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Magazine R148 Correspondence Differences in DNA methylation patterns between humans and chimpanzees Wolfgang Enard1,3*, Anne Fassbender2,3, Fabian Model2, Péter Adorján2, Svante Pääbo1 and Alexander Olek2 Methylation at CpG dinucleotides is important for gene regulation in mammals [1]. However, it is unknown how methylation patterns change during evolution. Here, we compare methylation patterns between humans and chimpanzees at 36 genes in the brain, the liver and in lymphocytes. We find that the extent of the change in methylation pattern is much more extensive in the brain than in the other tissues. Furthermore, of the 15 CpGs that have significantly changed methylation in the brain, 14 are more methylated in humans than in chimpanzees. This indicates that CpGs might generally be more methylated in human brains than in chimpanzee brains. Despite considerable phenotypic differences, humans and their closest living relatives, the chimpanzees, are on average 98.8% identical in their alignable genomic DNA sequences [2,3]. It is currently unknown which genotypic differences are responsible for the phenotypic differences. One possibility to tackle this question is to compare gene expression patterns between humans and chimpanzees using functional genomic approaches [4–6]. In this respect, it may also be useful to compare methylation patterns in regulatory DNA sequences, as the methylation status can be viewed as a “footprint” of the chromatin structures that are crucial for gene regulation [7,8]. In order to take a first step toward understanding the evolution of methylation patterns, we compared the methylation status of 145 CpGs in the presumed regulatory regions of 36 different genes between humans and chimpanzees in brain, liver and lymphocytes using a recently developed array technique [9–11]. Thereby, genomic DNA is treated with sodium bisulphite such that unmethylated CpGs are amplified as TpGs in the following PCR. For each CpG examined, the arrays contain two oligonucleotides: one complementary to a TpG, resembling a formerly unmethylated CpG and one complementary to a CpG, resembling a formerly methylated CpG. We identified 22 CpGs in which the ratio of the intensities of these two oligonucleotides differed significantly between human and chimpanzee in at least one tissue. By contrast, zero to three differences would be expected due to random experimental and biological variation, as is shown by permutating the species labels for each tissue (see supplemental data for all methodological details). Therefore, the differences between the two species are highly significant, whereas the differences between the individuals of the same species are within the range of the experimental error (data not shown). We also do not observe a strong correlation of methylation levels with age or time post mortem (see supplemental data), Thus, we conclude that most of the observed methylation differences between humans and chimpanzees are neither due to random measurement errors nor due to random or systematic differences in their environment. To exclude trivial genetic causes, we sequenced the region of the 22 CpG sites in the chimpanzee and excluded 4 CpGs that carried a sequence difference between the chimpanzee sequence and the human-based oligonucleotide sequence. The remaining 18 CpGs from 12 genes are shown in Figure 1. Three observations from these experiments are especially noteworthy: First, despite the limited number of CpGs studied, several significant differences in their methylation status can be found between humans and chimpanzees. Second, out of 18 differences, 15 are found between chimpanzee and human brain, whereas only six are found between the other two tissues. Third, 14 of the 15 sites differing in methylation in the brain show a higher degree of methylation in humans. The first observation indicates that — at least in humans and chimpanzees — the methylation status of many CpG sites changes during the course of evolution. The second observation indicates that more CpG sites have changed their methylation status in the brain than in liver or lymphocytes. Notably, DNA methylation seems to be especially important for the brain, as defects