Evolutionary Breakpoints in the Gibbon Suggest Association between Cytosine Methylation and Karyotype Evolution Lucia Carbone1*, R. Alan Harris2, Gery M. Vessere1¤a, Alan R. Mootnick3, Sean Humphray4¤b, Jane Rogers4¤c, Sung K. Kim5, Jeffrey D. Wall5, David Martin1, Jerzy Jurka6, Aleksandar Milosavljevic2, Pieter J. de Jong1 1 Children’s Hospital and Research Center Oakland, Oakland, California, United States of America, 2 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America, 3 Gibbon Conservation Center, Santa Clarita, California, United States of America, 4 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, United Kingdom, 5 Institute for Human Genetics, University of California San Francisco, San Francisco, California, United States of America, 6 Genetic Information Research Institute, Mountain View, California, United States of America Abstract Gibbon species have accumulated an unusually high number of chromosomal changes since diverging from the common hominoid ancestor 15–18 million years ago. The cause of this increased rate of chromosomal rearrangements is not known, nor is it known if genome architecture has a role. To address this question, we analyzed sequences spanning 57 breaks of synteny between northern white-cheeked gibbons (Nomascus l. leucogenys) and humans. We find that the breakpoint regions are enriched in segmental duplications and repeats, with Alu elements being the most abundant. Alus located near the gibbon breakpoints (,150 bp) have a higher CpG content than other Alus. Bisulphite allelic sequencing reveals that these gibbon Alus have a lower average density of methylated cytosine that their human orthologues. The finding of higher CpG content and lower average CpG methylation suggests that the gibbon Alu elements are epigenetically distinct from their human orthologues. The association between undermethylation and chromosomal rearrangement in gibbons suggests a correlation between epigenetic state and structural genome variation in evolution. Citation: Carbone L, Harris RA, Vessere GM, Mootnick AR, Humphray S, et al. (2009) Evolutionary Breakpoints in the Gibbon Suggest Association between Cytosine Methylation and Karyotype Evolution. PLoS Genet 5(6): e1000538. doi:10.1371/journal.pgen.1000538 Editor: Anne C. Ferguson-Smith, University of Cambridge, United Kingdom Received February 27, 2009; Accepted May 26, 2009; Published June 26, 2009 Copyright: 2009 Carbone et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: No funding was available to support this work. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lcarbone@chori.org ¤a Current address: Illumina, Hayward, California, United States of America ¤b Current address: Illumina Cambridge, Little Chesterford, Saffron Walden, Essex, United Kingdom ¤c Current address: Biotechnology and Biological Science Research Council (BBSRC), Swindon, Wiltshire, United Kingdom Introduction Gibbons (Hylobatidae) are small arboreal apes that inhabit the tropical and semi-deciduous forests of Southeast Asia and a portion of South- and East-Asia; their closest relatives are the great apes (human, chimpanzee, gorilla and orangutan). They are an excellent model in which to study mechanisms of chromosomal rearrangement during evolution, because their chromosomes have been accumulating changes at an accelerated rate in comparison to other apes [1–3]. As a result of this instability, the four genera of the gibbon family possess four different karyotypes (2n from 38 to 52). The genome shuffling observed in gibbons is in striking contrast to the high degree of karyotype conservation found in the other hominoids: there is only a single inter-chromosomal rearrangement separating humans from the great apes [4], but more than 40 such rearrangements have taken place on the gibbon lineage. Recent estimates based on the inferred karyotype of the common gibbon ancestor suggest that the rate of chromosomal rearrangements in these species is 20 times higher than in other primates [3]. Given the great taxonomic diversity found within the family (four genera and fifteen species), it is tempting to speculate that segregating chromosomal changes mediated the speciation events in a relatively short time. The cause of this abundance of chromosomal changes is still undefined [5]. Primate genomes harbor millions of interspersed repetitive elements [6], creating numerous opportunities for Non-Allelic Homologous Recombination (NAHR) events to produce deletions, duplications and chromosomal rearrangements. Chromosomal rearrangements caused by NAHR are nevertheless quite rare, and even on an evolutionary time scale mammalian chromosomes have proven to be very stable. Comparison of multiple mammalian karyotypes indicates that the average rate of gross chromosomal rearrangements is only approximately two events over 10 million years [7]. Many repetitive DNA elements are rich in CpGs, which in mammalian cells are typically methylated. CpG methylation is an essential component of epigenetic mechanisms that maintain repetitive elements in a transcriptionally repressed state, thereby suppressing their proliferation [8,9]. Cancer cells frequently exhibit a global decrease in genomic 5-methylcytosine, and it has been speculated that hypomethylation of repeat elements is an underlying factor in the high frequency of chromosomal rearrangements in cancer cells [10]. PLoS Genetics | www.plosgenetics.org 1 June 2009 | Volume 5 | Issue 6 | e1000538