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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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Allele-specific bias in H3K4me2 enrichment at the H19/Igf2 ICR in fetal germ cells.ChIP-SNuPE Sequenom assay results of H3K4me2-precipitated (A) 13.5 dpc and (B) 14.5 dpc fetal germ cell chromatin are shown. Other details are as in Figure 4.
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pgen-1001224-g005: Allele-specific bias in H3K4me2 enrichment at the H19/Igf2 ICR in fetal germ cells.ChIP-SNuPE Sequenom assay results of H3K4me2-precipitated (A) 13.5 dpc and (B) 14.5 dpc fetal germ cell chromatin are shown. Other details are as in Figure 4.

Mentions: We isolated male and control female germ cells from 13.5 and 14.5 gonads from the CS X OG2 mouse cross and performed ChIP-SNuPE assays using 100,000 germ cells per ChIP reaction. The control, nonspecific IgG-precipitated chromatin samples did not exhibit a clear pattern of allele-specific skewing (Figure S6B). The results did not show consistency between the −4 kb and −3 kb regions (A and B regions, respectively) or between the 13.5 and 14.5 dpc stages. Specific antibodies, on the other hand gave reproducible results using germ cell chromatin (Figure 4, Figure 5, Figure 6). CTCF binding was slightly biased toward the maternal ICR allele in male and female germ cells at 14.5 dpc (Figure 4). CTCF binding in the paternal allele would likely be inhibited by DNA methylation in PGCs similarly to somatic cells [23], [24], [27], but not in fetal prospermatogonia at 13.5–14.5 dpc in the lack of DNA methylation. The slight maternal bias is consistent with the possibility that allele-specific CTCF binding is not completely erased at 14.5 dpc, after DNA methylation erasure had been completed. The total level of CTCF binding at the ICR was very low in germ cells at 14.5 dpc compared to MEFs (Figure S7). This suggests that CTCF has been almost completely removed from both ICR alleles in germ cells by 14.5 dpc, consistent with biallelic Igf2 expression in the absence of insulation [31]–[33], [43]. The almost complete lack of CTCF binding, however is not due to the absence of CTCF from prospermatogonia at these stages. This would be expected based on that CTCF and CTCFL (BORIS) proteins exhibit mutually exclusive expression in adult male germ cells, round spermatids and spermatocytes, respectively [53] and that CTCFL is expressed in 14.5 dpc prospermatogonia [54]. It is not known whether CTCF is expressed in embryonic and fetal germ cells. We addressed this question by performing immunocytochemistry with anti-CTCF antibody using fetal germ cells (Figure S8). We found that CTCF staining in male and female germ cells was similar to that of control gonadal somatic cells at 12.5 dpc and 14.5 dpc. The mutually exclusive expression of CTCF and CTCFL, therefore, does not apply in germ cells at 14.5 dpc. CTCF may be inhibited to bind in the ICR at these stages because of changes in its covalent modifications [55], cofactors, or due to an RNA-dependent mechanism [56].


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)

Allele-specific bias in H3K4me2 enrichment at the H19/Igf2 ICR in fetal germ cells.ChIP-SNuPE Sequenom assay results of H3K4me2-precipitated (A) 13.5 dpc and (B) 14.5 dpc fetal germ cell chromatin are shown. Other details are as in Figure 4.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001224-g005: Allele-specific bias in H3K4me2 enrichment at the H19/Igf2 ICR in fetal germ cells.ChIP-SNuPE Sequenom assay results of H3K4me2-precipitated (A) 13.5 dpc and (B) 14.5 dpc fetal germ cell chromatin are shown. Other details are as in Figure 4.
Mentions: We isolated male and control female germ cells from 13.5 and 14.5 gonads from the CS X OG2 mouse cross and performed ChIP-SNuPE assays using 100,000 germ cells per ChIP reaction. The control, nonspecific IgG-precipitated chromatin samples did not exhibit a clear pattern of allele-specific skewing (Figure S6B). The results did not show consistency between the −4 kb and −3 kb regions (A and B regions, respectively) or between the 13.5 and 14.5 dpc stages. Specific antibodies, on the other hand gave reproducible results using germ cell chromatin (Figure 4, Figure 5, Figure 6). CTCF binding was slightly biased toward the maternal ICR allele in male and female germ cells at 14.5 dpc (Figure 4). CTCF binding in the paternal allele would likely be inhibited by DNA methylation in PGCs similarly to somatic cells [23], [24], [27], but not in fetal prospermatogonia at 13.5–14.5 dpc in the lack of DNA methylation. The slight maternal bias is consistent with the possibility that allele-specific CTCF binding is not completely erased at 14.5 dpc, after DNA methylation erasure had been completed. The total level of CTCF binding at the ICR was very low in germ cells at 14.5 dpc compared to MEFs (Figure S7). This suggests that CTCF has been almost completely removed from both ICR alleles in germ cells by 14.5 dpc, consistent with biallelic Igf2 expression in the absence of insulation [31]–[33], [43]. The almost complete lack of CTCF binding, however is not due to the absence of CTCF from prospermatogonia at these stages. This would be expected based on that CTCF and CTCFL (BORIS) proteins exhibit mutually exclusive expression in adult male germ cells, round spermatids and spermatocytes, respectively [53] and that CTCFL is expressed in 14.5 dpc prospermatogonia [54]. It is not known whether CTCF is expressed in embryonic and fetal germ cells. We addressed this question by performing immunocytochemistry with anti-CTCF antibody using fetal germ cells (Figure S8). We found that CTCF staining in male and female germ cells was similar to that of control gonadal somatic cells at 12.5 dpc and 14.5 dpc. The mutually exclusive expression of CTCF and CTCFL, therefore, does not apply in germ cells at 14.5 dpc. CTCF may be inhibited to bind in the ICR at these stages because of changes in its covalent modifications [55], cofactors, or due to an RNA-dependent mechanism [56].

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