Enhancers facilitate the birth of de novo genes and their integration into regulatory networks

Regulatory networks control the spatiotemporal gene expression patterns that give rise to and define the individual cell types of multicellular organisms. In eumetazoa, distal regulatory elements called enhancers play a key role in determining the structure of such networks, particularly the wiring diagram of \"who regulates whom.\" Mutations that affect enhancer activity can therefore rewire regulatory networks, potentially causing changes in gene expression that are adaptive. Here, we use whole-tissue and single-cell transcriptomic and chromatin accessibility data from mouse to show that enhancers play an additional role in the evolution of regulatory networks: They facilitate network growth by creating transcriptionally active regions of open chromatin that are conducive to de novo gene evolution. Specifically, our comparative transcriptomic analysis with three other mammalian species shows that young, mouse-specific intergenic open reading frames are preferentially located near enhancers, whereas older open reading frames are not. Mouse-specific intergenic open reading frames that are proximal to enhancers are more highly and stably transcribed than those that are not proximal to enhancers or promoters, and they are transcribed in a limited diversity of cellular contexts. Furthermore, we report several instances of mouse-specific intergenic open reading frames that are proximal to promoters that show evidence of being repurposed enhancers. We also show that open reading frames gradually acquire specific interactions with enhancers over macro-evolutionary timescales, helping integrate new genes into existing regulatory networks. Taken together, our results highlight a dual role of enhancers in expanding and rewiring gene regulatory networks.