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Epigenetic Modification Agents Improve Gene-Specific Methylation Reprogramming in Porcine Cloned Embryos.

Huan Y, Wu Z, Zhang J, Zhu J, Liu Z, Song X - PLoS ONE (2015)

Bottom Line: In this study, we found that compared with in vitro fertilized counterparts, cloned embryos displayed the disrupted patterns of Oct4 demethylation and Thy1 remethylation.In conclusion, our results demonstrated that cloned embryos displayed incomplete gene-specific methylation reprogramming and disrupted expression patterns of pluripotency and tissue specific genes, and epigenetic modification agents improved gene-specific methylation reprogramming and expression pattern by regulating epigenetic modification related genes.This work would have important implications in improving cloning efficiency.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, Shandong Province, China.

ABSTRACT
Incomplete DNA methylation reprogramming in cloned embryos leads to poor cloning efficiency. Epigenetic modification agents can improve genomic methylation reprogramming and the development of cloned embryos, however, the effect of epigenetic modification agents on gene-specific methylation reprogramming remains poorly studied. Here, we investigated DNA methylation reprogramming of pluripotency (Oct4) and tissue specific (Thy1) genes during early embryo development in pigs. In this study, we found that compared with in vitro fertilized counterparts, cloned embryos displayed the disrupted patterns of Oct4 demethylation and Thy1 remethylation. When 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (TSA) enhanced the development of cloned embryos, the transcripts of DNA methyltransferases (Dnmt1 and Dnmt3a), histone acetyltransferase 1 (Hat1) and histone deacetylase 1 (Hdac1) and the methylation and expression patterns of Oct4 and Thy1 became similar to those detected in in vitro fertilized counterparts. Further studies showed that Dnmt1 knockdown in cloned embryos enhanced the methylation reprogramming of Oct4 and Thy1 and promoted the activation of Oct4 and the silence of Thy1. In conclusion, our results demonstrated that cloned embryos displayed incomplete gene-specific methylation reprogramming and disrupted expression patterns of pluripotency and tissue specific genes, and epigenetic modification agents improved gene-specific methylation reprogramming and expression pattern by regulating epigenetic modification related genes. This work would have important implications in improving cloning efficiency.

No MeSH data available.


Related in: MedlinePlus

Oct4 methylation statuses in early embryos.A, the methylation statuses of Oct4 at 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages of IVF, NT-CON, NT-AZA and NT-TSA embryos, B, the methylation levels of Oct4 in the IVF, NT-CON, NT-AZA and NT-TSA groups, and C, the methylation levels of Oct4 at different stages of IVF, NT-CON, NT-AZA and NT-TSA embryos. Cloned embryos displayed incomplete methylation reprogramming of Oct4, while 5-aza-dC or TSA rescued the disrupted methylation pattern of Oct4 in cloned embryos. Black or white circles indicate methylated or unmethylated CpG sites, respectively, and gray circles represent mutated and/or single nucleotide polymorphism (SNP) variation at certain CpG sites. a-dValues in the same group or at a given stage with different superscripts differ significantly (P<0.05).
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pone.0129803.g001: Oct4 methylation statuses in early embryos.A, the methylation statuses of Oct4 at 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages of IVF, NT-CON, NT-AZA and NT-TSA embryos, B, the methylation levels of Oct4 in the IVF, NT-CON, NT-AZA and NT-TSA groups, and C, the methylation levels of Oct4 at different stages of IVF, NT-CON, NT-AZA and NT-TSA embryos. Cloned embryos displayed incomplete methylation reprogramming of Oct4, while 5-aza-dC or TSA rescued the disrupted methylation pattern of Oct4 in cloned embryos. Black or white circles indicate methylated or unmethylated CpG sites, respectively, and gray circles represent mutated and/or single nucleotide polymorphism (SNP) variation at certain CpG sites. a-dValues in the same group or at a given stage with different superscripts differ significantly (P<0.05).

Mentions: Then, the methylation statuses of Oct4 and Thy1 were examined in IVF embryos (Figs 1 and 2). Oct4 showed a gradual and significant demethylation from the 1-cell to 4-cell stage (P<0.05) and maintained a low methylation status from the 4-cell to blastocyst stage, while the methylation pattern of Thy1 took on an upward trend from the 2-cell to blastocyst stage, and the methylation level of Thy1 in blastocysts was significantly higher that those in 2-cell and 4-cell embryos (P<0.05). Thus, IVF embryos displayed a low methylation status of Oct4 and a high methylation level of Thy1.


Epigenetic Modification Agents Improve Gene-Specific Methylation Reprogramming in Porcine Cloned Embryos.

