Recent studies have demonstrated that the production of bidirectional enhancer-derived transcripts (eRNAs) is a characteristic of an active Cis-regulatory element (CRE). Higher levels of eRNA synthesis correlate with the activation of histone modifications, a potentially valuable tool for deciphering the complexity of the gene regulatory network. To understand the changes of CREs during gonadal development in mice, we collected gonadal WT1-positive cells from the piggyBac-Wt1-mCherry-2A-EGFP (PBWt1-RG) reporter strain at E13.5, E16.5, and P0 in both sexes and conducted Cap Analysis of Gene Expression analysis (CAGE) which is capable to capture transcriptional starting site (TSS). We compared the levels of intergenic bidirectional RNA, i.e, potentially eRNA, according to sex at each stage (testis somatic cells vs ovary somatic cells at E13.5, E16.5, and P0) and stage in each sex (E13.5 vs E16.5, E16.5 vs P0, and E13.5 vs P0 in testis somatic cells or ovary somatic cells). Intergenic RNAs with significant changes (|Log2FC| > 1, p < 0.05) were selected. The TSS profile of intergenic RNA changed more profoundly in testis somatic cells than in ovary somatic cells, suggesting embryonic testicular development is driven by larger changes in a transcriptional regulatory network than ovarian development. Based on the profiles of the predicted transcription factors (TFs) that would bind to the active CREs during gonadal development, the NR4A, EGR, and TCF3 families would be novel TFs to play pivotal roles in gonadal development. Identifying active CREs using eRNAs would provide a means to comprehensively understand the transcriptional regulatory system, leading to valuable insights into the gonadal development of male and female individuals.
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