Orphan CpG islands define a novel class of highly active enhancers.

CpG islands (CGI) are critical genomic regulatory elements that support transcriptional initiation and are associated with the promoters of most human genes. CGI are distinguished from the bulk genome by their high CpG density, lack of DNA methylation, and euchromatic features. While CGI are canonically known as strong promoters, thousands of 'orphan' CGI lie far from any known transcript, leaving their function an open question. We undertook a comprehensive analysis of the epigenetic state of orphan CGI across over 100 cell types. Here we show that most orphan CGI display the chromatin features of active enhancers (H3K4me1, H3K27Ac) in at least one cell type. Relative to classical enhancers, these enhancer CGI (ECGI) are stronger, as gauged by chromatin state and in functional assays, are more broadly expressed, and are more highly conserved. Likewise, ECGI engage in more genomic contacts and are enriched for transcription factor binding relative to classical enhancers. In human cancers, these epigenetic differences between ECGI vs. classical enhancers manifest in distinct alterations in DNA methylation. Thus, ECGI define a class of highly active enhancers, strengthened by the broad transcriptional activity, CpG density, hypomethylation, and chromatin features they share with promoter CGI. In addition to indicating a role for thousands of orphan CGI, these findings suggests that enhancer activity may be an intrinsic function of CGI in general and provides new insights into the evolution of enhancers and their epigenetic regulation during development and tumorigenesis.