Meta-analysis of human methylomes reveals stably methylated sequences surrounding CpG islands associated with high gene expression

DNA methylation is thought to play an important role in the regulation of mammalian gene expression. Part of the evidence for this role is the observation that lack of CpG island methylation in gene promoters is associated with high transcriptional activity. However, CpG island methylation level only accounts for a fraction of the variance in gene expression, and methylation in other domains is hypothesized to play a role (e.g., island shores and shelves). We set out to improve understanding of the human methylome through a meta-analysis approach, using 1737 samples from 30 publicly available studies. An initial screen identified 15224 CpGs that are “ultra-stable” in their state, being always fully methylated or unmethylated across diverse tissues, cell types and developmental stages (974 always methylated; 14250 always unmethylated). A further analysis of ultra-stable CpGs led us to identify a novel class of CpG islands, “ravines”, that exhibit a markedly consistent pattern of low methylation with highly methylated flanking shores and shelves. Our findings were validated using independent and heterogeneous datasets assayed on the same and different technologies. Building on additional existing data types such as gene expression microarrays, DNase hypersensitive sites, and histone modifications, we found that ravines are associated with higher gene expression, compared to typical unmethylated CpG islands. This finding suggests a novel role for methylation in promoters, markedly different from the traditional view that active promoters need to be unmethylated. We propose ravines are a new class of CpG islands, established early in development and maintained through differentiation, that mark universally active genes and provide new evidence that methylation beyond the CpG island could play a role in gene expression.Science, Faculty ofGraduat