Regulatory activity and evolutionary co-option of cis-regulatory elements underlying bear hibernation.

Uncovering the regulatory architectures that underlie complex phenotypes can provide insight into both the mechanisms and evolution of unique adaptations. In bears, thousands of genes are differentially expressed in a tissue-specific manner during hibernation, many of which are involved in major vertebrate metabolic signaling pathways. However, the precise regulatory mechanisms driving these gene expression changes, and the extent of their conservation in nonhibernating mammals, remain poorly understood. Using capped-small RNA-sequencing from brown bear adipose tissue, we identify putative enhancers that exhibit dynamic shifts in regulatory activity during hibernation. The majority of these enhancers share sequence homology with known human enhancers, yet many appear to target distinct genes, suggesting a role of regulatory co-option in the evolution of hibernation. Using these newly identified enhancers, we identify transcription factors putatively underlying hibernation gene expression, expanding our mechanistic understanding of hibernation physiology. Additionally, we find evidence for selection on cis-regulatory sequences associated with physiological adaptation across bears. Collectively, this study provides new perspectives on the mechanisms and evolution of mammalian hibernation and the roles of regulatory sequences in the evolution of complex physiological adaptations.