6806 Nucleic Acids Research, 2007, Vol. 35, No. 20 appears to be necessary for CG decay, it is not suﬃcient to cause local preservation of clustered CG dinucleotides. Furthermore, we can conclude that any annotation of the fugu genome to indicate the presence of CpG islands or CG clusters is inappropriate. DISCUSSION The approach of testing for CG clustering reveals the loci at which CG dinucleotide decay occurred at a markedly lower rate than it did in the rest of the genome. Unlike CpG islands, CG clusters occur with a large range of (G+C) content and O/E CG ratios, revealing the unusual CG density of loci such as alpha satellite DNA sequences, targets for the DNMT3B methyltransferase (27) that are very (A+T)-rich and are consequently not deﬁned as distinctive using traditional CpG island base composi- tional criteria. While this is an example of the CpG island deﬁnition being excessively restrictive, the deﬁnition also suﬀers from the problem of identifying a large number of sites located within transposable elements. It is certainly possible that these retroelements may have cis-regulatory eﬀects (28), but their overall tendency to be methylated (11) diminishes the usefulness of the CpG island annota- tion as a mark of unmethylated DNA in the genome, a major use of the annotation in cancer epigenomics, especially in deﬁning the CpG island methylator pheno- type (CIMP), which requires that methylation of a given CpG island be distinctive in neoplastic cells (29). The approach used in the creation of CpG island browser tracks involves using sequences from which repetitive elements have previously been removed, increasing the likelihood of identifying constitutively unmethylated, presumably cis-regulatory sequences, but even this approach deﬁnes a number of sites that are methylated in normal cells (12,13). Our HELP data indicate that CG clusters perform better than CpG islands at identifying unmethylated sites in the genome. However, our data do not support CG clusters being universally unmethylated, as we ﬁnd satellite DNA and young retrotransposons to encode CG clusters, and these sequences are normally methylated (11,27). We propose that what distinguishes clustered from non-clustered CGs in the genome is the greater stability of associated epigenetic marks, such as hypo- methylation at gene promoters or methylation of alpha satellite DNA. CpG islands have recently been described to be located at the bivalent domains of histone tail modiﬁcations in embryonic stem cells (30), reinforcing the rationale for using these loci as a means of identifying candidate cis-regulatory sites in the genome. CpG islands represent a foundation annotation of the genome on which other annotations are built, for example contributing to algo- rithms to identify gene promoters (31). However, we show (Figure 3) that the CG cluster annotation performs substantially better than the CpG island annotation in localizing to known promoters (as represented by refSeq transcription start sites), indicating that an improved foundation annotation like CG clusters may improve the performance of algorithms currently using CpG islands. Because identical criteria have been used to deﬁne CpG islands in diﬀerent species, in which CG clustering can have markedly diﬀerent characteristics, CpG islands have been thought to be poorly conserved between species, especially with the focus on human/mouse comparisons (21). We show that a species-speciﬁc deﬁnition of CG clusters reveals an unexpected degree of conservation of this annotation between human and mouse. We anticipate that conserved CG clusters will represent a subset of loci of exceptional functional importance in the genome. However, it is also clear (Figure 7) that it is inappropriate to annotate all methylating genomes for the presence of CG-dense regions. Fugu is annotated at genome browsers (UCSC and Ensembl) for the presence of CpG islands despite the fact that it does not have a distinctive population of loci maintaining CG content in an overall genomic context of CG decay. Zebraﬁsh, on the other hand, with a similar degree of CG decay, manifests the two populations of CG content. Interestingly, the methylation of cytosines in the zebraﬁsh genome includes a substantial proportion at non-CG dinucleotide sites (32), yet the selective preservation of CG content at a subset of loci occurs in this genome. CONCLUSIONS We show that CG clusters, when present in a genome, deﬁne themselves as a distinctive population of loci. This novel annotation performs better at identifying promoters and hypomethylated DNA than current CpG island deﬁnitions, and allows a species-speciﬁc deﬁnition of CG clusters that reveals a previously unsuspected degree of conservation of this sequence feature. The species speciﬁcity of what deﬁnes a CG cluster indicates that CG dinucleotides only need to be enriched within the context of their genome to be distinctive and presumably functional. We expect that the annotations of CG clusters will prove valuable to those studying the genome as well as the epigenome, so we have provided the human and mouse annotations as a resource for public use at http:// greallylab.aecom.yu.edu/cgClusters/ SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. URLS The UCSC Genome Browser (http://genome.ucsc.edu/), the ENSEMBL Genome Browser (http://www.ensembl. org/), the Greally lab CG cluster webpage (http:// greallylab.aecom.yu.edu/cgClusters/), the R project (http://www.r-project.org/) and the CpG island searcher website with the cpgi130 program (http://cpgislands.usc. edu/).