Abstract

Short contiguous regions of non-methylated DNA , known as CpG islands (CGIs), are found associated with around 70% of mammalian gene promoters. In cold-blooded vertebrates, CGI prediction algorithms indicate that these elements are rarely coincident with gene promoters, signifying a major divergence in vertebrate promoter architecture. Biochemical purification of non-methylated DNA in seven diverse vertebrates instead reveals that non-methylated islands of DNA (NMIs) are in fact a central feature of vertebrate gene promoters. Importantly, comparison of orthologous vertebrate gene promoters reveals that NMIs appear to be under an unexpected degree of selective pressure to be maintained across evolutionary time. Alongside ‘canonical’ promoter-associated NMIs, genome-wide profiling of non-methylated DNA revealed two further conserved classes of NMIs: ‘plastic’ and ‘broad’. ‘Plastic’ NMIs are distal to gene promoters and exhibit dynamic methylation between distinct tissues and developmental stages while a novel ‘broad’ class of NMIs encompass developmental transcription factors subject to polycomb-mediated repression. Together our findings demonstrate an ancient logic for NMI usage at gene regulatory elements and reveal an unprecedented level of epigenetic conservation across vertebrate evolution.