Broad complex negatively regulates Fibrohexamerin/P25 by binding to the cis-element BMFA in the silkworm, Bombyx mori

Silk proteins, as natural macromolecular substances, hold significant potential for applications in biomaterials and biomedical fields. The expression of silk protein genes exhibits spatiotemporal specificity. Broad Complex (BrC), a key primary response factor to 20-hydroxyecdysone, plays a crucial role in metamorphosis. Our previous study showed that overexpression of BmBrC-Z2 significantly reduced fibroin gene Fibrohexamerin/P25 expression in the posterior silk gland. However, the underlying regulatory mechanism remains unclear. BMFA, a widely expressed factor that inhibits silk protein gene expression by recognizing BMFA elements, remains unidentified. Notably, the binding sequence of BmBrC-Z2 on the P25 promoter aligns with the BMFA element. Dual-Luciferase Reporter Assays, EMSA, and ChIP-PCR confirmed that BmBrC-Z2 directly binds to the BMFA element, thereby inhibiting P25 promoter activity. Furthermore, we demonstrated that BmBrC-Z2 and its isoform BmBrC-Z4 jointly bind to the BMFA element on the P25 promoter during the molting stage, whereas BmBrC-Z4 contributes a secondary role. Knocking out BmBrC-Z2 using the CRISPR/Cas9 system led to significant upregulation of silk protein genes during the molting stage in mutant larvae. These findings deepen our understanding of the complex regulatory mechanisms governing silk production and highlight the interplay between hormonal signaling and transcriptional regulation.