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）是一种罕见的遗传性疾病，发病率为1/40000，可影响鳃、耳、肾等多器官的发育，约占儿童耳聋病因的2%。目前，针对EYA1、SIX1、SIX5等主要致病基因的编码区变异仅能解释该疾病半数的发病机制，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），将其作为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，通过比较基因组学技术对该基因上下游250kb范围内的候选增强子进行筛选，并借助Tol2转座子系统在斑马鱼体内验证这些候选序列的增强子活性。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，共有4个具备增强子活性。其中CNE16.39与CNE16.45在耳部呈现组织特异性增强子活性。CNE16.39需要完整的206bp序列才能实现内耳特异性表达，而CNE16.45的核心功能区域被确定为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（Green Fluorescent Protein, GFP）在斑马鱼内耳壶腹嵴感觉区域表达，该结果与eya1的表达模式相符。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思路。