Evolutionary Breakpoints in Gibbon Table 2. Mechanisms of double-strand repair for gibbon rearrangements. Rearrangement Putative mechanism Filling sequence Micro-homology BAC CH271-372B11 CH271-446I8 CH271-262E11 CH271-398E1 CH271-383H22 CH271-350B17 CH271-372B11 CH271-286K22 CH271-261K6 CH271-261A22 CH271-261L1 CH271-330D2 CH271-298N13 CH271-183B5 CH271-241J10 CH271-78K20 CH271-171B20 CH271-86M19 CH271-350B17 CH271-263C9 CH271-274L1 CH271-449L10 CH271-438C12 CH271-114O8 Traces 1744822164 CH271-228C1 t(HSA2;HSA9) Inv(HSA7) t(HSA17;HSA2) Inv(HSA17) Inv(HSA3) Inv(HSA16) t(HSA9;HSA6) Inv(HSA7) Inv(HSA3) Inv(HSA7) Inv(HSA1) t(HSA16;HSA5) t(HSA8;HSA18) t(HSA8;HSA5) Inv(HSA1) t(HSA4;HSA16) Inv(HSA9)_1 t(HSA7;HSA20) t(HSA4;HSA16) t(HSA22;HSA4) Inv(HSA17)_1 t(HSA12;HSA19) t(HSA10;HSA14) t(HSA5;HSA16) Inv(HSA17) t(HSA7;HSA20) Alu-Alu recombination Alu-Alu recombination Alu-Alu recombination Alu-Alu recombination Alu-Alu recombination NAHR (ABCC1-ABCC6) NAHR of gSD NAHR of hSD NAHR of hSD (in human) NAHR of hSD (in human) NHEJ NHEJ NHEJ NHEJ NHEJ NHEJ NHEJ NHEJ NHEJ NHEJ NHEJ NHEJ NHEJ NHEJ NHEJ Stem-Loop We were able to infer the mechanism for double strand repair on the bases of the molecular structure of the breakpoint for the 28 breakpoints that are listed in this table. The presence of long stretches of homology indicated that most likely NAHR recombination occurred while presence of micro-homologies or ‘‘filled in’’ sequences suggested NHEJ. doi:10.1371/journal.pgen.1000538.t002 we found that their values of dN/dS (average dN/dS = 0.56) were not as high as those reported by Girirajan et al. [5], subsequent analysis on the remaining dataset of all non-disrupted genes located within 50 Kbp of a break point, revealed a significantly reduced difference of dN/dS between gibbons and macaques (from p = 0.001 to p = 0.06) (Table S4). We hypothesize that some of these genes may still be functional, perhaps producing a smaller transcript, and that some may have become non-functional recently enough that non-synonymous substitutions have not had a chance to accumulate. Nevertheless, it appears that there are position effects on genes near to but not interrupted by breakpoints, perhaps due cis effects of chromatin in the breakpoint region, leading to changes in expression. A genome-wide expression assay would be needed to define the major trend for the genes that have been disrupted but this approach may be complicated by the scarcity of tissues available from this endangered species. Breakage regions were found to co-localize with repeats. Whereas the known link between simple repeats and fragile genomic regions makes this observation intriguing, it is difficult to predict a cause-effect relationship between these repeats and the gibbon breakpoints. For many breakpoints we could readily observe that simple repeats were the result of gibbon-specific insertions by the repair mechanism after the break occurred. We therefore defined them as ‘‘filling’’ (Table 2) and we can assume that they followed the double-strand breaks. Our data point to a role for both Non-Allelic Homologous Recombination (NAHR) and non-homologous end joining (NHEJ) in double-strand break repair, with a prevalence of the latter. In 9 cases NAHR was driven by either Alu-Alu or SD mediated recombination (Table S2). In additional 15 cases, where long stretches of homology were not detected, we observed micro-homology or ‘‘filling’’ sequences which are both signs of NHEJ [25]. In NHEJ the double-strand breaks are fused together without a requirement for extensive homology. For the remaining breakpoints it was not possible to pinpoint a mechanism, even though the absence of homology would lead us to speculate that NHEJ or some other complex mechanism occurred in most of them [25]. While seeking a mechanism associated to the chromosomal reshuffling of gibbon species, our approach was to investigate Alu elements in more detail, given their higher concentration at the breakpoints. Independent evidence shows that this family of retrotransposons is particularly active in gibbons [26], strength- ening our hypothesis. Our in silico and experimental data suggested that CpG cytosines in Alus are less methylated in gibbon than in human. CpG methylation has a major role in epigenetic PLoS Genetics | www.plosgenetics.org 7 June 2009 | Volume 5 | Issue 6 | e1000538 AAAAAAAAAATTTTCT AATTCCAA ATACTACA(TA)3GA(TA)5TCCT ATTCCAAGCCATATATTATTGG CTCCAACCTT GGGTTTCAGGG TGGTATGGAGCGAGCACCTCA AAAA AAC ATGATG GAAATAGAAATAAAAAC AAGGTG CA TG GA