Limits...
Sequences sufficient for programming imprinted germline DNA methylation defined.

Park YJ, Herman H, Gao Y, Lindroth AM, Hu BY, Murphy PJ, Putnam JR, Soloway PD - PLoS ONE (2012)

Bottom Line: Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus.We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans.Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutritional Science and Food Management, Ewha Womans University, Seoul, Republic of Korea. park.yoonjung@gmail.com

ABSTRACT
Epigenetic marks are fundamental to normal development, but little is known about signals that dictate their placement. Insights have been provided by studies of imprinted loci in mammals, where monoallelic expression is epigenetically controlled. Imprinted expression is regulated by DNA methylation programmed during gametogenesis in a sex-specific manner and maintained after fertilization. At Rasgrf1 in mouse, paternal-specific DNA methylation on a differential methylation domain (DMD) requires downstream tandem repeats. The DMD and repeats constitute a binary switch regulating paternal-specific expression. Here, we define sequences sufficient for imprinted methylation using two transgenic mouse lines: One carries the entire Rasgrf1 cluster (RC); the second carries only the DMD and repeats (DR) from Rasgrf1. The RC transgene recapitulated all aspects of imprinting seen at the endogenous locus. DR underwent proper DNA methylation establishment in sperm and erasure in oocytes, indicating the DMD and repeats are sufficient to program imprinted DNA methylation in germlines. Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus. We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans. Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization. When DR was paternally transmitted followed by maternal transmission, the unmethylated state that was properly established in the female germlines could not be maintained. This provides a model for transgenerational epigenetic inheritance in mice.

Show MeSH

Related in: MedlinePlus

Rasgrf1 expression is imprinted in neonatal brains of RC transgenic mice.Mice bearing the RC transgene were bred according the pedigree shown above the gel. Filled ♀ or ♂ symbols depict transgene positive mice, whereas unfilled symbols denote their non-transgenic mates. Progeny labeled 1 and 2 carry the transgene. To assay Rasgrf1 expression from the FVB/N or C57BL/6 alleles, we digested a 356 nt RT-PCR amplicon with AciI, which produced the distinct fragment sizes from the two alleles as shown below the gel. The endogenous FVB/N allele was expressed in all mice, whereas the C57BL/6 transgenic RC allele was expressed only upon paternal transmission. Inbred FVB/N (F) and C57BL/6 (B) were used as controls. M, marker; NTC, no template control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3293921&req=5

pone-0033024-g002: Rasgrf1 expression is imprinted in neonatal brains of RC transgenic mice.Mice bearing the RC transgene were bred according the pedigree shown above the gel. Filled ♀ or ♂ symbols depict transgene positive mice, whereas unfilled symbols denote their non-transgenic mates. Progeny labeled 1 and 2 carry the transgene. To assay Rasgrf1 expression from the FVB/N or C57BL/6 alleles, we digested a 356 nt RT-PCR amplicon with AciI, which produced the distinct fragment sizes from the two alleles as shown below the gel. The endogenous FVB/N allele was expressed in all mice, whereas the C57BL/6 transgenic RC allele was expressed only upon paternal transmission. Inbred FVB/N (F) and C57BL/6 (B) were used as controls. M, marker; NTC, no template control.

Mentions: To determine if the BAC carried sequences necessary for imprinted Rasgrf1 expression, RT-PCR was performed using RNA from brains of neonates with maternally or paternally transmitted RC transgenes. Neonatal brain is where Rasgrf1 shows imprinted expression. C57BL/6 and FVB/N have SNPs in the RT-PCR amplicon that spans exon 13 to 15, allowing us to distinguish Rasgrf1 expression from the endogenous FVB/N and C57BL/6 transgenic alleles, after AciI digestion of the RT-PCR product. Analysis of neonatal brain showed the RC transgene expressed Rasgrf1 only when transmitted through male germline, and was silenced upon maternal transmission (Fig. 2). Quantitative RT-PCR showed that mice with a paternally inherited RC transgene had 3.5 fold more Rasgrf1 expression relative to mice with a maternally inherited RC transgene, which is proportional to our estimate that there are three to five copies of the integrated transgene (data not shown). This demonstrates the RC transgenic line recapitulates the imprinting pattern and the level of expression per copy, of the endogenous locus, and that all regulatory elements necessary for proper imprinted expression are contained on the 242 kb transgene.


Sequences sufficient for programming imprinted germline DNA methylation defined.

