Structure and function of 5′-flanking regions of Bombyx mori fibroin heavy chain gene: Identification of a novel transcription enhancing element with a homeodomain protein-binding motif

We studied the promoter activity of a 5′-flanking region from −5000 to +24 (−5000/+24) in Bombyx mori fibroin heavy chain gene (fibH), fibH(−5000/+24). A luciferase reporter vector carrying fibH(−5000/+24) was bombarded to isolated posterior silk glands (PSGs). The PSGs showed a high luciferase activity when transplanted to larvae, indicating its potent promoter activity. Deletion experiments showed the requirement of fibH(−5000/−3844) and fibH(−2211/−542) for the promoter activity. These two regions and fibH(−541/+24) that contained the basal promoter were tandem fused to yield fibH(−5000/−3844:−2211/−542:−541/+24), which was found to retain 88% of the activity of fibH(−5000/+24). Germline transgenic silkworms bearing fibH(−5000/−3844:−2211/−542:−541/+24) as a promoter and enhanced green fluorescent protein (EGFP) gene as a reporter efficiently secreted EGFP in cocoons. The promoter activity of fibH(−2211/−542) was further investigated, because this contained a DNase I-hypersensitive site. The transient expression assay demonstrated that the activity of fibH(−2211/−542) required fibH(−1659/−1590), which contained the homeodomain protein-binding motif. Mutation experiments suggested a critical role of the motif for the promoter activity. Electrophoretic mobility shift assay (EMSA) demonstrated that a nuclear protein of PSGs bound to the motif. We propose fibH(−1659/−1590) as a novel transcription enhancer that plays a key role for the expression by recruiting a homeodomain protein.