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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.

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Bis-seq showing strong ASM in the ELK3, STEAP3-C2orf76, C3orf27-RPN1, and VTRNA2-1 index regions.A, Gene map and bis-seq of the index amplicon containing SNP rs2302902 in the first intron of the ELK3 gene. The strong asymmetry in CpG methylation, with the G allele consistently hypermethylated in the PBL samples, indicates haplotype-dependent non-imprinted ASM (additional data in Table 1; haplotype map in Figure S5). There is some tissue-specificity, with biallelic hypermethylation in the HMEC sample. The grey (lower) bars indicate the index amplicons for initial bis-seq, each tagged by the index SNP that showed recurrent ASM in the MSNP data. The green bars indicate CGIs and the black rectangles are exons. The X's indicate polymorphic CpG sites. B, Gene map and bis-seq of the index amplicon containing SNP rs1530562, between the STEAP3 and C2orf76 genes in chromosome band 2q14, showing strong ASM with the G allele consistently hypermethylated in multiple tissues consistent with haplotype-dependent non-imprinted ASM, but with biallelic hypermethylation in sperm DNA (additional data in Table 1). C, Gene map and bis-seq of the index amplicon containing SNP rs2811488 located downstream of the last exon of the RPN1 gene in chromosome band 3q21, showing strong ASM in placenta, with the G allele or A allele hypermethylated, depending on parent-of-origin, as proven in Figure 2. For this imprinted DMR the ASM is highly tissue-specific, being seen in placenta but not in PBL, liver, lung, brain, HMEC or sperm (additional data in Table 1 and Figure S2). D, Gene map and bilsufite sequencing of the index amplicon containing SNP rs2346019 downstream of the VTRNA2-1 vault-family RNA, located in chromosome band 5q31. Strong ASM is observed in multiple tissues with the A allele or G allele methylated, consistent with imprinting, which is proven by the data in Figure 2.
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pgen-1003622-g001: Bis-seq showing strong ASM in the ELK3, STEAP3-C2orf76, C3orf27-RPN1, and VTRNA2-1 index regions.A, Gene map and bis-seq of the index amplicon containing SNP rs2302902 in the first intron of the ELK3 gene. The strong asymmetry in CpG methylation, with the G allele consistently hypermethylated in the PBL samples, indicates haplotype-dependent non-imprinted ASM (additional data in Table 1; haplotype map in Figure S5). There is some tissue-specificity, with biallelic hypermethylation in the HMEC sample. The grey (lower) bars indicate the index amplicons for initial bis-seq, each tagged by the index SNP that showed recurrent ASM in the MSNP data. The green bars indicate CGIs and the black rectangles are exons. The X's indicate polymorphic CpG sites. B, Gene map and bis-seq of the index amplicon containing SNP rs1530562, between the STEAP3 and C2orf76 genes in chromosome band 2q14, showing strong ASM with the G allele consistently hypermethylated in multiple tissues consistent with haplotype-dependent non-imprinted ASM, but with biallelic hypermethylation in sperm DNA (additional data in Table 1). C, Gene map and bis-seq of the index amplicon containing SNP rs2811488 located downstream of the last exon of the RPN1 gene in chromosome band 3q21, showing strong ASM in placenta, with the G allele or A allele hypermethylated, depending on parent-of-origin, as proven in Figure 2. For this imprinted DMR the ASM is highly tissue-specific, being seen in placenta but not in PBL, liver, lung, brain, HMEC or sperm (additional data in Table 1 and Figure S2). D, Gene map and bilsufite sequencing of the index amplicon containing SNP rs2346019 downstream of the VTRNA2-1 vault-family RNA, located in chromosome band 5q31. Strong ASM is observed in multiple tissues with the A allele or G allele methylated, consistent with imprinting, which is proven by the data in Figure 2.

