© 2012 Landes Bioscience. Do not distribute. Figure 2. The assessment of a lu and LINE-1 DNa methylation is affected by evolutionary age and assay type. DNa methylation of (a ) a lu and (B) LINE-1 was assessed in two groups: merged villi (n = 31; 1st trimester, 2nd trimester and term villi) and merged somatic tissues (n = 39; brain, kidney, muscle and blood) usingpyrosequencing and probes from the Illumina Infinium h umanMethylation27 Beadc hip array. a rray probes that mapped to REs were divided into three age groups based on evolutionary emergence: old a lu (a luJ; n = 113), intermediate a lu (a lus; n = 272), young a lu (a luY; n = 58) and old LINE-1 (LINE-1M; n = 24), intermediate LINE-1 (LINE-1p; n = 160), young LINE-1 (LINE-1h ; n = 4). There was a trend for increased DNa methyla- tion from the old to young a lu and LINE-1 measured by the array. a lu and LINE-1 DNa methylation as assessed by pyrosequencing was significantly different from each age group measured by the array, except when comparing LINE-1 by pyrosequencing to young LINE-1s in the merged villi group. s ignificance is indicated by *p < 0.05, **p < 0.001, ***p < 0.0001. differences in DNA methylation by sex for any tissue or for any ReDS; thus, males and females were considered together for all analyses (Fig. S2). LINE-1 and weak island probe methylation were correlated in 2nd trimester villi (Table 1; r = 0.68, p = 0.025) and in brain (Table 1; r = 0.88, p = 0.007); however, these correlations did not withstand correction for multiple comparisons. Weak and non- island probe methylation were significantly correlated after cor- rection for multiple comparisons in 1st trimester villi (Table 1; r = 0.94, p < 0.001) and 2nd trimester villi (Table 1; r = 0.80, p = 0.005). In the merged somatic group, weak island probe methylation was also correlated with LINE-1 DNA methyla- tion (Table 1; r = 0.48, p = 0.002) and with non-island probe methylation (Table 1; r = 0.91, p < 0.0001). LINE-1 and Alu DNA methylation were correlated in 1st trimester villi (Table 1; r = 0.64, p = 0.054) and in brain (Table 1; r = 0.74, p = 0.046), but neither of these was significant after correction for multiple comparisons. Detailed correlation analyses within each tissue can be found in Figures S3–10. Since the correlation of DNA methylation between weak islands and non-islands in addition to weak islands and LINE-1s was present in several individual tissues and the merged somatic group, the association between these two pairs of ReDS may be more than inter-individual varia- tion. However, the inconsistency of correlation between DNA methylation of the other pairs of ReDS suggests that each mea- sure targets a genomic sequence with different trends in DNA methylation. Alternative dispersed DNA methylation assays each produce a distinct tissue-specific DNA methylation profile. The five ReDS examined here are functionally different and thus trends in tissue-specific DNA methylation at these regions may also be distinct. Second trimester tissues (villi, brain, kidney and muscle) www.landesbioscience.com Epigenetics 655