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...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2016-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4889044?pdf=render |
| _version_ | 1819100409280593920 |
|---|---|
| author | Joonghoon Park Jong Woo Park Hawmok Oh Fernanda S Maria Jaeku Kang Xiuchun Tian |
| author_facet | Joonghoon Park Jong Woo Park Hawmok Oh Fernanda S Maria Jaeku Kang Xiuchun Tian |
| author_sort | Joonghoon Park |
| collection | DOAJ |
| 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 7th and 9th 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. |
| first_indexed | 2024-12-22T01:02:19Z |
| format | Article |
| id | doaj.art-a5c36e9b3b7f440a895d4674a5bd6e2d |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-22T01:02:19Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-a5c36e9b3b7f440a895d4674a5bd6e2d2022-12-21T18:44:10ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01116e015579210.1371/journal.pone.0155792Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells.Joonghoon ParkJong Woo ParkHawmok OhFernanda S MariaJaeku KangXiuchun TianEpigenetics 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 7th and 9th 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.http://europepmc.org/articles/PMC4889044?pdf=render |
| spellingShingle | Joonghoon Park Jong Woo Park Hawmok Oh Fernanda S Maria Jaeku Kang Xiuchun Tian Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells. PLoS ONE |
| 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 |
| url | http://europepmc.org/articles/PMC4889044?pdf=render |
| work_keys_str_mv | AT joonghoonpark genespecificassessmentofguanineoxidationasanepigeneticmodulatorforcardiacspecificationofmouseembryonicstemcells AT jongwoopark genespecificassessmentofguanineoxidationasanepigeneticmodulatorforcardiacspecificationofmouseembryonicstemcells AT hawmokoh genespecificassessmentofguanineoxidationasanepigeneticmodulatorforcardiacspecificationofmouseembryonicstemcells AT fernandasmaria genespecificassessmentofguanineoxidationasanepigeneticmodulatorforcardiacspecificationofmouseembryonicstemcells AT jaekukang genespecificassessmentofguanineoxidationasanepigeneticmodulatorforcardiacspecificationofmouseembryonicstemcells AT xiuchuntian genespecificassessmentofguanineoxidationasanepigeneticmodulatorforcardiacspecificationofmouseembryonicstemcells |