Mentions: The MSNP procedure, an adaptation of SNP arrays for detecting ASM, was described in our initial report of haplotype-dependent ASM in human tissues [7]. As a starting point for comparing the structures of chromosomal domains with imprinted (parent-of-origin dependent) versus non-imprinted (haplotype dependent) ASM we applied higher resolution MSNP to several human tissue types from multiple individuals and identified 4 additional SNP-tagged StyI or NspI restriction fragments with CpG dinucleotides in HpaII sites that showed strongly asymmetrical methylation between the two alleles in multiple heterozygous samples (examples in Figure S1). These index fragments are tagged by SNPs rs1530562 between the STEAP3 and C2orf76 genes (chromosome band 2q14), rs2346019 near the VTRNA2-1 vault family small RNA gene (5q31.1), rs2811488 between the C3orf27 and RPN1 genes (3q21), and rs2302902 in the ELK3 gene (12q23.1). We used Sanger bisulfite sequencing (bis-seq) of amplicons containing these SNPs and multiple (non-polymorphic) adjacent CpG dinucleotides to confirm ASM in heterozygous individuals in a panel of primary tissues chosen for relevance to complex diseases and representation of multiple cell lineages: peripheral blood (PBL), whole fetal and adult lung and adult bronchial epithelial cells, adult liver, adult brain (cerebral cortical grey matter), placenta (chorionic villi taken from near the fetal surface), human mammary epithelial cells (HMEC), and sperm. These analyses showed that the ASM was reproducible for all 4 index fragments, with the allelic asymmetry in methylation affecting multiple (non-polymorphic) CpGs around and including the index HpaII sites (Fig. 1 and Figs. S2, S3, S4). Among these 4 regions, moderate ASM was observed in the ELK3 gene, while the other index regions showed stronger allelic asymmetries. ASM was seen in two or more tissues for each of the index regions except the C3orf27-RPN1 region, for which the index amplicon revealed highly tissue-specific ASM, which was very strong in the large majority of human placentas examined (33/34 cases; all tested using tissue from the fetal side of the organ, consisting of the free chorionic villi without maternal decidua; Table 1), but absent in the other tissues tested (Fig. 1, Table 1 and Figure S3).


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)

Bis-seq showing strong ASM in the ELK3, STEAP3-C2orf76, C3orf27-RPN1, and VTRNA2-1 index regions.A, Gene map and bis-seq of the index amplicon containing SNP rs2302902 in the first intron of the ELK3 gene. The strong asymmetry in CpG methylation, with the G allele consistently hypermethylated in the PBL samples, indicates haplotype-dependent non-imprinted ASM (additional data in Table 1; haplotype map in Figure S5). There is some tissue-specificity, with biallelic hypermethylation in the HMEC sample. The grey (lower) bars indicate the index amplicons for initial bis-seq, each tagged by the index SNP that showed recurrent ASM in the MSNP data. The green bars indicate CGIs and the black rectangles are exons. The X's indicate polymorphic CpG sites. B, Gene map and bis-seq of the index amplicon containing SNP rs1530562, between the STEAP3 and C2orf76 genes in chromosome band 2q14, showing strong ASM with the G allele consistently hypermethylated in multiple tissues consistent with haplotype-dependent non-imprinted ASM, but with biallelic hypermethylation in sperm DNA (additional data in Table 1). C, Gene map and bis-seq of the index amplicon containing SNP rs2811488 located downstream of the last exon of the RPN1 gene in chromosome band 3q21, showing strong ASM in placenta, with the G allele or A allele hypermethylated, depending on parent-of-origin, as proven in Figure 2. For this imprinted DMR the ASM is highly tissue-specific, being seen in placenta but not in PBL, liver, lung, brain, HMEC or sperm (additional data in Table 1 and Figure S2). D, Gene map and bilsufite sequencing of the index amplicon containing SNP rs2346019 downstream of the VTRNA2-1 vault-family RNA, located in chromosome band 5q31. Strong ASM is observed in multiple tissues with the A allele or G allele methylated, consistent with imprinting, which is proven by the data in Figure 2.
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Related In: Results  -  Collection

