Homeodomain POU and Abd-A proteins regulate the transcription of pupal genes during metamorphosis of the silkworm, Bombyx mori

A cascade of 20-hydroxyecdysone-mediated gene expression and repression initiates larva-to-pupa metamorphosis. We recently showed that two transcription factors, BmPOUM2 and Bm beta FTZ-F1, bind to the cis-regulatory elements in the promoter of the gene coding for cuticle protein, BmWCP4, and regulate its expression during Bombyx mori metamorphosis. Here we show that down-regulation of BmPOUM2 expression by RNA interference during the wandering stage resulted in failure to complete metamorphosis. The thorax epidermis of RNA interference-treated larvae became transparent, wing disc growth and differentiation were arrested, and the larvae failed to spin cocoons. Quantitative real-time PCR analysis showed that expression of the genes coding for pupal-specific wing cuticle proteins BmWCP1, BmWCP2, BmWCP3, BmWCP4, BmWCP5, BmWCP6, BmWCP8, and BmWCP9 were down-regulated in BmPOUM2 dsRNA-treated animals, whereas overexpression of BmPOUM2 protein increased the expression of BmWCP4, BmWCP5, BmWCP6, BmWCP7, and BmWCP8. Pull-down assays, far-Western blot, and electrophoretic mobility shift assay showed that the BmPOUM2 protein interacted with another homeodomain transcription factor, BmAbd-A, to induce the expression of BmWCP4. Immunohistochemical localization of BmPOUM2, BmAbd-A, and BmWCP4 proteins revealed that BmAbd-A and BmPOUM2 proteins are colocalized in the wing disc cell nuclei, whereas BmWCP4 protein is localized in the cytoplasm. Together these data suggest that BmPOUM2 interacts with the homeodomain transcription factor BmAbd-A and regulates the expression of BmWCP4 and probably other BmWCPs to complete the larva-to-pupa transformation. Although homeodomain proteins are known to regulate embryonic development, this study showed that these proteins also regulate metamorphosis.