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.
Shimizu
Katsuhiko
K
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.
Ogawa
Shingo
S
Hino
Rika
R
Adachi
Takahiro
T
Tomita
Masahiro
M
Yoshizato
Katsutoshi
K
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
D018398:Homeodomain Proteins; D019476:Insect Proteins; D014157:Transcription Factors; D005349:Fibroins