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CTCF-dependent chromatin bias constitutes transient epigenetic memory of the mother at the H19-Igf2 imprinting control region in prospermatogonia.

Lee DH, Singh P, Tsai SY, Oates N, Spalla A, Spalla C, Brown L, Rivas G, Larson G, Rauch TA, Pfeifer GP, Szabó PE - PLoS Genet. (2010)

Bottom Line: We found that the chromatin composition in fetal germ cells was biased at the ICR between the two alleles with the maternally inherited allele exhibiting more H3K4me3 and less H3K9me3 than the paternally inherited allele.The erasure of these allele-specific chromatin marks is not complete before the process of de novo methylation imprint establishment begins.CTCF-dependent allele-specific chromatin composition imposes a maternal allele-specific delay on de novo methylation imprint establishment at the H19/Igf2 ICR in prospermatogonia.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute, Duarte, CA, USA.

ABSTRACT
Genomic imprints-parental allele-specific DNA methylation marks at the differentially methylated regions (DMRs) of imprinted genes-are erased and reestablished in germ cells according to the individual's sex. Imprint establishment at paternally methylated germ line DMRs occurs in fetal male germ cells. In prospermatogonia, the two unmethylated alleles exhibit different rates of de novo methylation at the H19/Igf2 imprinting control region (ICR) depending on parental origin. We investigated the nature of this epigenetic memory using bisulfite sequencing and allele-specific ChIP-SNuPE assays. We found that the chromatin composition in fetal germ cells was biased at the ICR between the two alleles with the maternally inherited allele exhibiting more H3K4me3 and less H3K9me3 than the paternally inherited allele. We determined genetically that the chromatin bias, and also the delayed methylation establishment in the maternal allele, depended on functional CTCF insulator binding sites in the ICR. Our data suggest that, in primordial germ cells, maternally inherited allele-specific CTCF binding sets up allele-specific chromatin differences at the ICR. The erasure of these allele-specific chromatin marks is not complete before the process of de novo methylation imprint establishment begins. CTCF-dependent allele-specific chromatin composition imposes a maternal allele-specific delay on de novo methylation imprint establishment at the H19/Igf2 ICR in prospermatogonia.

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Methylation dynamics at the ICR in normal prospermatogonia.Bisulfite sequencing results are shown at fetal stages (in dpc). Prospermatogonia of CS X OG2 fetuses were analyzed. Unmethylated CpGs (white squares) and methylated CpGs (black squares) are shown along independent chromosomes (horizontal lines). Groups of chromosomes were derived from the same bisulfite reaction. CTCF sires 1 and 2 of the ICR are included in the analyzed region. CpG site 8 is polymorphic and is only present in the CS type allele. The percentage of methylated CpGs (methylated CpG/total CpG) at each developmental stage is indicated for each allele.
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pgen-1001224-g002: Methylation dynamics at the ICR in normal prospermatogonia.Bisulfite sequencing results are shown at fetal stages (in dpc). Prospermatogonia of CS X OG2 fetuses were analyzed. Unmethylated CpGs (white squares) and methylated CpGs (black squares) are shown along independent chromosomes (horizontal lines). Groups of chromosomes were derived from the same bisulfite reaction. CTCF sires 1 and 2 of the ICR are included in the analyzed region. CpG site 8 is polymorphic and is only present in the CS type allele. The percentage of methylated CpGs (methylated CpG/total CpG) at each developmental stage is indicated for each allele.

Mentions: In prospermatogonia, the paternally inherited ICR allele becomes methylated earlier than the maternally inherited allele in reciprocal crosses between C57BL/6J (B6) and JF1 [39], [41]. Similarly, when the ICR carries the B6 type allele in the maternal allele and the CAST/Ei type allele in the paternal allele, the B6 type maternal allele is delayed compared to the CAST/Ei type paternal allele in prospermatogonia between 14.5 and 18.5 dpc [37]. We tested the reciprocal situation when the CAST/Ei type ICR allele is inherited from the mother and the B6 type allele is inherited from the father. Females of FVB/NJ.CAST/Ei(N7), a distal chromosome 7 partial congenic strain for CAST/Ei (CS) [31] were mated with TgOG2 homozygous transgenic males [43] resulting in CS X OG2 fetuses. We isolated male and female germ cells from 13.5, 14.5, 15.5, 16.5 and 17.5 dpc gonads. We performed two or more independent bisulfite conversion reactions for each sample and sequenced at least twelve clones of each sample. A single nucleotide polymorphism in the CS strain was used to identify the parental alleles. We confirmed previous observations [37]–[40] that DNA methylation erasure is complete by 13.5–14.5 dpc at the ICR (Figure 2 and Figure S3). We found that primary oocytes exhibited no methylation of the ICR region between 13.5 and 16.5 dpc (Figure S3A) and prospermatogonia attained CpG methylation gradually (Figure 2) between 15.5 dpc and 17.5 dpc as expected [37], [39]. We confirmed that the maternal allele (CS type) was delayed compared to the paternal allele (B6 type) in CS X OG2 prospermatogonia (Figure 2) similar to the reciprocal B6 X CS situation [37]. Regardless of mouse strains used, there exists a time gap in methylation imprint establishment between the two chromosomes depending on the inheritance from the mother or father (M and P alleles in Figure 1) during spermatogenesis [37], [39], [41], [42]. Therefore, the two parental alleles must be distinguished from each other in 13.5–14.5 dpc prospermatogonia by epigenetic means other than DNA methylation.


