Aleksandra Turp - Academia.edu (original) (raw)

Uploads

Papers by Aleksandra Turp

Pharmacy Growth, Research, Innovation and Training, Jul 4, 2018

Aleksandra Turp Christchurch School of Medicine Otago Medical School University of Otago Aleksand... more Aleksandra Turp Christchurch School of Medicine Otago Medical School University of Otago Aleksandra Turp is a fifth year medical student at Christchurch School of Medicine. She has a research background in epigenetics and maintains a keen interest in medical research. This essay has won the Robin Carrell prize for an innovative research proposal, awarded by the Department of Pathology at the University of Otago.

DNA methylation is an epigenetic modification important in many cellular processes such as mainte... more DNA methylation is an epigenetic modification important in many cellular processes such as maintenance of genome stability, establishment and maintenance of imprinting, transposon silencing, chromatin remodelling and control of gene expression. It is therefore important to understand how this modification is established and erased. We set out to develop a sensitive, liquid–chromatography mass–spectrometry method to measure global levels of DNA methylation (5mdC), as well as hydroxymethylation (5hmdC), a potential intermediate of DNA demethylation. With the new Agilent 6490 QQQ LC–MS we were able to detect as little as 50 amol of 5mdC and 5hmdC. We used this method to quantify levels of DNA methylation from DNA extracted from only 100 cells, allowing us to compare DNA methylation levels in early zygote development. Given that the evidence for DNA demethylation in early zygotes comes from methods using either antibody staining or bisulfite sequencing, this is the first direct demonstration that global DNA demethylation occurs in zygotes. Myoblast differentiation is a well-known model of DNA demethylation, where locus-specific DNA demethylation at the promoters and enhancer elements of myogenic regulatory factors, such as myogenin and MyoD, induce muscle specification and differentiation. It has been proposed, however, that the DNA demethylation process involves a large proportion of the genome and that it occurs in the absence of replication, indicating an active process. Therefore, in the second part of this work I set out to further investigate the global scale of epigenetic events associated with myoblast differentiation. Whilst some of the myoblast differentiation experiments showed a marked wave of DNA demethylation (up to 51%) others did not show any changes in DNA methylation level, showing that myoblast differentiation and DNA demethylation are not co-dependent. Addition of a DNA demethylating agent, 5-Aza-3'-deoxycytidine, to the growth medium of differentiating myoblast enhanced the differentiation proces [...]

Pharmacy Growth, Research, Innovation and Training, 2018

Nature cell biology, Jan 11, 2016

Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Tet... more Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Tet methylcytosine dioxygenase 3 (Tet3)-driven oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC; refs ,,,). Here we demonstrate using detailed immunofluorescence analysis and ultrasensitive LC-MS-based quantitative measurements that the initial loss of paternal 5mC does not require 5hmC formation. Small-molecule inhibition of Tet3 activity, as well as genetic ablation, impedes 5hmC accumulation in zygotes without affecting the early loss of paternal 5mC. Instead, 5hmC accumulation is dependent on the activity of zygotic Dnmt3a and Dnmt1, documenting a role for Tet3-driven hydroxylation in targeting de novo methylation activities present in the early embryo. Our data thus provide further insights into the dynamics of zygotic reprogramming, revealing an intricate interplay between DNA demethylation, de novo methylation and Tet3-driven hydroxylation.

DNA methylation is an epigenetic modification important in many cellular processes such as mainte... more DNA methylation is an epigenetic modification important in many cellular processes such as maintenance of genome stability, establishment and maintenance of imprinting, transposon silencing, chromatin remodelling and control of gene expression. It is therefore important to understand how this modification is established and erased. We set out to develop a sensitive, liquid–chromatography mass–spectrometry method to measure global levels of DNA methylation (5mdC), as well as hydroxymethylation (5hmdC), a potential intermediate of DNA demethylation. With the new Agilent 6490 QQQ LC–MS we were able to detect as little as 50 amol of 5mdC and 5hmdC. We used this method to quantify levels of DNA methylation from DNA extracted from only 100 cells, allowing us to compare DNA methylation levels in early zygote development. Given that the evidence for DNA demethylation in early zygotes comes from methods using either antibody staining or bisulfite sequencing, this is the first direct demonstration that global DNA demethylation occurs in zygotes. Myoblast differentiation is a well-known model of DNA demethylation, where locus-specific DNA demethylation at the promoters and enhancer elements of myogenic regulatory factors, such as myogenin and MyoD, induce muscle specification and differentiation. It has been proposed, however, that the DNA demethylation process involves a large proportion of the genome and that it occurs in the absence of replication, indicating an active process. Therefore, in the second part of this work I set out to further investigate the global scale of epigenetic events associated with myoblast differentiation. Whilst some of the myoblast differentiation experiments showed a marked wave of DNA demethylation (up to 51%) others did not show any changes in DNA methylation level, showing that myoblast differentiation and DNA demethylation are not co-dependent. Addition of a DNA demethylating agent, 5-Aza-3'-deoxycytidine, to the growth medium of differentiating myoblast enhanced the differentiation proces [...]

Nature Structural & Molecular Biology, 2013

Naïve pluripotent embryonic stem (ESCs) cells and embryonic germ (EGCs) cells are derived from th... more Naïve pluripotent embryonic stem (ESCs) cells and embryonic germ (EGCs) cells are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however we find that EGCs exhibit equivalent levels of global DNA methylation to ESCs. Importantly, inhibition of MEK and Gsk3b by 2i conditions leads to a pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instils a naïve pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs. ACCESSION CODES The microarray data are available in the Gene Expression Omnibus (GEO) database (http:// www.ncbi.nlm.nih.gov/gds) under the accession number GSE43398.

