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 Engrailed 1 (EN1) transcription factor correlates with the onset of eccrine gland formation. In mice, regulation of ectodermal En1 expression is a major determinant of natural variation in eccrine gland density between strains, and increased En1 expression promotes the specification of more eccrine glands. Here, we show that regulation of EN1 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 EN1 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 EN1 expression, indicating this element positively regulates EN1 in this context. In a humanized enhancer knock-in mouse, hECE18 increased developmental En1 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.

人类通过出汗来冷却身体，并且在灵长类动物中拥有迄今为止最高的外分泌汗腺密度。长期以来，人类的高小汗腺密度一直被认为是人类进化适应的标志，但其遗传基础一直未知。在人类中，Engrailed 1 ( EN1 ) 转录因子的表达与小汗腺形成的开始相关。在小鼠中，外胚层En1表达的调节是品系之间小汗腺密度自然变化的主要决定因素，增加的En1表达促进更多小汗腺的规范化。在这里，我们展示了EN1的规定专门针对人类血统进化，以促进外分泌腺的形成。使用比较基因组学和小鼠外胚层增强剂活性的验证，我们确定了人类EN1皮肤增强剂 hECE18。我们表明，与培养的角质形成细胞中的非人猿直系同源物相比，人类 ECE18 增强子中的多个上位相互作用衍生替代增加了其活性。在人类培养的角质形成细胞中抑制 hECE18 会特异性减弱EN1表达，表明该元素在这种情况下正向调节EN1。在人源化增强子敲入小鼠中，hECE18 增加了发育En1在皮肤中表达以诱导更多外分泌腺的形成。我们的研究揭示了促成最奇异的人类适应性进化之一的遗传基础，并将单个增强子中的多个相互作用突变作为人类进化变化的机制。