Auricular Malformations Driven by Copy Number Variations in a Hierarchical Enhancer Cluster and Dominant Enhancer Recapitulates Human Pathogenesis

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 HMX1 downstream enhancer is associated to ear malformations, the mechanisms underlying bilateral constricted ear (BCE) remain unclear. Here, we identify a copy number variation (CNV) containing three enhancerscollectively 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 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. Using of scRNA-seq compare transcriptional difference between wild type and mEC1 knock-in transgenic mouse models.增强子（enhancers）通过与转录因子（transcription factors, TFs）的协同作用，决定基因表达的空间特异性与表达水平，其异常可引发疾病。尽管HMX1下游增强子与耳部畸形相关，但双侧缩耳症（bilateral constricted ear, BCE）的潜在致病机制仍不明确。本研究鉴定出一段包含三个增强子的拷贝数变异（copy number variation, CNV），该变异被命名为位置身份层级增强子簇（positional identity hierarchical enhancer cluster, PI-HEC），可通过协同调控HMX1的表达驱动BCE的发生。每个增强子均具有独特的活性-位置-结构特征；其中占主导地位的增强子分别携带高迁移率族（high mobility group, HMG）框基序与同源结构域转录因子基序，分别调控其活性与特异性。小鼠模型实验表明，耳廓基部中Hmx1表达异常的神经嵴来源成纤维细胞，加上异位的耳廓远端表达，会破坏外耳发育，影响软骨、肌肉与表皮组织。本研究阐明了哺乳动物耳部形态发生的机制，并凸显了增强子之间以及增强子与转录因子之间协同调控的复杂性。本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）技术，比较野生型与mEC1敲入转基因小鼠模型之间的转录组差异。