Pharmacy Growth, Research, Innovation and Training, Jul 4, 2018

Aleksandra Turp Christchurch School of Medicine Otago Medical School University of Otago Aleksand... more Aleksandra Turp Christchurch School of Medicine Otago Medical School University of Otago Aleksandra Turp is a fifth year medical student at Christchurch School of Medicine. She has a research background in epigenetics and maintains a keen interest in medical research. This essay has won the Robin Carrell prize for an innovative research proposal, awarded by the Department of Pathology at the University of Otago.

DNA methylation is an epigenetic modification important in many cellular processes such as mainte... more DNA methylation is an epigenetic modification important in many cellular processes such as maintenance of genome stability, establishment and maintenance of imprinting, transposon silencing, chromatin remodelling and control of gene expression. It is therefore important to understand how this modification is established and erased. We set out to develop a sensitive, liquid–chromatography mass–spectrometry method to measure global levels of DNA methylation (5mdC), as well as hydroxymethylation (5hmdC), a potential intermediate of DNA demethylation. With the new Agilent 6490 QQQ LC–MS we were able to detect as little as 50 amol of 5mdC and 5hmdC. We used this method to quantify levels of DNA methylation from DNA extracted from only 100 cells, allowing us to compare DNA methylation levels in early zygote development. Given that the evidence for DNA demethylation in early zygotes comes from methods using either antibody staining or bisulfite sequencing, this is the first direct demonstration that global DNA demethylation occurs in zygotes. Myoblast differentiation is a well-known model of DNA demethylation, where locus-specific DNA demethylation at the promoters and enhancer elements of myogenic regulatory factors, such as myogenin and MyoD, induce muscle specification and differentiation. It has been proposed, however, that the DNA demethylation process involves a large proportion of the genome and that it occurs in the absence of replication, indicating an active process. Therefore, in the second part of this work I set out to further investigate the global scale of epigenetic events associated with myoblast differentiation. Whilst some of the myoblast differentiation experiments showed a marked wave of DNA demethylation (up to 51%) others did not show any changes in DNA methylation level, showing that myoblast differentiation and DNA demethylation are not co-dependent. Addition of a DNA demethylating agent, 5-Aza-3'-deoxycytidine, to the growth medium of differentiating myoblast enhanced the differentiation proces [...]

Pharmacy Growth, Research, Innovation and Training, 2018

Nature cell biology, Jan 11, 2016

Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Tet... more Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Tet methylcytosine dioxygenase 3 (Tet3)-driven oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC; refs ,,,). Here we demonstrate using detailed immunofluorescence analysis and ultrasensitive LC-MS-based quantitative measurements that the initial loss of paternal 5mC does not require 5hmC formation. Small-molecule inhibition of Tet3 activity, as well as genetic ablation, impedes 5hmC accumulation in zygotes without affecting the early loss of paternal 5mC. Instead, 5hmC accumulation is dependent on the activity of zygotic Dnmt3a and Dnmt1, documenting a role for Tet3-driven hydroxylation in targeting de novo methylation activities present in the early embryo. Our data thus provide further insights into the dynamics of zygotic reprogramming, revealing an intricate interplay between DNA demethylation, de novo methylation and Tet3-driven hydroxylation.

DNA methylation is an epigenetic modification important in many cellular processes such as mainte... more DNA methylation is an epigenetic modification important in many cellular processes such as maintenance of genome stability, establishment and maintenance of imprinting, transposon silencing, chromatin remodelling and control of gene expression. It is therefore important to understand how this modification is established and erased. We set out to develop a sensitive, liquid–chromatography mass–spectrometry method to measure global levels of DNA methylation (5mdC), as well as hydroxymethylation (5hmdC), a potential intermediate of DNA demethylation. With the new Agilent 6490 QQQ LC–MS we were able to detect as little as 50 amol of 5mdC and 5hmdC. We used this method to quantify levels of DNA methylation from DNA extracted from only 100 cells, allowing us to compare DNA methylation levels in early zygote development. Given that the evidence for DNA demethylation in early zygotes comes from methods using either antibody staining or bisulfite sequencing, this is the first direct demonstration that global DNA demethylation occurs in zygotes. Myoblast differentiation is a well-known model of DNA demethylation, where locus-specific DNA demethylation at the promoters and enhancer elements of myogenic regulatory factors, such as myogenin and MyoD, induce muscle specification and differentiation. It has been proposed, however, that the DNA demethylation process involves a large proportion of the genome and that it occurs in the absence of replication, indicating an active process. Therefore, in the second part of this work I set out to further investigate the global scale of epigenetic events associated with myoblast differentiation. Whilst some of the myoblast differentiation experiments showed a marked wave of DNA demethylation (up to 51%) others did not show any changes in DNA methylation level, showing that myoblast differentiation and DNA demethylation are not co-dependent. Addition of a DNA demethylating agent, 5-Aza-3'-deoxycytidine, to the growth medium of differentiating myoblast enhanced the differentiation proces [...]

Nature Structural & Molecular Biology, 2013

Naïve pluripotent embryonic stem (ESCs) cells and embryonic germ (EGCs) cells are derived from th... more Naïve pluripotent embryonic stem (ESCs) cells and embryonic germ (EGCs) cells are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however we find that EGCs exhibit equivalent levels of global DNA methylation to ESCs. Importantly, inhibition of MEK and Gsk3b by 2i conditions leads to a pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instils a naïve pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs. ACCESSION CODES The microarray data are available in the Gene Expression Omnibus (GEO) database (http:// www.ncbi.nlm.nih.gov/gds) under the accession number GSE43398.