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Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting.

Suzuki S, Ono R, Narita T, Pask AJ, Shaw G, Wang C, Kohda T, Alsop AE, Marshall Graves JA, Kohara Y, Ishino F, Renfree MB, Kaneko-Ishino T - PLoS Genet. (2007)

Bottom Line: We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials.Surprisingly, the marsupial DMR was strictly limited to the 5' region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE.These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT
Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5' region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.

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DNA Methylation and Allelic Expression of PEG10 in 5-Aza-2′-deoxycytidine Treated Tammar Cells(A) The bars on the horizontal line represent each CpG site. Three CpG sites were examined for DNA methylation status using combined bisulphite restriction analysis indicated by the arrowheads. The intensity of the bands marked with the white and black circles show the amount of unmethylated and methylated CpG, respectively.(B) Repetitive experiments and quantification of the cut and uncut bands of combined bisulphite restriction analysis were performed for the most 3′ site indicated in Figure 4A. One black and two grey bars represent the results of positive control cells and of two independent 5-aza-2′-deoxycytidine treated cells, respectively. The decrease in methylation was statistically significant (**) (p < 0.01). Quantification of each samples was performed three times using independent PCR products.(C) Relative expression was calculated by quantifying the results of restriction fragment length polymorphism analysis. White and black bars represent the expression from active and inactive alleles of positive control cells, respectively. It should be noted that expression from the inactive alleles was negligible. Two grey bars represent induced expression from the inactive alleles of two independent 5-aza-2′-deoxycytidine treated cells. Statistically significant increase in expression after treatment is shown by * (p < 0.03) or ** (p < 0.01).
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pgen-0030055-g004: DNA Methylation and Allelic Expression of PEG10 in 5-Aza-2′-deoxycytidine Treated Tammar Cells(A) The bars on the horizontal line represent each CpG site. Three CpG sites were examined for DNA methylation status using combined bisulphite restriction analysis indicated by the arrowheads. The intensity of the bands marked with the white and black circles show the amount of unmethylated and methylated CpG, respectively.(B) Repetitive experiments and quantification of the cut and uncut bands of combined bisulphite restriction analysis were performed for the most 3′ site indicated in Figure 4A. One black and two grey bars represent the results of positive control cells and of two independent 5-aza-2′-deoxycytidine treated cells, respectively. The decrease in methylation was statistically significant (**) (p < 0.01). Quantification of each samples was performed three times using independent PCR products.(C) Relative expression was calculated by quantifying the results of restriction fragment length polymorphism analysis. White and black bars represent the expression from active and inactive alleles of positive control cells, respectively. It should be noted that expression from the inactive alleles was negligible. Two grey bars represent induced expression from the inactive alleles of two independent 5-aza-2′-deoxycytidine treated cells. Statistically significant increase in expression after treatment is shown by * (p < 0.03) or ** (p < 0.01).

Mentions: Even with the presence of a DMR, it is possible that the maternal copy of PEG10 in the tammar is silenced by another mechanism and is only secondarily methylated. We therefore examined whether the imprinted expression of tammar PEG10 was regulated by DNA methylation. A reduced level of DNA methylation was observed in three sites of the CpG island in cells cultured with 5-aza-2′-deoxycytidine, a DNA methylation inhibitor (Figure 4A). Repetitive experiments performed for the most 3′ site using three independent cell lines established from fetal lung and endometrium also showed statistically significant reductions in DNA methylation levels (Figure 4B), and increased PEG10 expression from normally repressed alleles was observed in each case (Figure 4C, black and grey bars), although the expression levels were still much lower than active alleles (Figure 4C, white bars). These results demonstrate the association between imprinted expression of PEG10 and DNA methylation in a marsupial, although it still remains unknown if the differential methylation originates in the germline as does a typical primary DMR in eutherians.


Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting.

Suzuki S, Ono R, Narita T, Pask AJ, Shaw G, Wang C, Kohda T, Alsop AE, Marshall Graves JA, Kohara Y, Ishino F, Renfree MB, Kaneko-Ishino T - PLoS Genet. (2007)

DNA Methylation and Allelic Expression of PEG10 in 5-Aza-2′-deoxycytidine Treated Tammar Cells(A) The bars on the horizontal line represent each CpG site. Three CpG sites were examined for DNA methylation status using combined bisulphite restriction analysis indicated by the arrowheads. The intensity of the bands marked with the white and black circles show the amount of unmethylated and methylated CpG, respectively.(B) Repetitive experiments and quantification of the cut and uncut bands of combined bisulphite restriction analysis were performed for the most 3′ site indicated in Figure 4A. One black and two grey bars represent the results of positive control cells and of two independent 5-aza-2′-deoxycytidine treated cells, respectively. The decrease in methylation was statistically significant (**) (p < 0.01). Quantification of each samples was performed three times using independent PCR products.(C) Relative expression was calculated by quantifying the results of restriction fragment length polymorphism analysis. White and black bars represent the expression from active and inactive alleles of positive control cells, respectively. It should be noted that expression from the inactive alleles was negligible. Two grey bars represent induced expression from the inactive alleles of two independent 5-aza-2′-deoxycytidine treated cells. Statistically significant increase in expression after treatment is shown by * (p < 0.03) or ** (p < 0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1851980&req=5

pgen-0030055-g004: DNA Methylation and Allelic Expression of PEG10 in 5-Aza-2′-deoxycytidine Treated Tammar Cells(A) The bars on the horizontal line represent each CpG site. Three CpG sites were examined for DNA methylation status using combined bisulphite restriction analysis indicated by the arrowheads. The intensity of the bands marked with the white and black circles show the amount of unmethylated and methylated CpG, respectively.(B) Repetitive experiments and quantification of the cut and uncut bands of combined bisulphite restriction analysis were performed for the most 3′ site indicated in Figure 4A. One black and two grey bars represent the results of positive control cells and of two independent 5-aza-2′-deoxycytidine treated cells, respectively. The decrease in methylation was statistically significant (**) (p < 0.01). Quantification of each samples was performed three times using independent PCR products.(C) Relative expression was calculated by quantifying the results of restriction fragment length polymorphism analysis. White and black bars represent the expression from active and inactive alleles of positive control cells, respectively. It should be noted that expression from the inactive alleles was negligible. Two grey bars represent induced expression from the inactive alleles of two independent 5-aza-2′-deoxycytidine treated cells. Statistically significant increase in expression after treatment is shown by * (p < 0.03) or ** (p < 0.01).
Mentions: Even with the presence of a DMR, it is possible that the maternal copy of PEG10 in the tammar is silenced by another mechanism and is only secondarily methylated. We therefore examined whether the imprinted expression of tammar PEG10 was regulated by DNA methylation. A reduced level of DNA methylation was observed in three sites of the CpG island in cells cultured with 5-aza-2′-deoxycytidine, a DNA methylation inhibitor (Figure 4A). Repetitive experiments performed for the most 3′ site using three independent cell lines established from fetal lung and endometrium also showed statistically significant reductions in DNA methylation levels (Figure 4B), and increased PEG10 expression from normally repressed alleles was observed in each case (Figure 4C, black and grey bars), although the expression levels were still much lower than active alleles (Figure 4C, white bars). These results demonstrate the association between imprinted expression of PEG10 and DNA methylation in a marsupial, although it still remains unknown if the differential methylation originates in the germline as does a typical primary DMR in eutherians.

Bottom Line: We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials.Surprisingly, the marsupial DMR was strictly limited to the 5' region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE.These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT
Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5' region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.

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