Genomic Analysis and Functional Validation of Bidirectional Promoters in Medaka (Oryzias latipes)

Abstract

Bidirectional promoters (BDPs) regulate the transcription of two adjacent, oppositely oriented genes, offering a compact structure with significant potential for multigene expression systems. Although BDPs are evolutionarily conserved, their regulatory roles and sequence characteristics vary across species, with limited studies in fish. Here, we systematically analyzed the distribution, sequence features, and expression patterns of BDPs in the medaka (Oryzias latipes) genome. A total of 1737 divergent gene pairs, representing 13% of medaka genes, were identified as potentially regulated by BDPs. These genes are enriched in essential biological processes, including organelle function, RNA processing, and ribosome biogenesis. Transcriptomic analysis revealed that co-regulation (co-expression and co-silencing) is a prominent feature of these gene pairs, with variability influenced by tissue and sex. Sequence analysis showed that medaka BDPs are compact, with most fragments under 400 bp and an average GC content of 42.06%. Validation experiments confirmed the bidirectional transcriptional activity of three histone-related BDPs in both medaka SG3 cells and embryos, demonstrating effective and robust regulatory efficiency. This study enhances our understanding of the genomic organization and transcriptional regulation in fish and provides a valuable reference for developing species-specific multigene expression systems in fish genetic engineering.

Keywords: bidirectional promoters; gene regulation; medaka; multigene expression.

Figure 1

The distribution of divergent gene pairs in the medaka genome. (A) Types of divergent gene pairs. TSS, transcription start site. (B) Proportion of different types of divergent gene pairs. (C) Correlation analysis between the number of divergent gene pairs and gene density.

Figure 2

Functional enrichment analysis of divergent gene pairs. (A) Top 10 enriched GO terms for Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The length of each bar represents the number of genes associated with each GO term. (B) KEGG pathway enrichment analysis. The y-axis shows the top 10 significantly enriched pathways, while the x-axis represents the gene ratio. The size of the dots indicates the number of genes, and the color gradient shows the adjusted p-value.

Figure 3

Expression patterns of divergent gene pairs across different tissues in male and female medaka (Oryzias latipes).

Figure 4

Venn diagrams showing the overlap of co-expressed and co-silent divergent gene pairs across tissues in male and female medaka (Oryzias latipes).

Figure 5

Length and GC content distribution of bidirectional promoter (BDP) fragments. (A) BDP fragment length distribution. (B) GC content distribution of BDP and non-BDP fragments.

Figure 6

Functional validation of histone bidirectional promoters (H-BDPs). (A) Schematic representation of the dual-reporter plasmid construct with GFP and RFP flanking the H-BDP fragments (H-BDP1, H-BDP2, H-BDP3). (B) Fluorescence microscopy images showing GFP and RFP expression driven by H-BDP1, H-BDP2, and H-BDP3 in SG3 cells. Green fluorescence represents GFP, red represents RFP, and the merged image shows co-expression. Scale bar, 100 μm. The white arrows indicate cells that simultaneously express RFP and GFP. (C) Quantitative analysis of relative GFP and RFP expression levels for H-BDP1, H-BDP2, and H-BDP3 in SG3 cells. ns, not significant. (D) Embryonic expression of GFP and RFP under the control of H-BDPs in medaka embryos. Scale bar, 500 μm. NC, negative control, represents embryo injected with promoter-free plasmid.

Figure 7

The distribution of core promoter elements (TATA, INR, DPE, and CCAAT) across H-BDP1, H-BDP2, and H-BDP3. Core promoter elements are color-coded and mapped to nucleotide positions. TSS, transcriptional start site.

Figure 8

The distribution of predicted transcription factor binding motifs across H-BDP1, H-BDP2, and H-BDP3. Different motifs are shown with distinct colors and mapped to nucleotide positions. TSS, transcriptional start site.