Identification and functional analysis of pointed homologs in Bombyx mori

• Published in: Gene 2017-03, Vol.604, p.22-32
• Main Authors: Tanaka, Hiromitsu, Sagisaka, Aki
• Format: Article
• Language: English
• Subjects: Alternative Splicing; Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides - genetics; Antimicrobial Cationic Peptides - immunology; Antimicrobial peptides; Bacillus subtilis; Bacillus subtilis - physiology; Base Sequence; Bombyx - genetics; Bombyx - immunology; Bombyx - microbiology; Bombyx mori; Cell Line; Conserved Sequence; DNA-Binding Proteins - genetics; DNA-Binding Proteins - immunology; Drosophila; Drosophila Proteins - genetics; Drosophila Proteins - immunology; Escherichia; Escherichia coli; Escherichia coli - physiology; Ets family proteins; Gene Expression; Hemocytes - immunology; Hemocytes - microbiology; Insect immunity; Insect Proteins - genetics; Insect Proteins - immunology; Larva - genetics; Larva - immunology; Larva - microbiology; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - immunology; Promoter Regions, Genetic; Protein Isoforms - genetics; Protein Isoforms - immunology; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - immunology; Sequence Alignment; Sequence Homology, Amino Acid; Silkworm; Transcription Factors - genetics; Transcription Factors - immunology
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/j.gene.2016.12.010

Using gene-knockdown techniques, we searched for endogenous Ets family proteins involved in the regulation of Escherichia coli-dependent lebocin promoter activation in the E. coli-responsive silkworm cell line NIAS-Bm-aff3. Results showed that the gene knockdown of BmPointeds (BmPNTs), Drosophila Pointed orthologs, enhanced E. coli-dependent lebocin promoter activation, suggesting that endogenous BmPNTs repress the activation of this promoter. Furthermore, we found that i) the BmPNT gene produced at least two alternative splicing isoforms, BmPNT1 and BmPNT2, both of which function as repressors; ii) BmPNTs were not associated with an already-reported repressor element, most proximal GGAA/T motif (EtsRE3), in lebocin promoter, which plays a role in the repression of E. coli- and BmRelish1-dependent lebocin promoter activation; iii) although BmPNTs did not directly affect BmRelish1-dependent lebocin promoter activation, they were able to directly repress its activation on the promoter lacking EtsRE3, probably because of competitive inhibition of binding of BmRelish1 to κB sites by BmPNTs; and iv) BmPNTs were mainly expressed in larval hemocytes, and the gene expression levels of BmPNT2, but not of BmPNT1, were decreased in response to E. coli and Bacillus subtilis. These findings suggest that endogenous BmPNTs are directly and indirectly involved in the repression of E. coli-mediated lebocin promoter activation in NIAS-Bm-aff3 cells. [Display omitted] •The BmPointeds (BmPNTs) gene produced at least two alternative splicing isoforms, BmPNT1 and BmPNT2.•BmPNTs repressed E. coli-dependent lebocin promoter activation, but not BmRelish1-dependent lebocin promoter activation.•BmPNTs directly repressed BmRelish1-dependent promoter activation in the promoter lacking the repressor element, EtsRE3.