Basic Helix-Loop-Helix Transcription Factor Bmsage Is Involved in Regulation of fibroin H-chain Gene via Interaction with SGF1 in Bombyx mori

Abstract

Silk glands are specialized in the synthesis of several secretory proteins. Expression of genes encoding the silk proteins in Bombyx mori silk glands with strict territorial and developmental specificities is regulated by many transcription factors. In this study, we have characterized B. mori sage, which is closely related to sage in the fruitfly Drosophila melanogaster. It is termed Bmsage; it encodes transcription factor Bmsage, which belongs to the Mesp subfamily, containing a basic helix–loop–helix motif. Bmsage transcripts were detected specifically in the silk glands of B. mori larvae through RT-PCR analysis. Immunoblotting analysis confirmed the Bmsage protein existed exclusively in B. mori middle and posterior silk gland cells. Bmsage has a low level of expression in the 4th instar molting stages, which increases gradually in the 5th instar feeding stages and then declines from the wandering to the pupation stages. Quantitative PCR analysis suggested the expression level of Bmsage in a high silk strain was higher compared to a lower silk strain on day 3 of the larval 5th instar. Furthermore, far western blotting and co-immunoprecipitation assays showed the Bmsage protein interacted with the fork head transcription factor silk gland factor 1 (SGF1). An electrophoretic mobility shift assay showed the complex of Bmsage and SGF1 proteins bound to the A and B elements in the promoter of fibroin H-chain gene(fib-H), respectively. Luciferase reporter gene assays confirmed the complex of Bmsage and SGF1 proteins increased the expression of fib-H. Together, these results suggest Bmsage is involved in the regulation of the expression of fib-H by being together with SGF1 in B. mori PSG cells.

A Juvenile Hormone Transcription Factor Bmdimm-Fibroin H Chain Pathway Is Involved in the Synthesis of Silk Protein in Silkworm, Bombyx mori

“…2C). The location and patterns of Bmdimm expression were consistent with that of the fib-H gene in PSG cells (2,15), implying that Bmdimm might be related to the fib-H gene.…”

“…Total RNA kit II according to the protocol provided by the manufacturer (Omega, Norcross, GA). Reverse transcription PCR (RT-PCR) was as described previously (15). Primers for amplifying Bmdimm, fib-H, BmKr-h1, BmMet2, BmMet1, BmSRC, and BmRpl3 are listed in Table 2.…”

“…Production of Recombinant Protein and Western Blots-The coding region of Bmdimm was amplified with specific primers (Table 3) and cloned into pET28a vector (Novagen, Germany). Positive clones were transformed into Escherichia coli strain BL21 (DE3) cells (TransGen, Beijing, China) to express Bmdimm recombinant protein, which was purified as described by Zhao et al (15). Purified protein was injected into New Zealand White rabbits for polyclonal antibody preparation.…”

“…Far-Western Blot and ELISA-A Bmdimm-glutathione S-transferase (GST) fusion protein expression vector was constructed by subcloning full-length Bmdimm cDNA into the GST gene fusion vector pGEX-4T-1 (Amersham Biosciences). Expression and purification of recombinant Bmdimm-GST were as described by Zhao et al (15). Site-directed mutants were made using QuikChange site-directed mutagenesis kits (Stratagene, La Jolla, CA).…”

“…The gene expression profiles of these factors in silk glands have been characterized individually (10,11) or by genome-wide analysis (12). A basic helix-loop-helix (bHLH) transcription factor, Bmsage, which is a homolog of Drosophila Sage (13,14), is involved in the regulation of the fib-H gene via interaction with SGF1 (15). The bHLH domain is ϳ60 amino acids and has a basic DNA-binding region of 15 amino acids followed by two ␣-helices separated by a variable loop region (16).…”