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Show All Figures
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pgen-1003622-g001: Bis-seq showing strong ASM in the ELK3, STEAP3-C2orf76, C3orf27-RPN1, and VTRNA2-1 index regions.A, Gene map and bis-seq of the index amplicon containing SNP rs2302902 in the first intron of the ELK3 gene. The strong asymmetry in CpG methylation, with the G allele consistently hypermethylated in the PBL samples, indicates haplotype-dependent non-imprinted ASM (additional data in Table 1; haplotype map in Figure S5). There is some tissue-specificity, with biallelic hypermethylation in the HMEC sample. The grey (lower) bars indicate the index amplicons for initial bis-seq, each tagged by the index SNP that showed recurrent ASM in the MSNP data. The green bars indicate CGIs and the black rectangles are exons. The X's indicate polymorphic CpG sites. B, Gene map and bis-seq of the index amplicon containing SNP rs1530562, between the STEAP3 and C2orf76 genes in chromosome band 2q14, showing strong ASM with the G allele consistently hypermethylated in multiple tissues consistent with haplotype-dependent non-imprinted ASM, but with biallelic hypermethylation in sperm DNA (additional data in Table 1). C, Gene map and bis-seq of the index amplicon containing SNP rs2811488 located downstream of the last exon of the RPN1 gene in chromosome band 3q21, showing strong ASM in placenta, with the G allele or A allele hypermethylated, depending on parent-of-origin, as proven in Figure 2. For this imprinted DMR the ASM is highly tissue-specific, being seen in placenta but not in PBL, liver, lung, brain, HMEC or sperm (additional data in Table 1 and Figure S2). D, Gene map and bilsufite sequencing of the index amplicon containing SNP rs2346019 downstream of the VTRNA2-1 vault-family RNA, located in chromosome band 5q31. Strong ASM is observed in multiple tissues with the A allele or G allele methylated, consistent with imprinting, which is proven by the data in Figure 2.
Mentions: The MSNP procedure, an adaptation of SNP arrays for detecting ASM, was described in our initial report of haplotype-dependent ASM in human tissues [7]. As a starting point for comparing the structures of chromosomal domains with imprinted (parent-of-origin dependent) versus non-imprinted (haplotype dependent) ASM we applied higher resolution MSNP to several human tissue types from multiple individuals and identified 4 additional SNP-tagged StyI or NspI restriction fragments with CpG dinucleotides in HpaII sites that showed strongly asymmetrical methylation between the two alleles in multiple heterozygous samples (examples in Figure S1). These index fragments are tagged by SNPs rs1530562 between the STEAP3 and C2orf76 genes (chromosome band 2q14), rs2346019 near the VTRNA2-1 vault family small RNA gene (5q31.1), rs2811488 between the C3orf27 and RPN1 genes (3q21), and rs2302902 in the ELK3 gene (12q23.1). We used Sanger bisulfite sequencing (bis-seq) of amplicons containing these SNPs and multiple (non-polymorphic) adjacent CpG dinucleotides to confirm ASM in heterozygous individuals in a panel of primary tissues chosen for relevance to complex diseases and representation of multiple cell lineages: peripheral blood (PBL), whole fetal and adult lung and adult bronchial epithelial cells, adult liver, adult brain (cerebral cortical grey matter), placenta (chorionic villi taken from near the fetal surface), human mammary epithelial cells (HMEC), and sperm. These analyses showed that the ASM was reproducible for all 4 index fragments, with the allelic asymmetry in methylation affecting multiple (non-polymorphic) CpGs around and including the index HpaII sites (Fig. 1 and Figs. S2, S3, S4). Among these 4 regions, moderate ASM was observed in the ELK3 gene, while the other index regions showed stronger allelic asymmetries. ASM was seen in two or more tissues for each of the index regions except the C3orf27-RPN1 region, for which the index amplicon revealed highly tissue-specific ASM, which was very strong in the large majority of human placentas examined (33/34 cases; all tested using tissue from the fetal side of the organ, consisting of the free chorionic villi without maternal decidua; Table 1), but absent in the other tissues tested (Fig. 1, Table 1 and Figure S3).

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