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Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells

Epigenetics have essential roles in development and human diseases. Compared to the complex histone modifications, epigenetic changes on mammalian DNA are as simple as methylation on cytosine. Guanine, however, can be oxidized as an epigenetic change which can undergo base-pair transversion, causing...

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Autores principales: Park, Joonghoon, Park, Jong Woo, Oh, Hawmok, Maria, Fernanda S., Kang, Jaeku, Tian, Xiuchun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889044/
https://www.ncbi.nlm.nih.gov/pubmed/27249188
http://dx.doi.org/10.1371/journal.pone.0155792
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author Park, Joonghoon
Park, Jong Woo
Oh, Hawmok
Maria, Fernanda S.
Kang, Jaeku
Tian, Xiuchun
author_facet Park, Joonghoon
Park, Jong Woo
Oh, Hawmok
Maria, Fernanda S.
Kang, Jaeku
Tian, Xiuchun
author_sort Park, Joonghoon
collection PubMed
description Epigenetics have essential roles in development and human diseases. Compared to the complex histone modifications, epigenetic changes on mammalian DNA are as simple as methylation on cytosine. Guanine, however, can be oxidized as an epigenetic change which can undergo base-pair transversion, causing a genetic difference. Accumulating evidence indicates that reactive oxygen species (ROS) are important signaling molecules for embryonic stem cell (ESC) differentiation, possibly through transient changes on genomic DNA such as 7,8-dihydro-8-oxoguanine (8-oxoG). Technical limitations on detecting such DNA modifications, however, restrict the investigation of the role of 8-oxoG in ESC differentiation. Here, we developed a Hoogsteen base pairing-mediated PCR-sequencing assay to detect 8-oxoG lesions that can subsequently cause G to T transversions during PCR. We then used this assay to assess the epigenetic and transient 8-oxoG formation in the Tbx5 gene of R1 mouse ESCs subjected to oxidative stress by removing 2-mercaptoethanol (2ME) from the culture media. To our surprise, significantly higher numbers of 8-oxoG-mediated G∙C to C∙G transversion, not G∙C to T∙A, were detected at 7(th) and 9(th) base position from the transcription start site of exon 1 of Tbx5 in ESCs in the (-)2ME than (+)2ME group (p < 0.05). This was consistent with the decrease in the amount of amplifiable of DNA harboring the 8-oxoG lesions at the Tbx5 promoter region in the oxidative stressed ESCs. The ESCs responded to oxidative stress, possibly through the epigenetic effects of guanine oxidation with decreased proliferation (p < 0.05) and increased formation of beating embryoid bodies (EBs; p < 0.001). Additionally, the epigenetic changes of guanine induced up-regulation of Ogg1 and PolB, two base excision repairing genes for 8-oxoG, in ESCs treated with (-)2ME (p < 0.01). Together, we developed a gene-specific and direct quantification assay for guanine oxidation. Using oxidative stressed mouse ESCs, we validated this assay and assessed the epigenetic effects of 8-oxoG by studying expression of DNA repair genes, ESC proliferation, and EB formation.
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spelling pubmed-48890442016-06-10 Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells Park, Joonghoon Park, Jong Woo Oh, Hawmok Maria, Fernanda S. Kang, Jaeku Tian, Xiuchun PLoS One Research Article Epigenetics have essential roles in development and human diseases. Compared to the complex histone modifications, epigenetic changes on mammalian DNA are as simple as methylation on cytosine. Guanine, however, can be oxidized as an epigenetic change which can undergo base-pair transversion, causing a genetic difference. Accumulating evidence indicates that reactive oxygen species (ROS) are important signaling molecules for embryonic stem cell (ESC) differentiation, possibly through transient changes on genomic DNA such as 7,8-dihydro-8-oxoguanine (8-oxoG). Technical limitations on detecting such DNA modifications, however, restrict the investigation of the role of 8-oxoG in ESC differentiation. Here, we developed a Hoogsteen base pairing-mediated PCR-sequencing assay to detect 8-oxoG lesions that can subsequently cause G to T transversions during PCR. We then used this assay to assess the epigenetic and transient 8-oxoG formation in the Tbx5 gene of R1 mouse ESCs subjected to oxidative stress by removing 2-mercaptoethanol (2ME) from the culture media. To our surprise, significantly higher numbers of 8-oxoG-mediated G∙C to C∙G transversion, not G∙C to T∙A, were detected at 7(th) and 9(th) base position from the transcription start site of exon 1 of Tbx5 in ESCs in the (-)2ME than (+)2ME group (p < 0.05). This was consistent with the decrease in the amount of amplifiable of DNA harboring the 8-oxoG lesions at the Tbx5 promoter region in the oxidative stressed ESCs. The ESCs responded to oxidative stress, possibly through the epigenetic effects of guanine oxidation with decreased proliferation (p < 0.05) and increased formation of beating embryoid bodies (EBs; p < 0.001). Additionally, the epigenetic changes of guanine induced up-regulation of Ogg1 and PolB, two base excision repairing genes for 8-oxoG, in ESCs treated with (-)2ME (p < 0.01). Together, we developed a gene-specific and direct quantification assay for guanine oxidation. Using oxidative stressed mouse ESCs, we validated this assay and assessed the epigenetic effects of 8-oxoG by studying expression of DNA repair genes, ESC proliferation, and EB formation. Public Library of Science 2016-06-01 /pmc/articles/PMC4889044/ /pubmed/27249188 http://dx.doi.org/10.1371/journal.pone.0155792 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication.
spellingShingle Research Article
Park, Joonghoon
Park, Jong Woo
Oh, Hawmok
Maria, Fernanda S.
Kang, Jaeku
Tian, Xiuchun
Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells
title Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells
title_full Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells
title_fullStr Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells
title_full_unstemmed Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells
title_short Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells
title_sort gene-specific assessment of guanine oxidation as an epigenetic modulator for cardiac specification of mouse embryonic stem cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889044/
https://www.ncbi.nlm.nih.gov/pubmed/27249188
http://dx.doi.org/10.1371/journal.pone.0155792
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