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. CNE16.39 required 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.

Branchio-oto-renal (BOR) syndrome is an autosomal dominant syndromic deafness disorder characterized by developmental malformations of the ear, hearing dysfunction, and associated abnormalities such as gill slit abnormalities and renal malformations [1]. The prevalence of BOR syndrome in the population is approximately 1 in 40,000, and it is responsible of 2% of cases of profound deafness in children [2]. The manifestation of ear and branchio defects is often observed during the preschool years, while renal defects may only become apparent in adulthood, leading to significant delays in therapeutic intervention [3]. Therefore, early identification and genetic diagnosis of the underlying cause of the disease are of utmost importance.

The known causative genes for this disease include EYA1, SIX1, SIX5, and SALL1 [4]. Among them, EYA1 is the most common causative gene for BOR syndrome, accounting for about 40% of the cases [5]. It is also a key gene involved in inner ear development [4]. This gene belongs to the EYA family and plays an important role in organ tissue development through protein phosphatases and transcriptional co-activators [4]. The expression of the eya1 gene is predominantly found in human embryonic kidney, as well as in the adult heart and skeletal muscle [7]. In situ hybridization studies conducted by Rauch et al. [8] revealed that the zebrafish Eya1 gene is widely expressed in substrate-derived sensory organs, with a significant concentration in the basal plate of the ear, the saccule, and the neural thalamus. Sahly et al. [9] found that the eya1 cDNA predicts a protein with 84.7% identity with the human homologue. Kozlowski DJ et al. [10] demonstrated that decreased gene expression of Eya1 affects the development of the otic capsule and leads to a decrease in sensory hair cells in five different sensory nerve regions. Xu et al. [11] discovered that Eya1 heterozygous knockout mice exhibit conductive deafness similarity to BOR syndrome, while homozygous knockout mice show arrested development of the inner ear and specific cranial sensory ganglia at the capsule stage. As early as 1997, Kumar et al. [12] mapped EYA1 gene mutation sites in patients with BOR syndrome, and in 2014, Castigline’s team summarized 92 Eya1-related mutations in exonic regions [13]. However, the numerous coding region variants in the main causative genes of BOR syndrome currently explain only half of the etiology [14].

In addition to variations in the gene coding region, temporal, spatial, and quantitative variations in gene expression are also closely related to the occurrence of diseases. Zou et al. [15] found that Eya1 plays a key role in the development of sensory organs in a dose-dependent manner using different alleles. Xu Pine-Xian’s team discovered that the degree of deletion of Eya1 gene expression level varies in mice, leading to differences in inner ear malformation and the severity of hearing impairment [11]. The severity of inner ear deformity and hearing impairment in mice varies depending on the level of Eya1 gene expression. The non-coding regions, which comprise the majority of the genome, especially the functional regulatory regions, play a crucial role in the temporal and spatial efficacy of gene expression. Variants in these regions may serve as novel pathogenic factors [16]. Therefore, the identification and analysis of the functional regulatory regions of EYA1 are extremely important for the etiological study of BOR syndrome. Mutations in enhancers, which act as spatiotemporal switches regulating gene expression, can also lead to disease phenotypes. For example, point mutations in enhancers can cause malaria drug resistance [17]. The absence of legs in snakes is due to the evolutionary deletion of the distant enhancer ZRS, the knockout of which in mice replicates the phenotype [18]. However, enhancers are scattered throughout the genome, and the distance of their action from target genes remains uncertain. Comparative genomics provides a method for identifying candidate enhancer regions on a genome-wide scale.

Comparative genomics is a powerful approach that involves comparing genomic sequences of different species during evolution. By identifying CNEs that are retained across multiple species, we can infer their importance in gene expression regulation [20]. Ovchinnikov et al. [22] compared the genome sequences of two limbless representatives and found a deletion of the ZRS enhancer. Further validation in mice confirmed that ZRS is essential for limb development. Indjeian et al. [23] used genetic crosses and comparative genomics to identify specific regulatory DNA alterations that control skeletal evolution. In addition to comparative genomics for screening of candidate CNEs, the zebrafish, a model organism with transparent embryos, easy reproduction, and simple manipulation, is commonly used for functional studies of CNEs. Zebrafish has a highly homologous genome sequence to humans and has been extensively employed in gene-related regulatory research [24].

