Pioneer factor BmFoxA modulates temporal gene regulatory networks via promoter selectivity in Bombyx mori

The silk gland is a specialized organ for silk protein synthesis in Bombyx mori. BmFoxA is considered an important transcription factor regulating silk protein gene expression. However, as a multifunctional transcription factor highly homologous to the mammalian pioneer factor FoxA1 and the Drosophila Fkh, large-scale identification of BmFoxA downstream genes and studies on its functional diversity in silkworms remain limited. In this study, we performed integrated multi-omics analyses, including RNA-seq, ChIP-seq (BmFoxA, H3K4me3, H3K27ac), and ATAC-seq, on silk gland tissues from three developmental stages: the fourth instar molting stage (4m), the fifth instar three days (5d3), and the prepupal stage (pp). Firstly, we mapped the temporal expression profile and genome-wide binding landscape of BmFoxA. Next, we annotated the promoter regions of 9473 silkworm genes using H3K4me3 and H3K27ac ChIP-seq data as promoter-associated markers, thereby constructing a more accurate promoter level stage-specific regulatory network for BmFoxA. Gene enrichment analysis showed that BmFoxA primarily regulates genes related to DNA binding, cell cycle, metabolism, and developmental processes across the three developmental stages. Using our refined regulatory network of BmFoxA, we identified and verified three stage-specific target genes MTHFD2L, MTHFS and Cdk20, which were involved in one-carbon metabolism, cell cycle progression and are positively regulated by BmFoxA at 5d3, contributing to the maintenance of material and energy metabolism. This study systematically delineates the transcriptional regulatory landscape of BmFoxA in silkworm, expanding its functional scope beyond silk protein gene expression to include stage-dependent coordination of metabolic programs. By constructing a high-confidence, promoter-level BmFoxA regulatory network, we provide a valuable resource and paradigm for future investigations into silk gland gene regulation, protein biosynthesis, and the broader functional potential of BmFoxA in specialized insect organs.