6804 Nucleic Acids Research, 2007, Vol. 35, No. 20 demonstrated hypomethylation (Figure 5a), the absolute number of hypomethylated loci diﬀered (Figure 5b), as the hypomethylated CpG islands represent a subset of the larger group of hypomethylated CG clusters. The CG clusters identify 50% more hypomethylated loci than do CpG islands. We conclude that the CG cluster annotation is not only identifying more transcription start sites, it is also deﬁning loci with comparable epigenetic characteristics. We next addressed the question of why CpG islands are often not conserved between human and mouse (16,22). This is a puzzling issue if the CG-rich nature of the promoter is of functional importance, for example conferring the ubiquitous expression patterns that deﬁne housekeeping genes (18). It would be expected that such functional promoter characteristics would be conserved between species despite diﬀerences in overall CG dinu- cleotide content [observed/expected (O/E) CG ratios of 0.19 and 0.24 for mouse and human, respectively (22)]. It is therefore surprising that the total number of CpG islands in mouse is only 58% of the number annotated for the human genome (Table 1). When we performed the CG clustering analysis of the mouse genome, we found it also generates two popula- tions with distinct CG density characteristics, but that the optimal CG cluster deﬁnition for the mouse genome is diﬀerent from that of the human, corresponding to 24 or more CG dinucleotides in a sequence of no more than 585 bp in length (Figure 6). By comparison, human CG clusters consist of 27 CGs in no more than 571 bp. When we calculated the total number of CG clusters for the mouse genome, it was strikingly similar to that for the human (42 971 and 44 165, respectively, Table 1). In addition, when we re-analyzed a sample of 23 loci originally published to demonstrate the failure of CpG island conservation between these species (15), we found that while only 18 conserve CpG islands, 22 out of 23 conserve CG clusters, the single exception in this limited sample being the alpha globin orthologs (HBA1/Hba-a1). We extended this study to test conservation of each annotation genome-wide. Of all of the 27 801 CpG islands annotated at the UCSC Genome Browser, 14 452 have orthologous sequences with CpG islands in the mouse genome, while there exist 19 410 sites of conserved CG clustering (Table 1). When studied using our genome- speciﬁc annotations, clustered CG dinucleotides are demonstrably much more conserved between species than previously appreciated. We extended the CG clustering histogram analysis to eight more genomes, including other organisms that are known to methylate their genomes, those that do so only transiently (Drosophila melanogaster) (23), and those that do not methylate at all. The surprising result of these analyses is that the fugu (Tiger Blowﬁsh, Takifugu rubripes) genome, which has been described to methylate its DNA (24), does not exhibit uniquely CG-dense regions. What may explain this diﬀerence is that the degree of decay of CG dinucleotide content in the fugu genome is less than that of most genomes in which unique CG-dense regions emerge (Figure 7). The zebraﬁsh (Danio rerio) genome, on the other hand, does display uniquely Figure 5. The HELP assay (13) was used on a custom promoter microarray to test cytosine methylation patterns in two samples each of CD34+ hematopoietic stem and progenitor cells (CD34) and human embryonic stem cells (ES). In panel (a) it is apparent that similar proportions of sites are categorized as hypomethylated for CG clusters (C) and CpG islands (I). However, the absolute number of hypomethylated sites detected by the CG cluster annotation is markedly larger than that for the CpG island annotation [panel (b)]. We conclude that the CG cluster annotation is not only detecting larger numbers of transcription start sites (Figure 4), it extends the ability of the CpG island annotation to identify more hypomethylated sites in the genome. CG-dense regions with only marginally greater CG dinucleotide decay (O/E CG 0.53 as opposed to 0.57 in fugu). The remaining major diﬀerence between these genomes is that of size, the fugu genome being substan- tially smaller than the other methylating genomes at only 365 Mb total (25), a variable already suggested to be related to the evolution of cytosine methylation (26). Our data demonstrate that while cytosine methylation