Sequences surrounding the transcription start sites of <i>Mef2C</i>, <i>Nkx2-5</i>, and <i>Gata4</i> genes in R1 mouse ESCs subjected to oxidative stress.

<p>Sequences surrounding the transcription start sites of <i>Mef2C</i>, <i>Nkx2-5</i>, and <i>Gata4</i> genes in R1 mouse ESCs subjected to oxidative stress.</p

G∙C to C∙G transversions at exon 1 of the <i>Tbx5</i> gene in R1 mouse ESCs subjected to oxidative stress.

<p>G∙C to C∙G transversions at exon 1 of the <i>Tbx5</i> gene in R1 mouse ESCs subjected to oxidative stress.</p

Occupancy of Ogg1 at the <i>Tbx5</i> promoter.

ChIP-qPCR results showed specific reduced occupancy by Ogg1 (white bars) at the Tbx5 promoter in oxidative stressed R1 mouse ESCs while occupancy by histone H3 did not change. Mean ± standard deviation (SD), ***p < 0.001, one-sided t-test.</p

Primer sequences for biomarker genes of cardiac specification.

Primer sequences for biomarker genes of cardiac specification.</p

Differential expression levels of 8-oxoG repair genes <i>Ogg1</i> and <i>PolB</i> in ESCs induced by oxidative stress.

<p>Relative mRNA levels of (A) <i>Ogg1</i> mRNA and (B) <i>PolB</i>. Gapdh was used as an internal control. Mean ± SD, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, one-sided t-test. (C) Levels of Ogg1 and PolB proteins by Western blotting. α-tubulin was used as an internal control.</p

Establishment of the Hoogsteen base pairing-mediated PCR-Sequencing assay for 8-oxoG lesions in DNA.

<p>(A) pUC19oxoG(+) was prepared by site-directed mutagenesis, (B) 8-oxoG lesion was identified by Hind III resistance, and (C) Hoogsteen base pairing-mediated 8-oxoG to T transversion. T4: T4 DNA ligase, HIII: Hind III restriction enzyme.</p

Effects of oxidative stress on R1 mouse ESC proliferation and differentiation.

<p>(A) Proliferation of ESCs after oxidative stress induced for two days by removing 2-mercaptoethanol (2ME) from ESC medium. *<i>p</i> < 0.05, Welch t-test. Means of fold changes with standard errors were presented. (B) Different EB morphologies from (+)2ME and (-)2ME media (bar = 100 μm) and (C) the percentages of beating EBs were observed from oxidative-stressed ESCs vs. controls (**<i>p</i> < 0.01, one-sided t-test).</p

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 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