BmFOXP1 and BmSGF1 divergently cooperate with BmGATAβ4 to regulate vitellogenin expression during vitellogenesis of...

Vitellogenin (Vg), a precursor to the egg yolk protein vitellin, is essential for reproduction in oviparous animals and is abundantly expressed in female silkworm (Bombyx mori) pupae. Although previous studies have demonstrated that Bombyx mori vitellogenin (BmVg) expression is modulated by 20-hydroxyecdysone (20E) and amino acids, the precise regulatory mechanisms remain incompletely elucidated. This study investigates the regulatory roles of two forkhead box (FOX) family transcription factors, FOXP1 and silk gland factor 1 (SGF1), in BmVg expression. Potential binding sites for FOXP1/FOXA and FOXP1 were predicted proximal to the GATA cis-regulatory element 1 (CRE1) and CRE2 elements, respectively in 499 bp BmVg promoter. Expression profiling and luciferase reporter assays demonstrated that BmSGF1 suppresses BmVg promoter activity and weakens the BmGATAβ4-induced up-regulation of BmVg expression, whereas BmFOXP1 enhances these inductive effects. Electrophoretic mobility shift assays (EMSA) and luciferase assays confirmed that BmSGF1 directly binds to the FOX CRE1 site to inhibit BmVg expression. Conversely, BmFOXP1 could promote the binding of BmGATAβ4 to GATAFOX CRE1 and CRE2 sites, thereby enhancing BmVg expression. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) experiments revealed that BmSGF1 diminishes the binding affinity of BmGATAβ to GATAFOX CRE1 and CRE2 sites, while BmFOXP1 enhances this interaction. Transgenic overexpression of BmSGF1 or RNA interference (RNAi)-mediated knockdown of BmFOXP1 or BmGATAβ4 resulted in significantly decreased BmVg expression in the fat body, accompanied by abnormal development of the oviduct and impaired egg formation. Notably, 20E significantly up-regulates BmFOXP1 expression, while down - regulate BmSGF1 expression. Consequently, the regulation of BmVg by 20E and nutritional factors is mediated through the coordinated interaction among of BmSGF1, BmFOXP1, and BmGATAβ4. These findings offer a more detailed understanding of BmVg being regulated in a stage-specific manner and provide insights into the regulatory mechanisms of Vg expression in other oviparous species.