Distal CpG islands can serve as alternative promoters to transcribe genes with silenced proximal promoters.

DNA methylation at the promoter of a gene is presumed to render it silent, yet a sizable fraction of genes with methylated proximal promoters exhibit elevated expression. Here, we show, through extensive analysis of the methylome and transcriptome in 34 tissues, that in many such cases, transcription is initiated by a distal upstream CpG island (CGI) located several kilobases away that functions as an alternative promoter. Specifically, such genes are expressed precisely when the neighboring CGI is unmethylated but remain silenced otherwise. Based on CAGE and Pol II localization data, we found strong evidence of transcription initiation at the upstream CGI and a lack thereof at the methylated proximal promoter itself. Consistent with their alternative promoter activity, CGI-initiated transcripts are associated with signals of stable elongation and splicing that extend into the gene body, as evidenced by tissue-specific RNA-seq and other DNA-encoded splice signals. Furthermore, based on both inter- and intra-species analyses, such CGIs were found to be under greater purifying selection relative to CGIs upstream of silenced genes. Overall, our study describes a hitherto unreported conserved mechanism of transcription of genes with methylated proximal promoters in a tissue-specific fashion. Importantly, this phenomenon explains the aberrant expression patterns of some cancer driver genes, potentially due to aberrant hypomethylation of distal CGIs, despite methylation at proximal promoters.