Building upon this background, our study aims to investigate the regulation of EYA1 gene expression, an important pathogenic gene in BOR syndrome. We will screen for CNEs within a 250-kb range upstream and downstream of the gene using bioinformatics and comparative genomics. Furthermore, we will characterize the enhancer activity of these identified CNEs in zebrafish. This research aims to provide new insights and methodologies for understanding the regulation of EYA1 gene expression and improving the molecular diagnosis of BOR syndrome.

Comparative Genomics Analyses of Zebrafish and Human Genomes

To perform the comparative genomics analysis, we compare the genomic sequences of human (hg19) and zebrafish (zv9) using the Comparative Genomics Online Database DCODE (http://www.dcode.org/). We selected a range of 250 kb upstream and downstream of the EYA1 gene and filtered for segments that were at least 50 bp in size, with at least 50% similarity to the human sequence, to identify candidate CNEs.

Transcription Factor Binding Site Prediction and Analysis

Since enhancer activity is primarily mediated by transcription factors (TFs), we utilized the JASPAR TF-prediction Website (http://jaspar.binf.ku.dk/) to predict the putative TFs binding to the identified DNA sequence. We used the JASPAR CORE Vertebrata database and kept the default parameters to maximize the accuracy of the predicted TFs. We then filtered the predicted TFs based on their known involvement in eye development.

Construction of Injection Plasmids Containing CNE

The zebrafish genome sequences corresponding to the identified candidate CNEs were obtained from the UCSC Website. SnapGene Primer software was used for PCR primer design. The complete list of primers can be found in online supplementary Table 1 (for all online suppl. material, see https://doi.org/10.1159/000536260), while the core primers are listed in Table 1. PCR amplification was carried out to obtain candidate CNEs, which then ligated to thepTol2-E1b-GFP plasmid (a backbone plasmid available in our laboratory). This plasmid contains the minimal Tol2 transposon, the minimal promoter E1b and the reporter gene GFP. The plasmid was linearized by XhoI and BgIII restriction enzymes and ligated by T4 ligase. The constructed plasmid was verified by sequencing.

Table 1.

Key primers used in this study

PrimersSequences (5′-3′)
CNE16.39-F aga​gct​cga​gCA​ACA​AAG​TCC​CAT​CTC​TCA​GCC​TTT​A 
CNE16.39-R aga​gag​atc​tGC​CAG​GAC​CCT​AAA​AAC​AAT​ATC​AGC 
CNE16.39-L-F aga​gct​cga​gCC​TAT​TCC​TGC​TTA​CCT​TGG​CTA​AG 
CNE16.39-L-R aga​gag​atc​tCC​TCC​ACC​GTT​GTT​TCC​TCC 
CNE16.39-R-F aga​gct​cga​gGG​TTG​AAC​CTA​CAG​ACA​ATG​TGC​C 
CNE16.39-R-R aga​gag​atc​tAA​TGG​GAA​GAG​AGG​AGT​CGG​T 
CNE16.45-F aga​gct​cga​gGT​AAG​CAG​TAG​CTC​TGA​AAG​GTT​TTC​AGC 
CNE16.45-R aga​gag​atc​tGC​AGA​CCA​ATC​ATC​CCT​CAG​TTT​GA 
CNE16.45-L-F aga​gct​cga​gTG​ATG​AGA​TGA​CAA​CTG​CCT​ATT​GAT​TTG​T 
CNE16.45-L-R aga​gag​atc​tTG​AGA​CAC​AGC​GAT​GGA​TTA​ATG​G 
CNE16.45-R-F aga​gct​cga​gCA​GAC​GTC​TGA​GAT​CAT​TGG​GCT 
CNE16.45-R-R aga​gag​atc​tTT​GGC​AGC​ACA​ATC​AAT​TAT​TTA​CGT​TGT 
PrimersSequences (5′-3′)
CNE16.39-F aga​gct​cga​gCA​ACA​AAG​TCC​CAT​CTC​TCA​GCC​TTT​A 
CNE16.39-R aga​gag​atc​tGC​CAG​GAC​CCT​AAA​AAC​AAT​ATC​AGC 
CNE16.39-L-F aga​gct​cga​gCC​TAT​TCC​TGC​TTA​CCT​TGG​CTA​AG 
CNE16.39-L-R aga​gag​atc​tCC​TCC​ACC​GTT​GTT​TCC​TCC 
CNE16.39-R-F aga​gct​cga​gGG​TTG​AAC​CTA​CAG​ACA​ATG​TGC​C 
CNE16.39-R-R aga​gag​atc​tAA​TGG​GAA​GAG​AGG​AGT​CGG​T 
CNE16.45-F aga​gct​cga​gGT​AAG​CAG​TAG​CTC​TGA​AAG​GTT​TTC​AGC 
CNE16.45-R aga​gag​atc​tGC​AGA​CCA​ATC​ATC​CCT​CAG​TTT​GA 
CNE16.45-L-F aga​gct​cga​gTG​ATG​AGA​TGA​CAA​CTG​CCT​ATT​GAT​TTG​T 
CNE16.45-L-R aga​gag​atc​tTG​AGA​CAC​AGC​GAT​GGA​TTA​ATG​G 
CNE16.45-R-F aga​gct​cga​gCA​GAC​GTC​TGA​GAT​CAT​TGG​GCT 
CNE16.45-R-R aga​gag​atc​tTT​GGC​AGC​ACA​ATC​AAT​TAT​TTA​CGT​TGT 

