BmBR-C Z4 is an upstream regulatory factor of BmPOUM2 controlling the pupal specific expression of BmWCP4 in the silkworm, Bombyx mori.

20-hydroxyecdysone (20E)-induced expression of the wing disc cuticle protein gene BmWCP4 was mediated by the transcription factor BmPOUM2, which binds to the cis-response elements (CREs) of BmWCP4 gene in Bombyx mori. In this study we report the regulation of BmPOUM2. RT-PCR analysis indicated that in response to 20E, BmPOUM2 was expressed at higher levels in the wing discs during the pre-pupal and mid-pupal stages than other stages and the expression pattern of BmBR-C Z1, BmBR-C Z2 and BmBR-C Z4 was in tandem with the expression of BmPOUM2. BmBR-C Z4 was induced by 20E in the wing discs, whereas BmBR-C Z1 and BmBR-C Z2 were not. Three potential BR-C Z4 cis-response elements (CREs) were identified in the promoter region of BmPOUM2. The expression of BmPOUM2 mRNA and protein was increased by the over-expression of BmBR-C Z4 in BmN cells, which acted at the promoter of BmPOUM2. Electrophoretic mobility shift assay (EMSA) and the luciferase activity analysis under the control of wild-type and mutants of the BR-C Z4 CREs suggested that BmBR-C Z4 protein bound to the predicted BRC-Z4 CRE C (-684 ∼ -660). Taken together the data suggest that BmBR-C Z4 is a direct upstream regulator of BmPOUM2 and regulates the pupal-specific expression of BmWCP4 through BmPOUM2.