Park YJ, Herman H, Gao Y, Lindroth AM, Hu BY, Murphy PJ, Putnam JR, Soloway PD - PLoS ONE (2012)

Rasgrf1 expression is imprinted in neonatal brains of RC transgenic mice.Mice bearing the RC transgene were bred according the pedigree shown above the gel. Filled ♀ or ♂ symbols depict transgene positive mice, whereas unfilled symbols denote their non-transgenic mates. Progeny labeled 1 and 2 carry the transgene. To assay Rasgrf1 expression from the FVB/N or C57BL/6 alleles, we digested a 356 nt RT-PCR amplicon with AciI, which produced the distinct fragment sizes from the two alleles as shown below the gel. The endogenous FVB/N allele was expressed in all mice, whereas the C57BL/6 transgenic RC allele was expressed only upon paternal transmission. Inbred FVB/N (F) and C57BL/6 (B) were used as controls. M, marker; NTC, no template control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293921&req=5

pone-0033024-g002: Rasgrf1 expression is imprinted in neonatal brains of RC transgenic mice.Mice bearing the RC transgene were bred according the pedigree shown above the gel. Filled ♀ or ♂ symbols depict transgene positive mice, whereas unfilled symbols denote their non-transgenic mates. Progeny labeled 1 and 2 carry the transgene. To assay Rasgrf1 expression from the FVB/N or C57BL/6 alleles, we digested a 356 nt RT-PCR amplicon with AciI, which produced the distinct fragment sizes from the two alleles as shown below the gel. The endogenous FVB/N allele was expressed in all mice, whereas the C57BL/6 transgenic RC allele was expressed only upon paternal transmission. Inbred FVB/N (F) and C57BL/6 (B) were used as controls. M, marker; NTC, no template control.
Mentions: To determine if the BAC carried sequences necessary for imprinted Rasgrf1 expression, RT-PCR was performed using RNA from brains of neonates with maternally or paternally transmitted RC transgenes. Neonatal brain is where Rasgrf1 shows imprinted expression. C57BL/6 and FVB/N have SNPs in the RT-PCR amplicon that spans exon 13 to 15, allowing us to distinguish Rasgrf1 expression from the endogenous FVB/N and C57BL/6 transgenic alleles, after AciI digestion of the RT-PCR product. Analysis of neonatal brain showed the RC transgene expressed Rasgrf1 only when transmitted through male germline, and was silenced upon maternal transmission (Fig. 2). Quantitative RT-PCR showed that mice with a paternally inherited RC transgene had 3.5 fold more Rasgrf1 expression relative to mice with a maternally inherited RC transgene, which is proportional to our estimate that there are three to five copies of the integrated transgene (data not shown). This demonstrates the RC transgenic line recapitulates the imprinting pattern and the level of expression per copy, of the endogenous locus, and that all regulatory elements necessary for proper imprinted expression are contained on the 242 kb transgene.

Bottom Line: Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus.We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans.Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutritional Science and Food Management, Ewha Womans University, Seoul, Republic of Korea. park.yoonjung@gmail.com

ABSTRACT
Epigenetic marks are fundamental to normal development, but little is known about signals that dictate their placement. Insights have been provided by studies of imprinted loci in mammals, where monoallelic expression is epigenetically controlled. Imprinted expression is regulated by DNA methylation programmed during gametogenesis in a sex-specific manner and maintained after fertilization. At Rasgrf1 in mouse, paternal-specific DNA methylation on a differential methylation domain (DMD) requires downstream tandem repeats. The DMD and repeats constitute a binary switch regulating paternal-specific expression. Here, we define sequences sufficient for imprinted methylation using two transgenic mouse lines: One carries the entire Rasgrf1 cluster (RC); the second carries only the DMD and repeats (DR) from Rasgrf1. The RC transgene recapitulated all aspects of imprinting seen at the endogenous locus. DR underwent proper DNA methylation establishment in sperm and erasure in oocytes, indicating the DMD and repeats are sufficient to program imprinted DNA methylation in germlines. Both transgenes produce a DMD-spanning pit-RNA, previously shown to be necessary for imprinted DNA methylation at the endogenous locus. We show that when pit-RNA expression is controlled by the repeats, it regulates DNA methylation in cis only and not in trans. Interestingly, pedigree history dictated whether established DR methylation patterns were maintained after fertilization. When DR was paternally transmitted followed by maternal transmission, the unmethylated state that was properly established in the female germlines could not be maintained. This provides a model for transgenerational epigenetic inheritance in mice.

Show MeSH
Related in: MedlinePlus