The lowercase letters are protective bases and enzyme restriction sites.

Zebrafish Feeding and Injection Experiments

Zebrafish (Danio rerio, TU strains) were raised in a standardized recirculation system according to Fisherman et al.’s [26] protocol. Zebrafish embryos were obtained from parental crosses of TU strains. Enhancer activity was assessed by a Tol2-GFP-containing reporter system. The plasmid containing the CNE, as described above, along with the tol2 mRNA, was co-injected into zebrafish embryos at the single-cell stage (refer to Fig. 1). If the CNE possessed enhancer activity, it would mediate GFP expression in zebrafish tissues. GFP expression was visualized by inverted fluorescence microscopy at 24 h post-fertilization (hpf), 48 hpf, and 72 hpf. At least 50–100 embryos were injected in each batch to count the fluorescence expression. The zebrafish experiments were approved by the Ethics Committee of Children’s Hospital of Fudan University.

Fig. 1.

Approach of Tol2-mediated microinjection in zebrafish. CNE was inserted into the enhancer activity detecting vector. Together with the tol2 mRNA, plasmid containing the CNE was co-injected into zebrafish embryos at the single-cell stage.

Fig. 1.

Approach of Tol2-mediated microinjection in zebrafish. CNE was inserted into the enhancer activity detecting vector. Together with the tol2 mRNA, plasmid containing the CNE was co-injected into zebrafish embryos at the single-cell stage.

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Screening of CNEs at the EYA1 Locus by Comparative Genomics

In this study, a total of 13 candidate CNEs were identified within the 250 kb upstream and downstream of the EYA1 gene by comparative genomics. Among these, 11 CNEs were successfully constructed into plasmids. Online supplementary Table 2 provides specific information regarding these constructs. Through the identification of enhancer activity in zebrafish, four CNEs were found to exhibit enhancer activity. Specifically, CNE16.39 (25%, N = 35) and CNE16.45 (47%, N = 53) showed specificity for ear tissue, while CNE16.41 (40%, N = 38) and CNE16.51 (41%, N = 34) exhibited specificity for heart and muscle tissues. CNE16.39, located 1.9 kb upstream of the eya1 gene, has a size of 206bp in the zebrafish genome sequence (446 bp after primer design). CNE16.45, positioned 61.2 kb upstream of the eya1 gene, has a size of 187 bp in the zebrafish genome sequence (423 bp after primer design). The schematic representation of the CNE locations can be found in Figure 2a, and detailed information is provided in Table 2.

Fig. 2.

Identification of tissue-specific CNEs around eya1 gene. a CNEs distribution near the zebrafish eya1 locus based on the danRer7 assembly. Genes are shown as blue rectangles and the orientation of arrows indicates the translational initiation sites of genes. The CNEs located near the eya1 gene are indicated by red rectangles. The horizontal line at the bottom indicates the studied range of the DNA sequence range. b Sequence conservation between zebrafish (danRer7 assembly) and humans (hg19 assembly). c CNE16.39 and CNE 16.45 regulate GFP expression mainly in zebrafish ear. Photos were taken with the GFP channel at 24 hpf, 48 hpf, and 72 hpf. Red arrow, location of ear.

Fig. 2.

Identification of tissue-specific CNEs around eya1 gene. a CNEs distribution near the zebrafish eya1 locus based on the danRer7 assembly. Genes are shown as blue rectangles and the orientation of arrows indicates the translational initiation sites of genes. The CNEs located near the eya1 gene are indicated by red rectangles. The horizontal line at the bottom indicates the studied range of the DNA sequence range. b Sequence conservation between zebrafish (danRer7 assembly) and humans (hg19 assembly). c CNE16.39 and CNE 16.45 regulate GFP expression mainly in zebrafish ear. Photos were taken with the GFP channel at 24 hpf, 48 hpf, and 72 hpf. Red arrow, location of ear.

