1,720,989 research outputs found
Kinetics of Passive Demethylation
No full textDNA methylation is an epigenetic modification established during cellular differentiation, and when removed by either active or passive means, can substantially improve the efficiency by which induced pluripotent stem cells are created for the sake of regenerative medicine. During the ‘active’ removal of DNA methylation, the Ten-Eleven Translocation (TET) enzymes catalyse the oxidation of cytosine methylation (5mC) to 5-hydroxymethylcytosine (5hmC) and further derivatives. Unpublished work from the Hore laboratory and collaborators has shown that TET differentially targets particular CG-containing hexamer motifs for rapid demethylation but leaves others unaffected.
Currently, it is unclear if ‘passive’ epigenetic memory loss, whereby demethylation results from the inhibition or inactivation of the DNA methylation maintenance machinery, also shows a preference for certain CG-containing hexamers. DNMT1 typically acts to maintain DNA methylation by catalysing the addition of methyl groups on hemimethylated DNA, enabling the transmission of methylation after DNA replication or repair. A cytosine-analogue called decitabine, which is also used in cancer treatment, can be utilised to block DNMT1 activity.
In this project, I used bisulfite sequencing data from decitabine-treated mouse embryonic stem cell samples to determine the kinetics of demethylation at all 256 CG-containing hexamers over a 48-hour period. A bioinformatic pipeline was implemented for the extraction and analysis of CG-containing hexamers from trimmed and mapped sequencing reads. Demethylation rates from these hexamers were examined both within and outside of CpG islands (CGI) and my analysis was performed in both absolute and relative terms according to similar experiments performed recently. In doing so, I found that once starting methylation levels and genomic location were taken into account, passive demethylation of DNA showed no preference to certain CG-containing hexamers, in stark contrast to that of active DNA demethylation.
In studying the kinetics of passive demethylation, I was able to answer a fundamental biological question regarding the mechanism by which passive demethylation occurs. However, in an applied sense, the value of this work will help to assess merits and risks of DNA demethylation strategies for the efficient creation of induced pluripotent stem cells that are safe for clinical use
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
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
Creating a New Code: Artificial Creation & Erasure of Epigenetic Memory in Mammalian Cells
No full textABSTRACT
Cytosine methylation is perhaps the most dynamic and best-studied form of epigenetic modification. Occurring predominantly in the CG dinucleotide context within mammalian genomes, it is essential for normal embryonic development, X chromosome inactivation, genomic imprinting and transposon silencing. The uniqueness of CG methylation lies in its ability to be maintained following cell division in the absence of the signal which created it. Together, the global distribution of CG methylation forms an “epigenetic memory” that contributes to the differentiation and maintenance of distinctive somatic cell fates. Artificial manipulation of cytosine methylation (known as “synthetic epigenetics”) could potentially improve the creation and differentiation of developmentally potent cells, which will be instrumental in the advancement of regenerative medicine.
I have undertaken two experimental projects investigating the potential of synthetic epigenetics, whereby epigenetic modifiers of non-mammalian provenance were overexpressed in mouse embryonic stem cells (ESCs). In plant genomes, methylation occurs at CHG nucleotides (where H is any base other than G) in an analogous manner to mammalian CG methylation, due to the action of the CMT3 methyltransferase. Thus, in the first project, I created transgenic cell-lines with inducible overexpression of Arabidopsis CMT3, with the aim of creating methylation at CHG nucleotides. Overexpression of CMT3 was performed in three mouse embryonic stem cells (mESC) lines; two wild-type lines (E14 and V6.5) and a DNA methylation depleted line known as (DNMT-TKO). My experimental results to date suggest that CMT3 alone is inadequate for CHG methylation establishment in mammalian genomes, however, increased levels of methylation in the CG context were detected, suggesting it may have previously unappreciated maintenance capacity in this context.
The second experimental project explored the nature of DNA methylation removal. Cytosine methylation can be actively removed from DNA by the Ten eleven translocation (TET) enzymes, which oxidise 5-methylcytosine (5mC) to 5 hydroxymethylcytosine (5hmC), and further oxidised derivatives 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Exactly how these analogues are returned to unmodified cytosine is unknown, partly because there are currently few molecular tools to control the transition between 5hmC, 5fC and 5caC. Unpublished in vitro observations suggest that Ten eleven translocation (TET) enzymes from the amœba Naegleria gruberi are far more effective at iteratively producing 5fC and 5caC, compared to mammalian homologues which produces 5hmC predominantly (T.P.
