Auricular malformations are driven by copy number variations in a hierarchical enhancer cluster and a dominant enhancer recapitulates human pathogenesis

Abstract

Abstract Enhancers, through the combinatorial action of transcription factors (TFs), dictate both the spatial specificity and the levels of gene expression, and their aberrations can result in diseases. While a HMX1 downstream enhancer is associated with ear malformations, the mechanisms underlying bilateral constricted ear (BCE) remain unclear. Here, we identify a copy number variation (CNV) containing three enhancers—collectively termed the positional identity hierarchical enhancer cluster (PI-HEC)—that drives BCE by coordinately regulating HMX1 expression. Each enhancer exhibits distinct activity-location-structure features, and the dominant enhancer with high mobility group (HMG)-box combined with Coordinator and homeodomain TF motifs modulating its activity and specificity, respectively. Mouse models demonstrate that neural crest-derived fibroblasts with aberrant Hmx1 expression in the basal pinna, along with ectopic distal pinna expression, disrupt outer ear development, affecting cartilage, muscle, and epidermis. Our findings elucidate mammalian ear morphogenesis and underscore the complexity of synergistic regulation among enhancers and between enhancers and transcription factors.