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Table 2.

Information about CNE16.39 and CNE16.45

CNEPositionLength, bpGC, %Identity (human/zebrafish)
CNE16.39 Human Chr8:72268760-72269081 226 50.4 74.8% 
Zebrafish Chr24:13836090-13836295 206 52.4 
CNE16.45 Human Chr8:72332253-72332584 201 30.3 68% 
Zebrafish Chr24:13776744-13777029 187 36.5 
CNEPositionLength, bpGC, %Identity (human/zebrafish)
CNE16.39 Human Chr8:72268760-72269081 226 50.4 74.8% 
Zebrafish Chr24:13836090-13836295 206 52.4 
CNE16.45 Human Chr8:72332253-72332584 201 30.3 68% 
Zebrafish Chr24:13776744-13777029 187 36.5 

Enhancers CNE16.39 and CNE16.45 Drive GFP Expression Mainly in the Zebrafish Ear

Using the Tol2 transposon system, we discovered that CNE16.39 and CNE16.45 can mediate GFP expression specifically in the zebrafish ear. The GFP expression in the zebrafish ear capsule was observed by inverted fluorescence microscopy at 24 hpf, 48 hpf, and 72 hpf, respectively, as shown in Figure 2c.

To identify the core region in the two CNEs that is sufficient for ear-specific activity in embryos, we employed a deletion strategy based on the distribution of TFs. Considering deletion efficiency, we dissected 2 plasmids (CNE16.39-F, CNE16.39-R) and (CNE16.45-F, CNE16.45-R) each according to the sequence similarity. Details about these four constructs are provided in Figure 3. Statistical analysis of GFP expression was as follows: CNE16.39 (62%, N = 53), CNE16.39 -L (12%, N = 59), CNE16.39-R (6%, N = 54) and CNE16.45 (37%, N = 57), CNE16.45-L (35%, N = 63), CNE16.45-R (77%, N = 70). These findings suggest that CNE16.39 requires the full length 206 bp for enhancer activity specifically expressed in the inner ear, while the core functional region of CNE16.45 spans 136 bp. The GFP expression rates mediated by the individual CNE in zebrafish are presented in Table 3 and the expression patterns are schematically illustrated in Figure 4.

Fig. 3.

Schematic diagram of dissection. Red color represents the region where sequences that are more than 80% similar in the human and zebrafish genome sequence alignment are located. a The case of CNE16.39 dissection. b The case of CNE16.45 dissection.

Fig. 3.

Schematic diagram of dissection. Red color represents the region where sequences that are more than 80% similar in the human and zebrafish genome sequence alignment are located. a The case of CNE16.39 dissection. b The case of CNE16.45 dissection.

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Table 3.

GFP expression rates of each full-length and dissected CNE plasmid in zebrafish embryos after the time of injection

CNENumber of GFP expression/injection embryosExpression rate in the ear, %
CNE16.39 33/53 62.26 
CNE16.39-L 7/59 11.86 
CNE16.39-R 3/54 5.56 
CNE16.45 21/57 36.84 
CNE16.45-L 22/63 34.92 
CNE16.45-R 54/70 77.14 
CNENumber of GFP expression/injection embryosExpression rate in the ear, %
CNE16.39 33/53 62.26 
CNE16.39-L 7/59 11.86 
CNE16.39-R 3/54 5.56 
CNE16.45 21/57 36.84 
CNE16.45-L 22/63 34.92 
CNE16.45-R 54/70 77.14 
Fig. 4.

Expression of each full-length and dissected CNE plasmid in zebrafish embryos at different hpf.

Fig. 4.

Expression of each full-length and dissected CNE plasmid in zebrafish embryos at different hpf.

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CNE16.39 and CNE16.45-R Drive GFP Expression in the Sensory Region of Crista of the Zebrafish Inner Ear

To further investigate the expression pattern of CNE16.39 and CNE16.45-R in the inner ear, we observed that CNE16.39 and CNE16.45-R drove GFP expression in the crista, anterior cristae, lateral cristae, and posterior cristae of the inner ear sensory region in the zebrafish using confocal microscopy (as shown in Fig. 5). The site where CNE16.39 and CNE16.45-R drove GFP expression precisely overlapped with the expression of the EYA1 gene, indicating that the eya1 gene is likely the target gene for CNE16.39 and CNE16.45-R. The results provide a theoretical basis for further investigation into the specific mechanisms by which these 2 CNEs affect inner ear development and function through the regulation of eya1 gene expression.

