Regulation of tissue-specific and lipopolysaccharide-inducible gene expression of an antibacterial protein from the silkworm, Bombyx mori

H. Tanaka 1, 3 , S. Furukawa 2 , H. Nakazawa 1 , J. Ishibashi 1 & M. Yamakawa 1, 2

1 Lab. of Biol. Defense, Natl. Inst. Seric. Entomol. Sci., Tukuba, Ibaraki 305-8634, Japan; 2 Doctoral program in Agric. Sci., University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; 3 Lab. of Genet. Resources, Natl. Inst. Seric. Entomol.Sci. Kobuchizawa, Yamanashi 408-0044, Japan (Present address)

Several antibacterial proteins such as cecropin, attacin, lebocin and moricin including their analogues are known to occur in the silkworm, Bombyx mori. cDNAs and genes encoding these antibacterial proteins have been cloned. Northern bolt analysis showed that tissue-specific gene expression of these antibacterial proteins occurs in the fat bodies and hemocytes, but not in other tissues such as the silk glands, midguts or Malpighian tubles. Antibacterial gene expression was found to be induced by bacteria or their cell wall components, e.g., lipopolysaccharide (LPS) and peptidoglycan. Although tissue-specific expression of insect antibacterial proteins is recognized as a general phenomenon, mechanisms still remain obscure. We analyzed in vitro gene expression of attacin using nuclear extracts from fat bodies (FB) and posterior silk glands (PSG). TATA box-dependent basal promoter activity was observed in vitro in FB and PSG, although the attacin gene is known to be expressed in vivo in the FB but not in the PSG. Comparison of nucleosomal structure around the attacin gene promoter regions between the 2 tissues indicated clear nucleosomal arrangement in the PSG but not in the FB, suggesting that regulatory tissue-specific attacin gene expression occurs at the chromatin level. We further analyzed the structure of the protein-attacin gene complex by in vivo footprinting using ligation-mediated PCR (LM-PCR). Protected and sensitive regions around the attacin promoter were detected when FB nuclei from LPS-injected larvae were compared with nontreated FB and/or LPS-treated PSG. Electrophoretic mobility shift assay (EMSA) was conducted using different regulatory regions of the attacin gene as probes. Results showed that nuclear proteins bind to a specific sequence (CATTT) in addition to a NF-?B site, suggesting the presence of a novel regulatory motif for attacin gene expression.

Index terms: attacin, chromatin structure, nuclear protein, EMSA.

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