Shigeharu Takiya

Spatial and temporal expression pattern of Bombyx fork head/SGF-1 gene in embryogenesis

Abstract The embryonic expression of Bombyx fkh/SGF-1 gene has been analysed using in situ hybridization and immunohistochemistry. Both transcripts and protein were first detected in the most anterior and posterior regions at the time of germ anlage formation, and were successively expressed in the foregut and hindgut at later stages. A weaker expression was also detected in the elongated midgut. By the time embryo retraction was finished transcripts and protein were also detectable in the invaginated whole silk glands, and after the blastokinesis stage the products were restricted to the middle and posterior silk glands achieving a state required for the SGF-1 distribution for later stages. Expression could also be detected in the central and peripheral nervous systems. From these observations, we propose that Bombyx Fkh/SGF-1 may play a role in organogenesis processes such as those of the gut, silk glands, and nervous systems, act as a region specific homeotic gene, and in spite of clear embryonic developmental differences between Drosophila and Bombyx, two terminals may be determined by region specific genes such as Bombyx fkh/SGF-1 as opposed to segmental development.

Fibroin-modulator-binding protein-1 (FMBP-1) contains a novel DNA-binding domain, repeats of the score and three amino acid peptide (STP), conserved from Caenorhabditis elegans to humans

The predicted transcriptional regulatory factor for the fibroin gene of the silkworm Bombyx mori, fibroin-modulator-binding protein-1 (FMBP-1), was purified by sequential DNA affinity column chromatography, and cDNA clones corresponding to FMBP-1 were isolated from a library. The N-terminal half of FMBP-1 has a weak similarity to the DNA-binding domain of several transcriptional regulatory factors in higher plants. The C-terminal half contains four tandem repeats of a novel 23 amino acid motif, which we named the score and three amino acid peptide (STP). Other genes containing STP repeats were found in Drosophila, Caenorhabditis elegans, mouse and human. Mutational analysis of FMBP-1 showed that the STP repeats form a novel DNA-binding domain. Sequences flanking STP repeats modulated DNA-binding activity. The FMBP-1 gene was expressed during the fourth to fifth instar. FMBP-1 activity appeared to be regulated at the transcriptional level and by the post-transcriptional modification.

Inhibition of the binding of MSG-intermolt-specific complex, MIC, to the sericin-1 gene promoter and sericin-1 gene expression by POU-M1/SGF-3

The sericin-1 gene encoding a glue protein is expressed in the middle silk gland (MSG) of the silkworm, Bombyx mori. A member of the class III POU domain transcription factors, POU-M1, was cloned as the factor bound to the SC site of the sericin-1 promoter and has been proposed to be a positive transcription factor. In this study, we analyzed the expression pattern of the POU-M1 gene in fourth and fifth instars in comparison with the pattern of the sericin-1 gene. The POU-M1 gene was expressed strongly in the region anterior to the sericin-1-expressing portion of the silk gland at both feeding stages. As the sericin-1-expressing region expands from the posterior to middle portions of the MSG in the fifth instar, the POU-M1-expressing region retreated from the middle to anterior portion. Introduction of the expression vector of POU-M1 into the silk glands by gene gun technology repressed promoter activity of the sericin-1 gene, suggesting that POU-M1 regulates the sericin-1 gene negatively. An in vitro binding assay showed that POU-M1 bound not only to the SC site but also to other promoter elements newly detected in vivo. Another spatiotemporal specific factor MIC binds to these elements, and POU-M1 competed with MIC to bind at the −70 site essential for promoter activity. These results suggest that POU-M1 is involved in restricting the anterior boundary of the sericin-1-expressing region in the silk gland by inhibiting the binding of the transcriptional activator to the promoter elements.

Silk Gland Factor-2, Involved in Fibroin Gene Transcription, Consists of LIM Homeodomain, LIM-interacting, and Single-stranded DNA-binding Proteins

Background: Silk gland factor-2 (SGF-2) is a key factor regulating tissue-specific expression of the fibroin gene. Results: SGF-2 is a 1.1-MDa heteromeric complex containing Awh, Ldb, Lcaf, and fibrohexamerin proteins. Conclusion: Awh, Ldb, and Lcaf interact functionally in SGF-2 to control fibroin gene expression. Significance: This study provides new insight into the functional role of single-stranded DNA-binding proteins in protein-protein interaction and transcriptional regulation. SGF-2 binds to promoter elements governing posterior silk gland-specific expression of the fibroin gene in Bombyx mori. We purified SGF-2 and showed that SGF-2 contains at least four gene products: the silkworm orthologues of LIM homeodomain protein Awh, LIM domain-binding protein (Ldb), a sequence-specific single-stranded DNA-binding protein (Lcaf), and the silk protein P25/fibrohexamerin (fhx). Using co-expression of these factors in Sf9 cells, Awh, Ldb, and Lcaf proteins were co-purified as a ternary complex that bound to the enhancer sequence in vitro. Lcaf interacts with Ldb as well as Awh through the conserved regions to mediate transcriptional activation in yeast. Misexpression of Awh in transgenic silkworms induces ectopic expression of the fibroin gene in the middle silk glands, where Ldb and Lcaf are expressed. Taken together, this study demonstrates that SGF-2 is a multisubunit activator complex containing Awh. Moreover, our results suggest that the Ldb·Lcaf protein complex serves as a scaffold to facilitate communication between transcriptional control elements.

