Genome-Wide Identification of Transcriptional Start Sites and Candidate Enhancers Regulating Worker Metamorphosis in Apis mellifera

Comparative genomics in bees has revealed correlations between transcription factor (TF) binding site (TFBS) abundance and social complexity; however, the activity of TFBS within enhancers is not experimentally examined during the caste developmental processes. In this study, we performed cap analysis of gene expression (CAGE) during worker metamorphosis in the honeybee Apis mellifera to identify TFBSs within active enhancers and to decipher the regulatory relationships between these enhancers and their target genes. We identified 17,349 transcription start sites (TSSs) and 842 candidate enhancers. Using CAGE profiles, we classified these elements into five clusters based on their expression patterns. Notably, genes associated with the canonical metamorphic regulators, Broad complex (Br-c) and E93, were found in specific clusters. By integrating correlations between enhancer and TSS activities with motif enrichment analysis, we identified 15 transcription factor–enhancer–TSS regulatory relationships. Among these, tramtrack (ttk) binding sites were identified in five enhancers associated with four target genes, including Br-c. Analysis of sequence conservation revealed that, across all target genes examined, perfect conservation of ttk binding sites was restricted to the genus Apis. These results suggest that gene regulatory relationships during worker metamorphosis are lineage-specific within the Apis genus.