Jurkowski, personal communication). I created mouse embryonic stem cells with inducible expression of Naegleria TET and mutated Naegleria TET variants, and used bisulphite sequencing to assess their relative ability to demethylate DNA. I found that the amœba TET variants were ineffective at demethylating DNA compared to mammalian TET controls. Unexpectedly, some constructs even showed significant increased methylation. I hypothesise that the Naegleria TET variants I created act as non-functional competitors of endogenous TET when overexpressed in mammalian cells. While this likely precludes their widespread use in the field of synthetic epigenetics, they still may provide a useful experimental tool for further study of TET enzyme biology
DNA methylation memory: Understanding epigenetic reprogramming in vertebrates
Full text linkDNA methylation is an epigenetic mark critical for vertebrate development and is associated with numerous cellular and organismal processes including X-chromosome inactivation, genomic imprinting, and regulation of gene expression. Importantly, DNA methylation patterns are faithfully inherited during cell division, providing an information memory module additional to the DNA code. This mark, along with other epigenetic modifications, plays an essential role in establishing and maintaining cell identity. DNA methylation dynamics has been studied in detail in eutherian mammals, where two major waves of demethylation, the first in the early embryo and the second during germline development, remove most marks. Erasure of epigenetic memory is associated with cell reprogramming, and in mammals, is inextricably linked to increased developmental potency. For divergent vertebrate models, this dynamic is largely untested and indirect evidence suggests epigenetic memory may be retained in the germline. Furthermore, the role of epigenetic memory and reprogramming in major cell fate transitions, such as sex determination and sex change, is underexplored despite being apparently driven by epigenetic mechanisms in at least some species.
In order to understand how epigenetic memory is maintained, erased, and reprogrammed in divergent vertebrates, I have focused on two fish species. I have analysed the epigenome of the germline during gonad development in zebrafish (Danio rerio), and the transcriptome and methylome of bluehead wrasse (Thalassoma bifasciatum) during female-to-male sex change. Using a combination of techniques, including isolation of germline cells, whole genome bisulfite sequencing, and comparative epigenomics and transcriptomics, I explored epigenetic memory and reprogramming in these species. This thesis is presented as a collection of research and review papers, as well as a discussion synthesising my results
Balancing Safety and Developmental Potency: Assessing the Effect of Antioxidants and TET on Genomic Imprinting Stability.
Full text linkDevelopmental potency represents the ability of undifferentiated cells to undergo transformation into differentiated cells, with specialised functions. The inner cell mass (ICM) of a blastocyst represents the most developmentally potent cells of the body. The ICM, along with the naïve embryonic stem cells derived from it, possess remarkably low levels of global DNA methylation; a trait that is intrinsic to their pluripotent phenotype and when artificially induced can help re-program differentiated cells back into a naïve state.
Ten-eleven translocation (TET) enzymes actively remove DNA methylation in a Fe2+-dependent fashion. By assisting Fe2+ recycling, the antioxidant ascorbate has been shown to increase TET enzymatic activity and enhance reprogramming of differentiated cells to the naïve state. However, it is currently unclear if TET activation via ascorbate, or other antioxidants, can cause unwanted demethylation at imprint control regions; regulatory elements that control parent-of-origin specific gene expression. Loss of methylation at these regulatory groups results in the biallelic expression of the genes under their control. This phenomenon has been implicated in the formation of a range of cancers, as well as Beckwith-Wiedemann syndrome, a congenital overgrowth disorder.
In this project, I aimed to test what effect the antioxidants ascorbate and hydroquinone have on the stability of imprint control regions through the activation of TET proteins, and in doing so, assess the safety and practicality of using naïve embryonic stem cells for future medical applications. I grew several naïve embryonic stem cell lines, including a TET triple knockout line with inducible TET expression, in varying concentrations of ascorbate or hydroquinone. Bisulfite amplicon sequencing and florescence activated cell sorting techniques were used to assess methylation patterns at the KCNQ1ot1 imprinted loci in the presence or absence of increased TET activity.
While the study has been hampered by technical difficulties (i.e. clonal amplification was detected, particularly in TET knockout cell lines), I have shown that ascorbate causes significant demethylation of the KCNQ1ot1 imprinted region at 25 ng/μL of ascorbate. While other concentrations, (12.5 ng/μL and 50 ng/μL of ascorbate) were not statistically significant there appeared to be a trend of decreasing methylation which may have become more obvious had the cells been cultured with ascorbate for longer. Interestingly this effect was not seen at 100 ng/μL of ascorbate, indicating there is an optimal concentration range for TET-induced demethylation at the KCNQ1ot1 locus. In contrast, hydroquinone had no significant effect.