CTCF-dependent chromatin bias constitutes transient epigenetic memory of the mother at the H19-Igf2 imprinting control region in prospermatogonia.

Lee DH, Singh P, Tsai SY, Oates N, Spalla A, Spalla C, Brown L, Rivas G, Larson G, Rauch TA, Pfeifer GP, Szabó PE - PLoS Genet. (2010)

Methylation dynamics at the ICR in normal prospermatogonia.Bisulfite sequencing results are shown at fetal stages (in dpc). Prospermatogonia of CS X OG2 fetuses were analyzed. Unmethylated CpGs (white squares) and methylated CpGs (black squares) are shown along independent chromosomes (horizontal lines). Groups of chromosomes were derived from the same bisulfite reaction. CTCF sires 1 and 2 of the ICR are included in the analyzed region. CpG site 8 is polymorphic and is only present in the CS type allele. The percentage of methylated CpGs (methylated CpG/total CpG) at each developmental stage is indicated for each allele.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991272&req=5

pgen-1001224-g002: Methylation dynamics at the ICR in normal prospermatogonia.Bisulfite sequencing results are shown at fetal stages (in dpc). Prospermatogonia of CS X OG2 fetuses were analyzed. Unmethylated CpGs (white squares) and methylated CpGs (black squares) are shown along independent chromosomes (horizontal lines). Groups of chromosomes were derived from the same bisulfite reaction. CTCF sires 1 and 2 of the ICR are included in the analyzed region. CpG site 8 is polymorphic and is only present in the CS type allele. The percentage of methylated CpGs (methylated CpG/total CpG) at each developmental stage is indicated for each allele.
Mentions: In prospermatogonia, the paternally inherited ICR allele becomes methylated earlier than the maternally inherited allele in reciprocal crosses between C57BL/6J (B6) and JF1 [39], [41]. Similarly, when the ICR carries the B6 type allele in the maternal allele and the CAST/Ei type allele in the paternal allele, the B6 type maternal allele is delayed compared to the CAST/Ei type paternal allele in prospermatogonia between 14.5 and 18.5 dpc [37]. We tested the reciprocal situation when the CAST/Ei type ICR allele is inherited from the mother and the B6 type allele is inherited from the father. Females of FVB/NJ.CAST/Ei(N7), a distal chromosome 7 partial congenic strain for CAST/Ei (CS) [31] were mated with TgOG2 homozygous transgenic males [43] resulting in CS X OG2 fetuses. We isolated male and female germ cells from 13.5, 14.5, 15.5, 16.5 and 17.5 dpc gonads. We performed two or more independent bisulfite conversion reactions for each sample and sequenced at least twelve clones of each sample. A single nucleotide polymorphism in the CS strain was used to identify the parental alleles. We confirmed previous observations [37]–[40] that DNA methylation erasure is complete by 13.5–14.5 dpc at the ICR (Figure 2 and Figure S3). We found that primary oocytes exhibited no methylation of the ICR region between 13.5 and 16.5 dpc (Figure S3A) and prospermatogonia attained CpG methylation gradually (Figure 2) between 15.5 dpc and 17.5 dpc as expected [37], [39]. We confirmed that the maternal allele (CS type) was delayed compared to the paternal allele (B6 type) in CS X OG2 prospermatogonia (Figure 2) similar to the reciprocal B6 X CS situation [37]. Regardless of mouse strains used, there exists a time gap in methylation imprint establishment between the two chromosomes depending on the inheritance from the mother or father (M and P alleles in Figure 1) during spermatogenesis [37], [39], [41], [42]. Therefore, the two parental alleles must be distinguished from each other in 13.5–14.5 dpc prospermatogonia by epigenetic means other than DNA methylation.

Bottom Line: We found that the chromatin composition in fetal germ cells was biased at the ICR between the two alleles with the maternally inherited allele exhibiting more H3K4me3 and less H3K9me3 than the paternally inherited allele.The erasure of these allele-specific chromatin marks is not complete before the process of de novo methylation imprint establishment begins.CTCF-dependent allele-specific chromatin composition imposes a maternal allele-specific delay on de novo methylation imprint establishment at the H19/Igf2 ICR in prospermatogonia.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute, Duarte, CA, USA.

ABSTRACT
Genomic imprints-parental allele-specific DNA methylation marks at the differentially methylated regions (DMRs) of imprinted genes-are erased and reestablished in germ cells according to the individual's sex. Imprint establishment at paternally methylated germ line DMRs occurs in fetal male germ cells. In prospermatogonia, the two unmethylated alleles exhibit different rates of de novo methylation at the H19/Igf2 imprinting control region (ICR) depending on parental origin. We investigated the nature of this epigenetic memory using bisulfite sequencing and allele-specific ChIP-SNuPE assays. We found that the chromatin composition in fetal germ cells was biased at the ICR between the two alleles with the maternally inherited allele exhibiting more H3K4me3 and less H3K9me3 than the paternally inherited allele. We determined genetically that the chromatin bias, and also the delayed methylation establishment in the maternal allele, depended on functional CTCF insulator binding sites in the ICR. Our data suggest that, in primordial germ cells, maternally inherited allele-specific CTCF binding sets up allele-specific chromatin differences at the ICR. The erasure of these allele-specific chromatin marks is not complete before the process of de novo methylation imprint establishment begins. CTCF-dependent allele-specific chromatin composition imposes a maternal allele-specific delay on de novo methylation imprint establishment at the H19/Igf2 ICR in prospermatogonia.

Show MeSH