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，下称BOR综合征）是一种罕见的遗传性疾病，发病率为1/40000，可累及鳃、耳与肾脏等多器官的发育，约占儿童耳聋病因的2%。目前，导致该病的主要致病基因（如EYA1、SIX1及SIX5）的编码区变异仅能解释半数的发病机制。因此，亟需探索占基因组绝大多数的非编码区，尤其是调控区域，以发掘潜在的新型致病因素。方法：本研究聚焦于在BOR综合征病例中占比超40%的EYA1基因，通过比较基因组学方法对该基因上下游250 kb范围内的候选增强子进行筛选。我们采用Tol2转座子系统（Tol2 transposon system），在斑马鱼（zebrafish）中对这些候选元件的增强子活性进行鉴定。结果：本次研究共检测11个保守非编码元件（conserved non-coding elements，下称CNEs），其中4个展现出增强子活性。值得注意的是，CNE16.39与CNE16.45在耳部呈现组织特异性增强子活性。CNE16.39需完整的206 bp序列方能实现内耳特异性表达，而CNE16.45的核心功能区域为136 bp。共聚焦显微镜结果显示，CNE16.39与CNE16.45均可驱动绿色荧光蛋白（GFP）在斑马鱼内耳壶腹感觉区表达，这与eya1的表达模式一致。结论：本研究有助于阐明调控EYA1表达的网络机制，为进一步明确EYA1在BOR综合征中的致病作用提供了新的研究视角。