6798–6807 Nucleic Acids Research, 2007, Vol. 35, No. 20 doi:10.1093/nar/gkm489 Published online 10 October 2007 CG dinucleotide clustering is a species-specific property of the genome Jacob L. Glass1, Reid F. Thompson 1 , Batbayar Khulan 1 , Maria E. Figueroa 2 , Emmanuel N. Olivier3, Erin J. Oakley4, Gary Van Zant4, Eric E. Bouhassira3,5, Ari Melnick 2 , Aaron Golden 6 , Melissa J. Fazzari7 and John M. Greally1,5,* 1Department of Molecular Genetics, 2Department of Developmental and Molecular Biology and 3Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA, 4Division of Hematology/Oncology, University of Kentucky, Markey Cancer Center, 800 Rose Street, Lexington KY 40536, USA, 5Department of Medicine (Hematology), Albert Einstein College of Medicine, Bronx, NY 10461, USA, 6Department of Information Technology, National University of Ireland Galway, Newcastle Road, Galway, Republic of Ireland and 7Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA Received February 9, 2007; Revised May 23, 2007; Accepted June 6, 2007 ABSTRACT Cytosines at cytosine-guanine (CG) dinucleotides are the near-exclusive target of DNA methyltrans- ferases in mammalian genomes. Spontaneous deamination of methylcytosine to thymine makes methylated cytosines unusually susceptible to mutation and consequent depletion. The loci where CG dinucleotides remain relatively enriched, presumably due to their unmethylated status during the germ cell cycle, have been referred to as CpG islands. Currently, CpG islands are solely defined by base compositional criteria, allowing annotation of any sequenced genome. Using a novel bioinformatic approach, we show that CG clusters can be identified as an inherent property of genomic sequence without imposing a base compositional a priori assumption. We also show that the CG clusters co-localize in the human genome with hypomethylated loci and annotated transcription start sites to a greater extent than annotations produced by prior CpG island definitions. Moreover, this new approach allows CG clusters to be identified in a species-specific manner, revealing a degree of orthologous conservation that is not revealed by current base compositional approaches. Finally, our approach is able to identify methylating genomes (such as Takifugu rubripes) that lack CG clustering entirely, in which it is inappropriate to annotate CpG islands or CG clusters. INTRODUCTION Several observations have converged to focus attention on cytosine-guanine (CG) dinucleotide clusters in mamma- lian genomes. Digestion of genomic DNA with HpaII allowed the isolation of loci where these restriction sites cluster and are unmethylated in cis, deﬁning a population of loci referred to as HpaII tiny fragments (1). Upon sequencing, these loci were found to be unusually rich in CG dinucleotides and (G+C) content when compared with other sequences in the 1985 Genbank database (2). The CpG island base compositional criteria now used for genomic annotation are derived from this original set of experiments. With the appreciation that methylcytosine is unusually susceptible to mutation through deamination to thymine (3), a logical conclusion was that the absence of methylation at cytosines in CpG islands protected them from mutational decay during evolution. The implicit assumption is that these loci are universally unmethylated in normal cells but can be the target of abnormal methylation in cancer, or in unusual epigenetic regulatory processes such as genomic imprinting or X chromosome inactivation (4,5). CpG islands have proven valuable in focusing the study of the widespread genomic changes that occur in these processes, and are commonly used in designing custom microarrays for that purpose. CpG islands have also been used as a foundation for bioinfor- matic analyses such as ﬁnding gene promoters (6) and identifying sequence features that distinguish imprinted genes (7). Despite its proven utility, there are problems with the original deﬁnition of CpG islands, including its lack of speciﬁcity. Using base compositional criteria alone, CpG island annotations identify over 350 000 sites in the *To whom correspondence should be addressed. Tel: +1 718 430 2875; Fax: +1 718 824 3153; Email: jgreally@aecom.yu.edu 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.