Limits...
Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

Paliwal A, Temkin AM, Kerkel K, Yale A, Yotova I, Drost N, Lax S, Nhan-Chang CL, Powell C, Borczuk A, Aviv A, Wapner R, Chen X, Nagy PL, Schork N, Do C, Torkamani A, Tycko B - PLoS Genet. (2013)

Bottom Line: Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb.Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR.Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Genetics, Columbia University Medical Center, New York, New York, United States of America.

ABSTRACT
Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.

Show MeSH
Local mapping of the imprinted C3orf27-RPN1 and VTRNA2-1 DMRs shows precise overlap with CTCF binding sites.A, Bis-seq of heterozygous placenta samples for amplicons immediately downstream of the RPN1 gene shows ASM localized to a discrete region of about 1 kb in length (chr3: 128,336,485–128,337,414) spanning a strong CTCF binding site and a CGI. Amplicons with ASM are colored red. B, Bis-seq of heterozygous PBL samples for amplicons surrounding the VTRNA2-1 vault RNA gene shows ASM localized to a 1.9 kb region (chr5: 135,414,670–135,416,821) spanning one CTCF binding site, two small CGIs, and the VTRNA2-1 RNA gene, while another CTCF binding site upstream is hypomethylated. ASM for both genes was evaluated visually and by T-tests on the percent methylation of individual clones, comparing the sets of clones for the two alleles.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3757050&req=5

pgen-1003622-g005: Local mapping of the imprinted C3orf27-RPN1 and VTRNA2-1 DMRs shows precise overlap with CTCF binding sites.A, Bis-seq of heterozygous placenta samples for amplicons immediately downstream of the RPN1 gene shows ASM localized to a discrete region of about 1 kb in length (chr3: 128,336,485–128,337,414) spanning a strong CTCF binding site and a CGI. Amplicons with ASM are colored red. B, Bis-seq of heterozygous PBL samples for amplicons surrounding the VTRNA2-1 vault RNA gene shows ASM localized to a 1.9 kb region (chr5: 135,414,670–135,416,821) spanning one CTCF binding site, two small CGIs, and the VTRNA2-1 RNA gene, while another CTCF binding site upstream is hypomethylated. ASM for both genes was evaluated visually and by T-tests on the percent methylation of individual clones, comparing the sets of clones for the two alleles.

Mentions: The most striking finding from this long-range analysis, and from additional intensive short-range mapping by Sanger bis-seq (Figs. 5 and 6A), is the discrete nature of all three DMRs; for all three regions, two with imprinted ASM and one with non-imprinted haplotype-dependent ASM, the allelic asymmetry in DNA methylation is restricted to one or two adjacent amplicons, with all three DMRs being less than 2 kb in length (Figs. 5 and 6 and Table 1). Within each of the regions examined, the flanking DNA outside of the DMRs showed varying levels of net CpG methylation, without asymmetry between the two alleles. Our mapping does not rule out additional DMRs farther away, but for the STEAP3-C2orf76 region, which shows haplotype-dependent ASM, any DMRs farther away would be in separate haplotype blocks and therefore be independently regulated domains. We additionally carried out short-range mapping of ASM around the ELK3 intragenic index fragment, which showed that it too is discrete and <2 kb in size (Figure S6).


Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

Paliwal A, Temkin AM, Kerkel K, Yale A, Yotova I, Drost N, Lax S, Nhan-Chang CL, Powell C, Borczuk A, Aviv A, Wapner R, Chen X, Nagy PL, Schork N, Do C, Torkamani A, Tycko B - PLoS Genet. (2013)

Local mapping of the imprinted C3orf27-RPN1 and VTRNA2-1 DMRs shows precise overlap with CTCF binding sites.A, Bis-seq of heterozygous placenta samples for amplicons immediately downstream of the RPN1 gene shows ASM localized to a discrete region of about 1 kb in length (chr3: 128,336,485–128,337,414) spanning a strong CTCF binding site and a CGI. Amplicons with ASM are colored red. B, Bis-seq of heterozygous PBL samples for amplicons surrounding the VTRNA2-1 vault RNA gene shows ASM localized to a 1.9 kb region (chr5: 135,414,670–135,416,821) spanning one CTCF binding site, two small CGIs, and the VTRNA2-1 RNA gene, while another CTCF binding site upstream is hypomethylated. ASM for both genes was evaluated visually and by T-tests on the percent methylation of individual clones, comparing the sets of clones for the two alleles.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757050&req=5

pgen-1003622-g005: Local mapping of the imprinted C3orf27-RPN1 and VTRNA2-1 DMRs shows precise overlap with CTCF binding sites.A, Bis-seq of heterozygous placenta samples for amplicons immediately downstream of the RPN1 gene shows ASM localized to a discrete region of about 1 kb in length (chr3: 128,336,485–128,337,414) spanning a strong CTCF binding site and a CGI. Amplicons with ASM are colored red. B, Bis-seq of heterozygous PBL samples for amplicons surrounding the VTRNA2-1 vault RNA gene shows ASM localized to a 1.9 kb region (chr5: 135,414,670–135,416,821) spanning one CTCF binding site, two small CGIs, and the VTRNA2-1 RNA gene, while another CTCF binding site upstream is hypomethylated. ASM for both genes was evaluated visually and by T-tests on the percent methylation of individual clones, comparing the sets of clones for the two alleles.
Mentions: The most striking finding from this long-range analysis, and from additional intensive short-range mapping by Sanger bis-seq (Figs. 5 and 6A), is the discrete nature of all three DMRs; for all three regions, two with imprinted ASM and one with non-imprinted haplotype-dependent ASM, the allelic asymmetry in DNA methylation is restricted to one or two adjacent amplicons, with all three DMRs being less than 2 kb in length (Figs. 5 and 6 and Table 1). Within each of the regions examined, the flanking DNA outside of the DMRs showed varying levels of net CpG methylation, without asymmetry between the two alleles. Our mapping does not rule out additional DMRs farther away, but for the STEAP3-C2orf76 region, which shows haplotype-dependent ASM, any DMRs farther away would be in separate haplotype blocks and therefore be independently regulated domains. We additionally carried out short-range mapping of ASM around the ELK3 intragenic index fragment, which showed that it too is discrete and <2 kb in size (Figure S6).

Bottom Line: Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb.Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR.Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Genetics, Columbia University Medical Center, New York, New York, United States of America.

ABSTRACT
Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.

Show MeSH