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
Non-imprinted ASM in the STEAP3-C2orf76 region and imprinted ASM in the RPN1-C3orf27 regions are both associated with ASE.A, ASE of the C2orf76 gene, associated with the C2orf76-STEAP3 DMR, is shown in duplicate for four PBL samples (rs6542522). In each case the C allele is preferentially expressed in the cDNA when compared to the genomic DNA, consistent with the sequence dependent nature of ASM in this locus. Overall, 12 informative PBL samples were analyzed in duplicate for ASE and 7 PBL samples showed preferential expression of the C allele. Likewise, preferential expression of the C allele was identified in 7 out of the 22 informative liver samples (data not shown). B, ASE of the lncRNA corresponding to the AK097792 EST, overlapping the index SNP rs2811488 in the RPN1-C3orf27 intergenic DMR are shown on the left and those overlapping two additional informative SNPs, i.e. rs35604103 and rs61112519, are shown on the right for four placenta samples. Overall, the assays were performed in duplicates for 13 informative placenta samples. Ten of them showed a definite or complete ASE. In each of 6 informative samples assayed for both ASE and ASM, the hypermethylated maternal allele was the relatively repressed one.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003622-g008: Non-imprinted ASM in the STEAP3-C2orf76 region and imprinted ASM in the RPN1-C3orf27 regions are both associated with ASE.A, ASE of the C2orf76 gene, associated with the C2orf76-STEAP3 DMR, is shown in duplicate for four PBL samples (rs6542522). In each case the C allele is preferentially expressed in the cDNA when compared to the genomic DNA, consistent with the sequence dependent nature of ASM in this locus. Overall, 12 informative PBL samples were analyzed in duplicate for ASE and 7 PBL samples showed preferential expression of the C allele. Likewise, preferential expression of the C allele was identified in 7 out of the 22 informative liver samples (data not shown). B, ASE of the lncRNA corresponding to the AK097792 EST, overlapping the index SNP rs2811488 in the RPN1-C3orf27 intergenic DMR are shown on the left and those overlapping two additional informative SNPs, i.e. rs35604103 and rs61112519, are shown on the right for four placenta samples. Overall, the assays were performed in duplicates for 13 informative placenta samples. Ten of them showed a definite or complete ASE. In each of 6 informative samples assayed for both ASE and ASM, the hypermethylated maternal allele was the relatively repressed one.

Mentions: One of the biologically important consequences of ASM is allele-specific RNA expression (ASE). We previously demonstrated strong haplotype-dependent ASE of the CYP2A7 gene [7], and we were interested to ask whether ASE could be detected in the other chromosomal regions with ASM analyzed in this report. Assays comparing the representation of SNPs in genomic versus cDNA PCR products showed a definite and recurrent bias in allele-specific mRNA expression of the C2orf76 gene, associated with the STEAP3-C2orf76 DMR (Fig. 8). The untranslated AK097792 RNA transcript, associated with RPN1-C3orf27 DMR also showed a recurrent bias in ASE: from 13 tested individuals informative for ASE, 6 showed a definite but not complete allele-specific bias, four had complete ASE and three were biallelically expressed (Fig. 8 and Fig. S3). In each of 6 informative samples with definite or complete ASE, and data for ASM, the relatively hypermethylated allele was the repressed one, suggesting a functional link between ASE and ASM at this locus. Interestingly, current ENCODE data support our findings from RT-PCR of a long non-coding RNA (lncRNA) traversing the DMR, and also suggest that a micro-RNA, as yet unnamed, arises from very close to this DMR (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)

Non-imprinted ASM in the STEAP3-C2orf76 region and imprinted ASM in the RPN1-C3orf27 regions are both associated with ASE.A, ASE of the C2orf76 gene, associated with the C2orf76-STEAP3 DMR, is shown in duplicate for four PBL samples (rs6542522). In each case the C allele is preferentially expressed in the cDNA when compared to the genomic DNA, consistent with the sequence dependent nature of ASM in this locus. Overall, 12 informative PBL samples were analyzed in duplicate for ASE and 7 PBL samples showed preferential expression of the C allele. Likewise, preferential expression of the C allele was identified in 7 out of the 22 informative liver samples (data not shown). B, ASE of the lncRNA corresponding to the AK097792 EST, overlapping the index SNP rs2811488 in the RPN1-C3orf27 intergenic DMR are shown on the left and those overlapping two additional informative SNPs, i.e. rs35604103 and rs61112519, are shown on the right for four placenta samples. Overall, the assays were performed in duplicates for 13 informative placenta samples. Ten of them showed a definite or complete ASE. In each of 6 informative samples assayed for both ASE and ASM, the hypermethylated maternal allele was the relatively repressed one.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003622-g008: Non-imprinted ASM in the STEAP3-C2orf76 region and imprinted ASM in the RPN1-C3orf27 regions are both associated with ASE.A, ASE of the C2orf76 gene, associated with the C2orf76-STEAP3 DMR, is shown in duplicate for four PBL samples (rs6542522). In each case the C allele is preferentially expressed in the cDNA when compared to the genomic DNA, consistent with the sequence dependent nature of ASM in this locus. Overall, 12 informative PBL samples were analyzed in duplicate for ASE and 7 PBL samples showed preferential expression of the C allele. Likewise, preferential expression of the C allele was identified in 7 out of the 22 informative liver samples (data not shown). B, ASE of the lncRNA corresponding to the AK097792 EST, overlapping the index SNP rs2811488 in the RPN1-C3orf27 intergenic DMR are shown on the left and those overlapping two additional informative SNPs, i.e. rs35604103 and rs61112519, are shown on the right for four placenta samples. Overall, the assays were performed in duplicates for 13 informative placenta samples. Ten of them showed a definite or complete ASE. In each of 6 informative samples assayed for both ASE and ASM, the hypermethylated maternal allele was the relatively repressed one.
Mentions: One of the biologically important consequences of ASM is allele-specific RNA expression (ASE). We previously demonstrated strong haplotype-dependent ASE of the CYP2A7 gene [7], and we were interested to ask whether ASE could be detected in the other chromosomal regions with ASM analyzed in this report. Assays comparing the representation of SNPs in genomic versus cDNA PCR products showed a definite and recurrent bias in allele-specific mRNA expression of the C2orf76 gene, associated with the STEAP3-C2orf76 DMR (Fig. 8). The untranslated AK097792 RNA transcript, associated with RPN1-C3orf27 DMR also showed a recurrent bias in ASE: from 13 tested individuals informative for ASE, 6 showed a definite but not complete allele-specific bias, four had complete ASE and three were biallelically expressed (Fig. 8 and Fig. S3). In each of 6 informative samples with definite or complete ASE, and data for ASM, the relatively hypermethylated allele was the repressed one, suggesting a functional link between ASE and ASM at this locus. Interestingly, current ENCODE data support our findings from RT-PCR of a long non-coding RNA (lncRNA) traversing the DMR, and also suggest that a micro-RNA, as yet unnamed, arises from very close to this DMR (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