Regulation of Gene Expression of Attacin, an Antibacterial Protein in the Silkworm, Bombyx mori

Abstract

In vitro gene expression of attacin, an antibacterial protein from the silkworm, Bombyx mori, was analyzed using nuclear extracts from the fat body (FB) and posterior silk gland (PSG). TATA box-dependent basal promoter activity was observed in vitro in the FB and PSG, even though the Attacin gene is known to be expressed in vivo in the FB but not in the PSG. Comparison of nucleosomal structure near the Attacin gene promoter regions between the two tissues indicated clear nucleosomal arrangements in the PSG but not in the FB, suggesting that regulation of tissue-specific Attacin gene expression occurs at the chromatin level. Electrophoresis mobility shift assay (EMSA) was conducted using different regulatory regions of the Attacin gene as probes. Results showed that nuclear proteins bind tissue-specifically to the sequence CATTT in addition to a NF-κB-like (κB) motif, suggesting the presence of an additional regulatory motif for Attacin gene expression. Nuclear protein binding to CATTT was not competitively prevented by the Cecropin B1 (CecB1) regulatory region containing two CATTA sequences, suggesting different nuclear protein(s) bind to the CATT(T/A) motif. Functional analysis of the CATTT motif of the Attacin gene showed that a mutation of this motif still retains 78.9% of the activity of the wild type control, suggesting that the CATT(T/A) motif plays a different role in Attacin and CecB1 even though they are similarly involved in immune reactions against bacterial infection.