show Abstracthide AbstractChanges in gene regulation have long been though to underlie most phenotypic differences between species. Subterranean rodents, and in particular the naked mole-rat (NMR), have attracted substantial attention due to their proposed phenotypic adaptations, which include hypoxia tolerance, metabolic changes and cancer resistance. However, it is largely unknown what regulatory changes may associate with these phenotypic traits, and whether these are unique to the NMR, the mole-rat clade or also present in other mammals. Here, we undertook a comparative genomics approach to identify genome-wide promoter and enhancer regions harbouring epigenomic hallmarks of regulatory activity, in heart and liver from two mole-rat species (NMR and DMR) and two rodent outgroups. To identify promoters and enhancers displaying robust shifts in regulatory activity in the mole-rat clade, we adapted and applied a phylogenetic modeling approach to quantitatively compare epigenomic signals at orthologous locations, while accounting for phylogenetic distance and inter-species variation. This method identified thousands of orthologous promoter and enhancer regions with increased activity in ancestral or single-species mole-rat branches, as well as hundreds of promoters and enhancers with reduced activity in mole-rats versus other rodents. These elements underlie both shared tissue-specific changes in gene regulation associated with mole-rat evolution, which include metabolic and functional adaptations in heart and liver. Moreover, by comparing mole-rat specific changes in promoters and enhancers between ancestral and single-species branches, our data revealed a number of candidate pathways with stepwise regulatory changes during mole-rat evolution. Lastly, we analysed the genomic properties of non-alignable promoters and enhancers in mole-rats, and report (i) their overlap with specific repetitive elements and transcription factor binding sites; and (ii) their association with metabolic gene functions. On the whole, these comparative analyses reveal mole-rat specific epigenomic changes across orthologous and non-mappable promoters and enhancers - which inform previously reported mole-rat adaptations from a gene regulation perspective. Overall design: Chromatin immunoprecipitation sequencing (ChIP-seq) for histone postranslational modifications H3K27ac, H3K4me3 and H3K4me1 in liver and heart tissues from naked mole-rat, damaraland mole-rat, guinea pig and mouse (with three to six biological replicates)