in methylation lead to mental retardation in humans [8] and a mouse model for one of these diseases — Rett syndrome — indicates that the symptoms can be caused solely by a defect in postmitotic neurons [12,13]. Our third observation, namely that 14 of 15 CpG sites differently methylated in the brain show a higher degree of methylation in humans, might reflect a general up-methylation of genes in the human brain, rather than several independent, gene-specific methylation changes. Although it is unclear at this point whether this up-methylation directly translates into observable changes in gene expression (supplemental data), it is tempting to speculate that such an upmethylation was important for the evolution of the human brain. However, we cannot exclude that a general tendency towards a lower degree of methylation occurred on the chimpanzee lineage. It is furthermore unclear if the change in methylation patterns is especially pronounced in the human brain or if a rapid change in methylation patterns is typical of brain evolution in many mammals. Further work has to clarify these issues. Supplemental Data Supplemental data containing experimental procedures are Magazine R149 Liver Humans Chimpanzees Humans 6m 6m 12m 12m 18f 25f 47m 48m 56m 69m 74f 85m 1m 6m 12m 12m 34m 36f 59f 27f 29f 49m unkn. unkn. Chimpanzees Brain C4B_3 CD63_4 MYCN_2 BAT1_2 MYCL1_3 UBC_1 MYCL1_2 HLA-F_4 ABL1_4 SLC25A4_3 CDC25A_2 MYCN_3 EGFR_3 ABL1_2 CD63_2 MYC_8 POMC_2 GP1BB_1 GP1BB_2 SLC25A4_1 DAD1_1 LTA_2 MYCL1_3 DBCCR1_3 UNG_3 CTLA4_4 UBC_2 EGFR_4 MYCL1_2 LTA_1 GP1BB_2 CDC25A_1 UBC_3 POMC_3 EGR4_4 SLC25A4_1 GP1BB_4 CD63_4 SLC25A4_3 GP1BB_3 5. 6. Lymphocytes Humans 3f 4m 9m 10f 10f 10m 11m 12m 14f 18f 18m 19f 19m 21f 23m 33f 33f 34m 37f 39m 5m 8f 10f 11m 16f 16m 18m 23f 27f 29m 35m 41f 41m 46m 55f 59m 70f 71m 78f 93f Chimpanzees 7. HLA-F_2 EGR4_2 MYCL1_4 MYC_3 EGFR_2 BAT1_2 CDK4_1 LY6G6D_3 ABL1_4 ABL1_2 ERBB2_3 LY6G6D_2 EGFR_7 HSPA1A_7 UNG_2 GP1BB_3 CSNK2B_3 HLA-F_4 DBCCR1_3 SLC25A4_1 8 4 2 1 2 4 8. 9. 8 10. -fold less methylated than median -fold more methylated than median Current Biology Figure 1. Methylation patterns of humans and chimpanzees in brain, liver and lymphocyte samples. Each row corresponds to a single CpG, each column to one sample of one individual. For each cell the logarithm of the ratio of the intensity of the oligonucleotide representing the methylated CpG to the intensity of the oligonucleotide representing the unmethylated CpG is calculated and log2 transformed. The color represents the methylation level calculated as the distance from the median of all values in a row. As indicated in the legend, more methylation than the median is depicted in red and less methylation in green. Each CpG is named by the official gene symbol of the associated gene and a consecutive number. For each individual the age and sex is indicated. The 20 CpG sites with the highest effect size of the t-statistic (t2) are shown, sorted by increasing t2-values from top to bottom. CpG sites classified as differently methylated are indicated in bold. available at http://www.currentbiology.com/supplemental Acknowledgments We thank Victor Wiebe for help with the DNA sequencing and the Bundesminsterium für Bildung und Forschung and the Max Planck for financial support. The authors at Epigenomics AG disclose financial conflict of interest. References 1. Jones, P.A., and Takai, D. (2001). The role of DNA methylation in mammalian epigenetics. Science 293, 1068–1070. 2. Ebersberger, I., Metzler, D., Schwarz, C., and Paabo, S. (2002). Genomewide comparison of DNA sequences between humans and chimpanzees. Am. J. Hum. Genet. 70, 1490–1497. 3. Fujiyama, A., Watanabe, H., Toyoda, A., Taylor, T.D., Itoh, T., Tsai, S.F., Park, H.S., Yaspo, M.L., Lehrach, H., Chen, Z., et al. (2002). Construction and analysis of a human-chimpanzee comparative clone map. Science 295, 131–134. 4. 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A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat. Genet. 27, 322–326. 1Max Planck Institute for evolutionary Anthropology, Deutscher Platz 6, D04103 Leipzig, Germany. *E-mail: [email protected] 2Epigenomics AG, Kleine Präsidentenstr. 1, D-10178 Berlin, Germany. 3These authors contributed equally to this work.