This study identifies three periods of regulatory innovation during vertebrate evolution. Using conserved non-exonic elements (CNEEs), the researchers analyzed genome-wide regulatory regions in five vertebrates, including humans, to determine when and where regulatory elements evolved. The first period, from the vertebrate ancestor to about 300 million years ago, was characterized by regulatory gains near transcription factors and developmental genes. The second period, from 300 to 100 million years ago, saw innovations near extracellular signaling genes, with a decline in innovations near trans-dev genes. The third period, in placental mammals, showed a rise in innovations near genes involved in post-translational protein modification, while innovations near trans-dev and receptor genes declined to background levels. The study found that regulatory innovations are often associated with specific gene functions. For example, genes involved in post-translational protein modification showed increased regulatory innovations, while genes related to development and transcription factors showed decreased innovations. The analysis also revealed that regulatory elements are often associated with ancient genes, as seen in the case of the PRKD1 gene, which dates back to at least the tetrapod ancestor. The study used various methods, including genome-wide alignments and experimental techniques like ChIP-seq and DNase hypersensitivity, to identify and analyze regulatory elements. These methods confirmed that CNEEs are enriched for regions under purifying selection and are often functional regulatory elements. The results were consistent across multiple vertebrate lineages, including mammals, fish, and birds. The findings suggest that regulatory innovations have played a significant role in vertebrate evolution, influencing traits such as pigmentation, bristle patterns, and skeletal differences. The study also highlights the importance of evolutionary conservation in identifying regulatory elements and the role of different functional gene categories in regulatory innovation over time. The results provide insights into the evolutionary dynamics of gene regulation and the factors that drive regulatory changes in vertebrates.This study identifies three periods of regulatory innovation during vertebrate evolution. Using conserved non-exonic elements (CNEEs), the researchers analyzed genome-wide regulatory regions in five vertebrates, including humans, to determine when and where regulatory elements evolved. The first period, from the vertebrate ancestor to about 300 million years ago, was characterized by regulatory gains near transcription factors and developmental genes. The second period, from 300 to 100 million years ago, saw innovations near extracellular signaling genes, with a decline in innovations near trans-dev genes. The third period, in placental mammals, showed a rise in innovations near genes involved in post-translational protein modification, while innovations near trans-dev and receptor genes declined to background levels. The study found that regulatory innovations are often associated with specific gene functions. For example, genes involved in post-translational protein modification showed increased regulatory innovations, while genes related to development and transcription factors showed decreased innovations. The analysis also revealed that regulatory elements are often associated with ancient genes, as seen in the case of the PRKD1 gene, which dates back to at least the tetrapod ancestor. The study used various methods, including genome-wide alignments and experimental techniques like ChIP-seq and DNase hypersensitivity, to identify and analyze regulatory elements. These methods confirmed that CNEEs are enriched for regions under purifying selection and are often functional regulatory elements. The results were consistent across multiple vertebrate lineages, including mammals, fish, and birds. The findings suggest that regulatory innovations have played a significant role in vertebrate evolution, influencing traits such as pigmentation, bristle patterns, and skeletal differences. The study also highlights the importance of evolutionary conservation in identifying regulatory elements and the role of different functional gene categories in regulatory innovation over time. The results provide insights into the evolutionary dynamics of gene regulation and the factors that drive regulatory changes in vertebrates.