Journal: PLoS Genetics

Article Title: Comparative Anatomy of Chromosomal Domains with Imprinted and Non-Imprinted Allele-Specific DNA Methylation

doi: 10.1371/journal.pgen.1003622

Figure Lengend Snippet: Trios consisting of matched parents (PBL) and offspring (placental chorionic villi from the fetal surface) were analyzed for ASM to determine the parental origin of the hypermethylated and hypomethylated alleles in the C3orf27-RPN1 and VTRNA2-1 index regions. A, Hpa II-predigestion/PCR/RFLP assay for ASM at non-polymorphic Hpa II sites in an amplicon including SNP rs12487604 (located immediately downstream of RPN1 ) which creates a Bts I restriction site. Undigested genomic DNA and genomic DNA pre-digested with the methylation-sensitive restriction enzyme Hpa II were amplified by PCR and then digested with Bts I. In the samples pre-digested with Hpa II (+) one allele drops out, indicating hypomethylation. A homozygous case for each allele is shown, as well as four heterozygotes; two cases show T-allele hypermethylation and two show A-allele hypermethylation. B, Trios analyzed by this assay showing parental imprinting of the index region downstream of RPN1 . A total of 13 informative trios (two shown) were analyzed and each showed hypermethylation of the maternally-derived allele in the placenta (p<0.00311). C, Examples of bis-seq of trios for the index amplicon including SNP rs2346019 in the VTRNA2-1 locus. The hypermethylated allele was found to be maternally derived in each of 10 informative trios (p<0.001565).

Article Snippet: To compare the structures of chromosomal domains with imprinted versus non-imprinted ASM we used high throughput bisulfite PCR (Fluidigm AccessArray) with sample bar-coding, followed long-read 454 Pyrosequencing of amplicons bracketing 3 of the index regions; two with imprinted ASM ( VTRNA2-1 and C3orf27 - RPN1 ) and one with non-imprinted/haplotype dependent ASM ( STEAP3 - C2orf76 ).

Techniques: RFLP Assay, Amplification, Methylation, Derivative Assay

Journal: PLoS Genetics

Article Title: Comparative Anatomy of Chromosomal Domains with Imprinted and Non-Imprinted Allele-Specific DNA Methylation

doi: 10.1371/journal.pgen.1003622

Figure Lengend Snippet: A, Bis-seq of the STEAP3 - C2orf76 intergenic DMR in heterozygous PBL samples shows localization of ASM to a discrete region about 450 bp in length (chr2: 120,048,634–120,049,081) spanning the strong CTCF binding site. *No heterozygous samples were found for rs55740427. B, PCR of the STEAP3 - C2orf76 intergenic DMR performed on non-immunoprecipitated input DNA, negative control IgG IP DNA and CTCF ChIP DNA. As expected, the non-immunoprecipitated input sample results in a strong amplification while the negative control IgG IP sample shows no amplification. The observed amplification in the CTCF immunoprecipitated sample demonstrates CTCF binding at this region. Direct sequencing of end-point PCR products shows that the non-immunoprecipitated input DNA is heterozygous at the index SNP rs1530562 while the CTCF ChIP sample shows a strong enrichment for the A-allele indicating that CTCF is bound selectively to this (hypomethylated) allele. C, Maps of 4 representative haplotype blocks containing both a CTCF binding site with at least two ASM CpG sites (as defined by Chen et al. ) within a 1 kb window and SNPs associated with important human traits or disease susceptibility from GWAS (supra-threshold or strong sub-threshold peaks, all p<10 −6 ; http://www.genome.gov/gwastudies ). The relevant traits or diseases are indicated next to each SNP. The CpG sites that were analyzed for ASM (vertical lines) and located near the CTCF binding sites (horizontal black bars below the lines) are color-coded as orange for ASM sites that do not overlap with a CpG SNP, green for CpG SNP ASM sites, gray for non ASM sites. See also .

Article Snippet: To compare the structures of chromosomal domains with imprinted versus non-imprinted ASM we used high throughput bisulfite PCR (Fluidigm AccessArray) with sample bar-coding, followed long-read 454 Pyrosequencing of amplicons bracketing 3 of the index regions; two with imprinted ASM ( VTRNA2-1 and C3orf27 - RPN1 ) and one with non-imprinted/haplotype dependent ASM ( STEAP3 - C2orf76 ).

Techniques: Binding Assay, Immunoprecipitation, Negative Control, Amplification, Sequencing