cell lines compared to paired, primary cell lines. This increase in methylation was also found to be associated with a subsequent increase in gene expression. These findings suggest that EBF3 in human melanoma cell lines is a good candidate gene to investigate this complex relationship.
Here, I aim to establish a methylation editing system and apply it to the EBF3 promoter region. This is a three-component CRISPR system incorporating the SUperNova Tag (SunTag), which allows for the recruitment of multiple effector proteins. In this project, I use the DNA methyltransferase 3A (DNMT3A) effector protein to induce active methylation at a target locus within the EBF3 promoter region in melanoma cell lines.
Methylation changes have been evaluated using methylation-specific Illumina iSeq sequencing. Variable levels of targeted methylation have been observed, of up to 38.0% absolute methylation change between edited and unedited samples. This project represents important progress in investigating the promoter methylation of EBF3 in more detail, as well as laying out an essential platform to further investigate methylation changes and causal mechanisms of gene expression alteration in future studies
Targeted Epigenetic Editing using a dCas9-SunTag Demethylation system
No full textDNA methylation is a stable epigenetic modification, playing a critical role in the regulation of gene expression. Dysregulation of DNA methylation is implicated in the pathogenesis of numerous diseases including cancer. High level of promoter DNA methylation is associated with transcriptional silencing. However, we identified Early B-cell Factor 3 (EBF3) as a potential epigenetic driver of melanoma metastasis displaying high promoter methylation associated with increased mRNA expression. To establish the causal role of DNA methylation in directly altering gene transcription, it is necessary to exclusively edit the methylation status of the target loci.
The emergence of CRISPR/dCas9-based editing systems have shown a more specific and efficient method for targeted manipulation of DNA methylation compared to traditional epigenomic editing technologies. However, despite the recent improvements in CRISPR/dCas9 technology, off-target effects are still a major concern.
Here, I describe the initial construction of plasmid components of CRISPR-dCas9-SunTag demethylation system required for the editing the EBF3 promoter region. Moreover, an evaluation of the system’s potential off-target effects in relation with locus-specific guide- RNA (gRNA) targeting was analysed by the presence of Cas9 activity. Analysis of Cas9 activity on putative off-target locations was performed using targeted Illumina Miseq Sequencing. Limited Cas9 activity have been observed in these off-target sites confirming the specificity of gRNA targeting.
Overall, these work has laid an initial platform for further investigation of the relationship between DNA methylation and transcriptional regulation
Targeted Epigenetic Editing Using a dCas9- SunTag Methylation System
No full textDNA methylation is a stable and somatically heritable epigenetic process. It is classically understood as a gene silencing mechanism, whereby hypermethylation of gene promoters is largely associated with transcriptional silencing. However, emerging research in the past decade has found a different association, particularly in the context of cancer and metastasis. These studies have demonstrated a seemingly paradoxical correlation between high levels of promoter methylation and subsequent transcriptional activation. As such, we are yet to fully unravel the complex mechanism underlying this relationship. This warrants further investigations to better understand this dynamic molecular mechanism and thus its implications in cancer pathobiology and metastasis.
Previously established methods of manipulating DNA methylation have largely involved the use of chemical drugs, such as decitabine. These drugs act globally on the epigenome and are thus non-specific. Importantly, as they are inhibitors of DNA methylation, they cannot definitively demonstrate that an increase in promoter methylation is directly responsible for an increase in gene expression. Therefore, by using a CRISPR-based system for targeted epigenetic editing, the precise mechanism of paradoxical gene activation by DNA methylation can now be investigated.
Our group has recently demonstrated this paradoxical correlation in a study characterising epigenetic changes in metastatic melanoma. The Early B-cell Factor 3 (EBF3) gene promoter region was found to be substantially hypermethylated in metastatic cell lines compared to paired, primary cell lines. This increase in methylation was also found to be associated with a subsequent increase in gene expression. These findings suggest that EBF3 in human melanoma cell lines is a good candidate gene to investigate this complex relationship.
Here, I aim to establish a methylation editing system and apply it to the EBF3 promoter region. This is a three-component CRISPR system incorporating the SUperNova Tag (SunTag), which allows for the recruitment of multiple effector proteins. In this project, I use the DNA methyltransferase 3A (DNMT3A) effector protein to induce active methylation at a target locus within the EBF3 promoter region in melanoma cell lines.
Methylation changes have been evaluated using methylation-specific Illumina iSeq sequencing. Variable levels of targeted methylation have been observed, of up to 38.0% absolute methylation change between edited and unedited samples. This project represents important progress in investigating the promoter methylation of EBF3 in more detail, as well as laying out an essential platform to further investigate methylation changes and causal mechanisms of gene expression alteration in future studies
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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