Fig. 5.

Ear-specific enhancers CNE16.39 and CNE16.45-R mediate GFP-specific expression in zebrafish inner ear crista.

Fig. 5.

Ear-specific enhancers CNE16.39 and CNE16.45-R mediate GFP-specific expression in zebrafish inner ear crista.

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With the continuous advancement of whole-genome sequencing technology and the integration of bioinformatics, the study and interpretation of non-coding regions, particularly the functionally regulated regions within them, are progressively deepening. The combination of comparative genomics and molecular genetics employed in this study represents a valuable exploration. It significantly reduces labor and time costs while facilitating the discovery of new genes and gene regulatory elements from the vast genome landscape. Once candidate regulatory regions are identified, they can be targeted for further mechanistic investigations, thereby offering a new direction for the etiological diagnosis of diseases.

Enhancers, as important regulatory elements in cell growth and development, typically bind to TFs to regulate the transcriptional activity of target genes. This process influences the unique spatiotemporal pattern of target gene expression, thereby impacting gene regulation. Shigeru Sato et al. [27] in 2015 focused on the sensory ganglia-specific Six1 enhancer (Six1–8), which is conserved between chick and mouse, and report the establishment of a new transgenic mouse line under the control of mouse Six1–8. In addition to enhancer identification, numerous studies have focused on the involvement of enhancer variants in disease development. Soldner et al. [29] identified a common Parkinson’s disease-associated risk variant in an enhancer element that regulates the expression of SNCA gene. Even single-nucleotide changes in enhancer sequences can affect embryonic development and human disease [30‒32]. However, the currently known enhancers of the EYA1 gene are limited. Tadashi Ishihara’s [33] study reported that 24 CNEs conserved between four species, with 10 of them exhibiting tissue-specific enhancer activity in chick embryo. Furthermore, in 2017, Santosh KM identified two novel cis-regulatory elements of Eya1 in Xenopus laevis using BAC recombineering [34]. None of the studies identified enhancers in zebrafish. In our study, we identified two ear-specific enhancers with expression patterns consistent with eya1 through comparative genomics combined with zebrafish for in vivo validation. This finding lays the foundation for further investigation into the interaction mechanisms of these two enhancers.

However, the specific mechanism by which these two enhancers act on eya1 to exert tissue specificity remains unclear. The next step could involve predicting the reciprocal TFs and conducting knockdown or knockout experiments on the enhancers to study their effect on the expression of the target genes and phenotypic changes. Another approach would be to analyze clinical samples to identify the presence of these two enhancers at the mutation site and comprehensively investigate the mechanism of BOR syndrome caused by EYA1. This comprehensive and multi-dimensional approach would provide new insights into the abnormalities in the EYA1 regulatory region associated with BOR syndrome.

In this study, we performed comparative genomics analysis of the upstream and downstream 250-kb sequences of EYA1 gene and identified four CNEs capable of regulating tissue-specific expression in zebrafish, two of which are specific to the ear. Through dissection experiments, we confirmed that CNE16.39 and CNE16.45-R are ear-specific enhancer elements, and their expression profiles align with those of eya1, laying a groundwork for the functional study. This study presents a novel approach for the clinical screening of the etiology of BOR syndrome.

We are grateful to Yinglan Zhang and Jia Lin for their technical support.

This study protocol was reviewed and approved by the Ethics Committee of Children’s Hospital of Fudan University, approval number (2017) 87.

The authors have no conflicts of interest to declare.

This work was supported by grants from the National Natural Science Foundations of China (Grant No. 881771632) and the Shanghai Key Laboratory of Birth Defects (Grant No. 21ZR1410100) to Qiang Li and the National Natural Science Foundations of China (Grant No. 82300272) to Feng Wang.

Feng Wang and Ruizhi Zhang performed the experiments, analyzed the data, prepared figures and/or tables, and wrote the manuscript. Jing Jian and Yanhe Sun offered technical support and approved the final draft. Qiang Li conceived and designed the project and approved the final draft. All authors commented on and approved the manuscript.

All data generated or analyzed during this study are included in this article and its online supplementary material files. Further inquiries can be directed to the corresponding author.

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