least one interspersed repeat. Alus and L1 LINEs are the most frequently represented, followed by simple repeats, as illustrated in Table 1. In 11 instances, one or more repeats span the breakpoint site in the gibbon. This could result either from an insertion after the breakage, or from a recombination event (Figure 2A). In the remaining cases, the repeats flank the breakpoint, and they are frequently truncated by the rearrangement event. Moreover, three breakpoints are next to blocks of repeats that were inserted sequentially in the gibbon genome, creating complex arrange- ments (Table S2). Out of the 57 breakpoints, 11 co-localize with simple repeats of various types. Most of these breakpoints (6 out of 11) overlap with (AT)n-rich repeats which are either gibbon specific (CH271- 254H12, CH271-171B20 and CH271-122E24) or shared by human (CH271-228C1, CH271-86M19, CH271-40A18). A different case is the breakpoint of a translocation HSA 3–5 that falls in the intra-genic tandemly repeated region (TR) of the mucin gene MUC4 (3q29). Analysis of Alu CpG content and methylation We were intrigued by the predominance of Alus at the breakpoint sites, as Alu-Alu recombination events have been reported as examples of Non Allelic Homologous Recombination (NAHR) [20,21]. We verified that the proportion of Alus associated with breakpoints was significant when compared to other repeats by using a random sampling simulation (Figure S3A) (p = 0.001). At the same time this method showed that the association with LINE L1 in human was lower than expected by chance (Figure S3B). We then looked for features of Alus that may be distinctive in gibbon compared to other hominoids. To carry out this analysis we used the 23 assembled BACs to represent portions of the gibbon genome surrounding the breakpoints. First, we observed a decline in Alu density within the BACs with increasing distance from the breakpoints (Figure 2B). Furthermore, Alu fragments at Table 1. Detailed count of interspersed and simple repeats at the breakpoints. Family SINE LINE LTR SIMPLE Other Repeat Alu S Alu J Alu Y MIR L1 L2 LTR (AT)n (CA)n Other simple SVA HERVL Charlie Tigger 3b HSMAR 2 Count 17 17 4 3 3 3 8 6 2 3 1 1 1 1 1 Total 27 20 8 11 5 The regions that were analyzed for repeat content extended for 500 bp on each side of the breakpoint site. Only repeats at a distance ,150 bp were counted and reported in this table and in Table S2. doi:10.1371/journal.pgen.1000538.t001 Evolutionary Breakpoints in Gibbon or near (,150 bp) the breakpoints were almost twice as CpG-rich as the remaining Alu sequences in the same BAC (4.5 CpGs/ 100 bp compared to 2.4 CpGs/100 bp; t-test p,0.001). As shown in Figure 2C, the number of CpG doublets per 100 bp of Alu sequence declines rapidly as the distance from the breakpoint increases. Active Alus contain a relatively high number of CpG dinucleotides, which are linked to active retrotransposition [9]. Normally, the epigenetic apparatus of the cell suppresses the activity of retrotransposons by adding methyl groups to cytosines in CpGs [22,23]. Methyl-C tends to decay to T or A (therefore CpG become TpG/CpA) through a process known as CpG decay [22]. Our data thus suggest a higher concentration of ‘‘active’’ Alus associated with breakpoints. We hypothesized that the higher rate of chromosomal breakage observed in gibbons is due to an active epigenetic state of these elements in the gibbon as compared to the common ancestor of the hominoids; the higher CpG content of these Alus suggests that they have been less methylated and consequently that they may have a different epigenetic state. The hypothesis predicts reduced CpG methylation of the gibbon breakpoint Alus in comparison to their human orthologues. We tested this prediction by performing bisulfite allelic sequencing of 14 orthologous Alus in human and gibbon, 8 of which were located near the breakpoint sites (,150 bp from the breakpoint) and 6 Alus outside of breakpoint regions but with similar CpG content to the breakpoint Alus (Materials and Methods and Table S5). As orthologous Alus are inserted in the genome of the common ancestor, we can safely assume that the CpG groups had the same amount of time to be methylated. Our results (Figure 3) demonstrate a significant reduction of CpG methylation in gibbon compared to human (p,0.001, Mann-Whitney U test). Discussion Gibbon species carry an extraordinary number of chromosomal rearrangements, accumulated in a relatively short evolutionary time (15–18 mya). In order to uncover a possible genetic source for the genomic reshuffling observed in these species, we carried on a detailed analysis of 57 sequenced synteny breakpoints between the northern white cheeked gibbon (NLE) and human. Our molecular analysis revealed a scenario which, at a first glance, is similar to that described in other primates [19], where segmental duplications and repeats play a major role in chromosomal rearrangements (Figure 4). But a broader analysis, which took into account epigenetic modifications, uncovered a possible explana- tion for the high frequency of evolutionary chromosomal changes. The gibbon breakpoints are associated with Alu elements that have an unusually high CpG content, and in the gibbon these Alu elements are less methylated than their human orthologues. This may indicate that the epigenetic state of these Alus has predisposed them to recombination. In this study we were able to confirm the correlation between breakpoints and human SD which we had reported previously [11]. The higher resolution achieved in the present study, and the availability of gibbon sequences, allowed us to confirm association of the breakpoints with gibbon-specific SDs. As many breakpoints could not be mapped, due to the presence of these duplications (Table S2), the overlap is very likely to be more frequent than we have been able to demonstrate. It is noteworthy that we found only two breakpoints where SDs were present in both gibbon and human. As the intersection between gSD and hSD over the whole genome is much higher (32%), this observation suggests that the chromosomal rearrangements are mainly associated with ‘‘species- specific’’ duplications. The two cases of breakpoints in shared PLoS Genetics | www.plosgenetics.org 4 June 2009 | Volume 5 | Issue 6 | e1000538