Ultimately, these results add to the current literature describing the effects of different culture media constituents on naïve embryonic stem cells. This may allow for the rational design of culture media that can improve both the efficiency and safety of naïve embryonic stem cells and embryos used in regenerative medicine, mammalian transgenics and IVF
Balancing Safety and Developmental Potency: Assessing the Effect of Antioxidants and TET on Genomic Imprinting Stability.
No full textDevelopmental potency represents the ability of undifferentiated cells to undergo transformation into differentiated cells, with specialised functions. The inner cell mass (ICM) of a blastocyst represents the most developmentally potent cells of the body. The ICM, along with the naïve embryonic stem cells derived from it, possess remarkably low levels of global DNA methylation; a trait that is intrinsic to their pluripotent phenotype and when artificially induced can help re-program differentiated cells back into a naïve state.
Ten-eleven translocation (TET) enzymes actively remove DNA methylation in a Fe2+-dependent fashion. By assisting Fe2+ recycling, the antioxidant ascorbate has been shown to increase TET enzymatic activity and enhance reprogramming of differentiated cells to the naïve state. However, it is currently unclear if TET activation via ascorbate, or other antioxidants, can cause unwanted demethylation at imprint control regions; regulatory elements that control parent-of-origin specific gene expression. Loss of methylation at these regulatory groups results in the biallelic expression of the genes under their control. This phenomenon has been implicated in the formation of a range of cancers, as well as Beckwith-Wiedemann syndrome, a congenital overgrowth disorder.
In this project, I aimed to test what effect the antioxidants ascorbate and hydroquinone have on the stability of imprint control regions through the activation of TET proteins, and in doing so, assess the safety and practicality of using naïve embryonic stem cells for future medical applications. I grew several naïve embryonic stem cell lines, including a TET triple knockout line with inducible TET expression, in varying concentrations of ascorbate or hydroquinone. Bisulfite amplicon sequencing and florescence activated cell sorting techniques were used to assess methylation patterns at the KCNQ1ot1 imprinted loci in the presence or absence of increased TET activity.
While the study has been hampered by technical difficulties (i.e. clonal amplification was detected, particularly in TET knockout cell lines), I have shown that ascorbate causes significant demethylation of the KCNQ1ot1 imprinted region at 25 ng/μL of ascorbate. While other concentrations, (12.5 ng/μL and 50 ng/μL of ascorbate) were not statistically significant there appeared to be a trend of decreasing methylation which may have become more obvious had the cells been cultured with ascorbate for longer. Interestingly this effect was not seen at 100 ng/μL of ascorbate, indicating there is an optimal concentration range for TET-induced demethylation at the KCNQ1ot1 locus. In contrast, hydroquinone had no significant effect.
Ultimately, these results add to the current literature describing the effects of different culture media constituents on naïve embryonic stem cells. This may allow for the rational design of culture media that can improve both the efficiency and safety of naïve embryonic stem cells and embryos used in regenerative medicine, mammalian transgenics and IVF
DNA methylation memory: Understanding epigenetic reprogramming in vertebrates
No full textDNA methylation is an epigenetic mark critical for vertebrate development and is associated with numerous cellular and organismal processes including X-chromosome inactivation, genomic imprinting, and regulation of gene expression. Importantly, DNA methylation patterns are faithfully inherited during cell division, providing an information memory module additional to the DNA code. This mark, along with other epigenetic modifications, plays an essential role in establishing and maintaining cell identity. DNA methylation dynamics has been studied in detail in eutherian mammals, where two major waves of demethylation, the first in the early embryo and the second during germline development, remove most marks. Erasure of epigenetic memory is associated with cell reprogramming, and in mammals, is inextricably linked to increased developmental potency. For divergent vertebrate models, this dynamic is largely untested and indirect evidence suggests epigenetic memory may be retained in the germline. Furthermore, the role of epigenetic memory and reprogramming in major cell fate transitions, such as sex determination and sex change, is underexplored despite being apparently driven by epigenetic mechanisms in at least some species.
In order to understand how epigenetic memory is maintained, erased, and reprogrammed in divergent vertebrates, I have focused on two fish species. I have analysed the epigenome of the germline during gonad development in zebrafish (Danio rerio), and the transcriptome and methylome of bluehead wrasse (Thalassoma bifasciatum) during female-to-male sex change. Using a combination of techniques, including isolation of germline cells, whole genome bisulfite sequencing, and comparative epigenomics and transcriptomics, I explored epigenetic memory and reprogramming in these species. This thesis is presented as a collection of research and review papers, as well as a discussion synthesising my results
- …
