title: 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.
abstract: 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.
authors.affiliation: Yoshizato Project, Cooperative Link of Unique Science and Technology for Economy Revitalization, Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Shimizu
authors.forename: Shingo
authors.initials: S
authors.lastname: Ogawa
authors.forename: Rika
authors.initials: R
authors.lastname: Hino
authors.forename: Takahiro
authors.initials: T
authors.lastname: Adachi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tomita
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Yoshizato
mesh_terms: D024506:5' Flanking Region / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D012831:Bombyx / Q000235:genetics* / Q000378:metabolism; D005349:Fibroins / Q000235:genetics* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D005801:Genes, Homeobox; D018398:Homeodomain Proteins / Q000378:metabolism*; D019476:Insect Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D018398:Homeodomain Proteins; D019476:Insect Proteins; D014157:Transcription Factors; D005349:Fibroins