Supplementary Material for: Identification and functional study of enhancers of EYA1, the causative gene of branchio-oto-renal syndrome

Introduction: Branchio-oto-renal syndrome (BOR syndrome) is a rare genetic disorder with an incidence of 1 in 40,000, affecting the development of multiple organs, including the branchio, ear and kidney. It is responsible for 2% of childhood deafness. Currently, variants in the coding regions of the main causative genes, such as EYA1, SIX1, and SIX5, explain only half of the disease’s etiology. Therefore, there is a need to explore the non-coding regions, which constitute the majority of the genome, especially the regulatory regions, as potential new causative factors. Method: In this study, we focused on the EYA1 gene, which accounts for over 40% of BOR syndrome cases, and conducted a screening of candidate enhancers within a 250 kb region upstream and downstream of the gene using comparative genomics. We characterized the enhancer activities of these candidates in zebrafish using the Tol2 transposon system. Results: Our findings revealed that out of the 11 conserved non-coding elements (CNEs) examined, four exhibited enhancer activity. Notably, CNE16.39 and CNE16.45 displayed tissue-specific enhancer activity in the ear. CNE16.39required the full-length 206 bp sequence for inner-ear-specific expression, while the core functional region of CNE16.45 was identified as 136 bp. Confocal microscopy results demonstrated that both CNE16.39 and CNE16.45 drove the expression of GFP in the sensory region of the crista of the inner ear in zebrafish, consistent with the expression pattern of eya1. Conclusion: This study contributes to the understanding of the regulatory network governing EYA1 expression and offers new insights to further clarify the pathogenic role of EYA1 in BOR syndrome.

引言：鳃-耳-肾综合征（Branchio-oto-renal syndrome, BOR综合征）是一种罕见遗传性疾病，发病率为1/40000，可影响鳃、耳、肾等多个器官的发育，约占儿童耳聋病因的2%。目前，针对该病主要致病基因（如EYA1、SIX1及SIX5）编码区的变异仅能解释约半数的疾病发病机制，因此亟需探索占基因组绝大多数的非编码区域，尤其是调控区域，将其作为潜在的新型致病因素。研究方法：本研究聚焦于占BOR综合征病例超40%的EYA1基因，采用比较基因组学方法对该基因上下游250kb区域内的候选增强子开展筛选，并利用Tol2转座子系统在斑马鱼中对这些候选元件的增强子活性进行表征。研究结果：本研究发现，在检测的11个保守非编码元件（conserved non-coding elements, CNEs）中，有4个表现出增强子活性。其中，CNE16.39与CNE16.45在耳部展现出组织特异性增强子活性。CNE16.39实现内耳特异性表达需要完整的206bp序列，而CNE16.45的核心功能区域为136bp。共聚焦显微镜检测结果显示，CNE16.39与CNE16.45均可驱动绿色荧光蛋白（GFP）在斑马鱼内耳壶腹嵴的感觉区域表达，该表达模式与eya1的内源表达模式一致。研究结论：本研究有助于解析调控EYA1基因表达的调控网络，为进一步阐明EYA1在BOR综合征中的致病作用提供了新的研究视角。