Repeated mutation of a developmental enhancer contributed to human thermoregulatory evolution.

Abstract

Humans sweat to cool their bodies and have by far the highest eccrine sweat gland density among primates. Humans' high eccrine gland density has long been recognized as a hallmark human evolutionary adaptation, but its genetic basis has been unknown. In humans, expression of the <i>Engrailed 1</i> (<i>EN1</i>) transcription factor correlates with the onset of eccrine gland formation. In mice, regulation of ectodermal <i>En1</i> expression is a major determinant of natural variation in eccrine gland density between strains, and increased <i>En1</i> expression promotes the specification of more eccrine glands. Here, we show that regulation of <i>EN1</i> has evolved specifically on the human lineage to promote eccrine gland formation. Using comparative genomics and validation of ectodermal enhancer activity in mice, we identified a human <i>EN1</i> skin enhancer, hECE18. We showed that multiple epistatically interacting derived substitutions in the human ECE18 enhancer increased its activity compared with nonhuman ape orthologs in cultured keratinocytes. Repression of hECE18 in human cultured keratinocytes specifically attenuated <i>EN1</i> expression, indicating this element positively regulates <i>EN1</i> in this context. In a humanized enhancer knock-in mouse, hECE18 increased developmental <i>En1</i> expression in the skin to induce the formation of more eccrine glands. Our study uncovers a genetic basis contributing to the evolution of one of the most singular human adaptations and implicates multiple interacting mutations in a single enhancer as a mechanism for human evolutionary change.