50-year-old B EcoRV fragment es +5.4 kb Notl/EcoRV fragment 41.9 kb methylation 24 20 37 3-year-old 400::6 ll H Fig 4. Differences in promoter CpG island DNA methylation and expression profiles in MZ twins are associated with aging. (A)Example of an RLGS pi D-year-old twin pair showing the pr of an RLGS fragment C14orf162 in Twin A, indicated by the arrow, in whose sister (Twin B)it is significantl (B)Southern blot analysis confirms the CpG island methylation difference in C14orf162 between twins. All lines contain DNa digested with EcoRV. Th o digested with Notl, as indicated by + (Q Bisulfite genomic sequencing of 12 clones of the C14orf162 promoter CpG island in the 50-year-old twi and white dots indicate methylated and unmethylated CpGs, respectively. Below the sequences are two representative electropherograms showing 三 specific DNA methylation changes at the C14orf162 promoter in the 50-year-old twin pair. (D) The older MZ twins display the greatest difference expression profiles between siblings. Left) Scatter plots showing expression profiles of 3- and 50-year-old twin pairs (Right) Number of overexpressed and repressed genes obtained by DNa array analyses in the 3-year-old compared with the 50-year-old twin pair. dentification of Chromosomal Regions with DNA Methylation Differ- distributed DNA methylation events(hypermethylation and ences in MZ Twin Pairs. We also investigated the"geographical hypomethylation) throughout the chromosomes described distribution of DNA methylation differences within the chro- above nosomes of MZ twin pairs by competitively hybridizing the AIMS PCR Products to metaphases to determine the differen- Global CpG island Methylation Differences in MZ Twin Pairs Charac- tially methylated DNA regions( 9). The AIMS products from terized by RLGS. Twins'differentially methylated sequences ob- competitively hybridized with those of his or her sibling. The tained by the AIMS approach also included CpG islands located in lata obtained demonstrated that the twin pairs with similar 5mC the promoter regions of single-copy genes. We implemented a methylation patterns shared a common distribution of dna ation differences in these important DNA regions. We subje However,those twins with different global epigenetic patterns youngest 3- and 22-year-old vs. those completely epigenetically also showed a distinct profile of DNA methylation signal in their different from the 50-year-old, to methylation-oriented RLGS(10) metaphase chromosomes. In particular, these differences were This approach allows global determination of the methylation located in almost all telomeres and a few selected gene-rich status of sl, 800 unselected CpG islands. We observed that the gions(such as 1p36, 1q21, 3p21, 8q21, 9q21, and 22q11.2 older twin pairs presented 2.5 times as many DNA methylation Furthermore, our DNA methylation-competitive genomic hy mic hy- differences in the CpG islands of single-copy genes as did the bridization results showed that MZ twins who were younger twin-pairs(Fig 4A). These DNA methylation differences unger, had similar lifestyles, and had spent more of their observed in the older twins also were demonstrated for these lifetimes together displayed minimal DNA methylation changes particular gene-associated CpG islands by methylation-sensitive in all chromosomes, whereas those who were older, had different Southern blot(Fig. 4B)and bisulfite genomic sequencing of mul- lifestyles, and had spent less of their lives together had unevenly tiple clones(Fig. 4C) 10608iwww.pnas.org/cgi/doi/10.1073/pnas.0500398102 Fraga et al.Identification of Chromosomal Regions with DNA Methylation Differ￾ences in MZ Twin Pairs. We also investigated the ‘‘geographical’’ distribution of DNA methylation differences within the chro￾mosomes of MZ twin pairs by competitively hybridizing the AIMS PCR products to metaphases to determine the differen￾tially methylated DNA regions (9). The AIMS products from each component of one individual of an MZ twin pair were competitively hybridized with those of his or her sibling. The data obtained demonstrated that the twin pairs with similar 5mC DNA content, acetylation levels of H3 and H4, and AIMS DNA methylation patterns shared a common distribution of DNA methylation signal throughout all chromosomal regions (Fig. 3). However, those twins with different global epigenetic patterns also showed a distinct profile of DNA methylation signal in their metaphase chromosomes. In particular, these differences were located in almost all telomeres and a few selected gene-rich regions (such as 1p36, 1q21, 3p21, 8q21, 9q21, and 22q11.2). Furthermore, our DNA methylation–competitive genomic hy￾bridization results again showed that MZ twins who were younger, had similar lifestyles, and had spent more of their lifetimes together displayed minimal DNA methylation changes in all chromosomes, whereas those who were older, had different lifestyles, and had spent less of their lives together had unevenly distributed DNA methylation events (hypermethylation and hypomethylation) throughout the chromosomes described above. Global CpG Island Methylation Differences in MZ Twin Pairs Charac￾terized by RLGS. Twins’ differentially methylated sequences ob￾tained by the AIMS approach also included CpG islands located in the promoter regions of single-copy genes. We implemented a specific strategy for further characterizing global genomic methyl￾ation differences in these important DNA regions. We subjected the two opposite sites of the MZ twin spectrum, those from the youngest 3- and 22-year-old vs. those completely epigenetically different from the 50-year-old, to methylation-oriented RLGS (10). This approach allows global determination of the methylation status of 1,800 unselected CpG islands. We observed that the older twin pairs presented 2.5 times as many DNA methylation differences in the CpG islands of single-copy genes as did the younger twin-pairs (Fig. 4A). These DNA methylation differences observed in the older twins also were demonstrated for these particular gene-associated CpG islands by methylation-sensitive Southern blot (Fig. 4B) and bisulfite genomic sequencing of mul￾tiple clones (Fig. 4C). Fig. 4. Differences in promoter CpG island DNA methylation and expression profiles in MZ twins are associated with aging. (A) Example of an RLGS profile from a 50-year-old twin pair showing the presence of an RLGS fragment C14orf162 in Twin A, indicated by the arrow, in whose sister (Twin B) it is significantly reduced. (B) Southern blot analysis confirms the CpG island methylation difference in C14orf162 between twins. All lines contain DNA digested with EcoRV. The DNA was also digested with NotI, as indicated by . (C) Bisulfite genomic sequencing of 12 clones of the C14orf162 promoter CpG island in the 50-year-old twin pair. Black and white dots indicate methylated and unmethylated CpGs, respectively. Below the sequences are two representative electropherograms showing sequence￾specific DNA methylation changes at the C14orf162 promoter in the 50-year-old twin pair. (D) The older MZ twins display the greatest differences in gene expression profiles between siblings. (Left) Scatter plots showing expression profiles of 3- and 50-year-old twin pairs. (Right) Number of overexpressed and repressed genes obtained by DNA array analyses in the 3-year-old compared with the 50-year-old twin pair. 10608 www.pnas.orgcgidoi10.1073pnas.0500398102 Fraga et al.