Huan Y, Wu Z, Zhang J, Zhu J, Liu Z, Song X - PLoS ONE (2015)

Oct4 methylation statuses in early embryos.A, the methylation statuses of Oct4 at 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages of IVF, NT-CON, NT-AZA and NT-TSA embryos, B, the methylation levels of Oct4 in the IVF, NT-CON, NT-AZA and NT-TSA groups, and C, the methylation levels of Oct4 at different stages of IVF, NT-CON, NT-AZA and NT-TSA embryos. Cloned embryos displayed incomplete methylation reprogramming of Oct4, while 5-aza-dC or TSA rescued the disrupted methylation pattern of Oct4 in cloned embryos. Black or white circles indicate methylated or unmethylated CpG sites, respectively, and gray circles represent mutated and/or single nucleotide polymorphism (SNP) variation at certain CpG sites. a-dValues in the same group or at a given stage with different superscripts differ significantly (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465902&req=5

pone.0129803.g001: Oct4 methylation statuses in early embryos.A, the methylation statuses of Oct4 at 1-cell, 2-cell, 4-cell, 8-cell and blastocyst stages of IVF, NT-CON, NT-AZA and NT-TSA embryos, B, the methylation levels of Oct4 in the IVF, NT-CON, NT-AZA and NT-TSA groups, and C, the methylation levels of Oct4 at different stages of IVF, NT-CON, NT-AZA and NT-TSA embryos. Cloned embryos displayed incomplete methylation reprogramming of Oct4, while 5-aza-dC or TSA rescued the disrupted methylation pattern of Oct4 in cloned embryos. Black or white circles indicate methylated or unmethylated CpG sites, respectively, and gray circles represent mutated and/or single nucleotide polymorphism (SNP) variation at certain CpG sites. a-dValues in the same group or at a given stage with different superscripts differ significantly (P<0.05).
Mentions: Then, the methylation statuses of Oct4 and Thy1 were examined in IVF embryos (Figs 1 and 2). Oct4 showed a gradual and significant demethylation from the 1-cell to 4-cell stage (P<0.05) and maintained a low methylation status from the 4-cell to blastocyst stage, while the methylation pattern of Thy1 took on an upward trend from the 2-cell to blastocyst stage, and the methylation level of Thy1 in blastocysts was significantly higher that those in 2-cell and 4-cell embryos (P<0.05). Thus, IVF embryos displayed a low methylation status of Oct4 and a high methylation level of Thy1.

Bottom Line: In this study, we found that compared with in vitro fertilized counterparts, cloned embryos displayed the disrupted patterns of Oct4 demethylation and Thy1 remethylation.In conclusion, our results demonstrated that cloned embryos displayed incomplete gene-specific methylation reprogramming and disrupted expression patterns of pluripotency and tissue specific genes, and epigenetic modification agents improved gene-specific methylation reprogramming and expression pattern by regulating epigenetic modification related genes.This work would have important implications in improving cloning efficiency.

View Article: PubMed Central - PubMed

Affiliation: College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, Shandong Province, China.

ABSTRACT
Incomplete DNA methylation reprogramming in cloned embryos leads to poor cloning efficiency. Epigenetic modification agents can improve genomic methylation reprogramming and the development of cloned embryos, however, the effect of epigenetic modification agents on gene-specific methylation reprogramming remains poorly studied. Here, we investigated DNA methylation reprogramming of pluripotency (Oct4) and tissue specific (Thy1) genes during early embryo development in pigs. In this study, we found that compared with in vitro fertilized counterparts, cloned embryos displayed the disrupted patterns of Oct4 demethylation and Thy1 remethylation. When 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (TSA) enhanced the development of cloned embryos, the transcripts of DNA methyltransferases (Dnmt1 and Dnmt3a), histone acetyltransferase 1 (Hat1) and histone deacetylase 1 (Hdac1) and the methylation and expression patterns of Oct4 and Thy1 became similar to those detected in in vitro fertilized counterparts. Further studies showed that Dnmt1 knockdown in cloned embryos enhanced the methylation reprogramming of Oct4 and Thy1 and promoted the activation of Oct4 and the silence of Thy1. In conclusion, our results demonstrated that cloned embryos displayed incomplete gene-specific methylation reprogramming and disrupted expression patterns of pluripotency and tissue specific genes, and epigenetic modification agents improved gene-specific methylation reprogramming and expression pattern by regulating epigenetic modification related genes. This work would have important implications in improving cloning efficiency.

No MeSH data available.


Related in: MedlinePlus