Fibroin Gene Transcription in the Embryonic Stages of the Silkworm, Bombyx mori

Using a modified RNase mapping method the transcription of the fibroin gene in Bombyx mori embryos was analyzed. It is known that the silk gland development begins at stage 19 of the 30 embryonic stages and its morphological development completes by stage 25. RNA samples obtained from embryos of a Chinese strain C108 from stages 4 through 23 did not give a positive signal except a faint and transient transcript detected at stage 22. In RNA samples from later stage embryos of a Kanebo commercial strain Kin‐Shu X Sho‐Wa, a faint and ambiguous fibroin transcript was detected at stages 25 and 26, and a clear and accurate initiation of transcription of the gene was detected from stage 27 and increased greatly at stages 29 and 30 reaching a level of about 0.3 ng/embryo or about 1% of total RNA presumably in the posterior silk gland. These results indicate that the fibroin gene transcription begins for the first time after the completion of the embryonic silk gland development, and also suggest that around stages 25 to 27 are probably a critical time to search for the production and accumulation of a factor(s) governing the transcriptional regulation of the fibroin gene.

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.

Hox transcription factor Antp regulates sericin-1 gene expression in the terminal differentiated silk gland of Bombyx mori

Hox genes are well-known master regulators in developmental morphogenesis along the anteroposterior axis of animals. However, the molecular mechanisms by which Hox proteins regulate their target genes and determine cell fates are not fully understood. The silk gland of Bombyx mori is a tubular tissue divided into several subparts along the anteroposterior axis, and the silk genes are expressed with specific patterns. The sericin-1 gene (ser1) is expressed in the middle silk gland (MSG) with sublocal specificity. Here we show that the Hox protein Antp is a component of the middle silk gland-specific complex, MIC (MSG-intermolt-specific complex), binds to the essential promoter element of ser1, and activates its expression. Ectopic expression of Antp in transgenic silkworms induced the expression of ser1 in the posterior silk gland (PSG), but not in the anterior part of MSG (MSG-A). Correspondingly, a MIC-like complex was formed by the addition of recombinant Antp in extracts from PSG with its cofactors Exd and Hth, but not in extracts from MSG-A. Splicing patterns of ser1 mRNA induced by the ectopic expression of Antp in PSG were almost the same as those in MSG at the fifth instar and altered depending on the induction timing of Antp. Other Hox genes were expressed with sublocal specificity in the silk gland. The Bombyx silk gland might provide a useful system for understanding how Hox proteins select and regulate their target genes.

The DNA binding of insect Fork head factors is strongly influenced by the negative cooperation of neighbouring bases.

The Drosophila melanogaster Fork head and Bombyx mori SGF1/Fork head proteins are key regulators of tissue specific gene expression in the modified larval labial glands. Here we use the competitive electrophoretic mobility shift assay to create a detailed Fork head binding matrix and we investigate some unusual features of the Fork head interaction with DNA. We found that the Fork head-DNA interaction is context dependent--the binding specificity of the protein is partly determined by specific combinations of neighbouring bases. Although the total number of the sub-optimal dinucleotide steps is not high, the negative cooperation of neighbouring bases significantly contributes to the overall binding site specificity. Our results allow efficient recognition of insect Fork head binding sites and we show that the putative Fork head cognate elements preferentially accumulate in the near upstream region of genes abundantly expressed in the labial gland.

Differential Transcription of the Fibroin and Sericin‐1 Genes in Cell‐Free Extracts

Devices of the extraction procedues and a recovery of the final supernatant as tiers accompanied with a transcription ability assessment of the individual tiers have enabled us to develop a variety of cell‐free transcription systems. The latter step revealed that the factors necessary for faithful transcription as well as enhanced transcription form aggregates or complexes indicating an intrinsic nature of these factors to interact each other. Altogether 14 cell‐free transcription systems have been developed from Bombyx mori embryos and tissues of various developmental stages and a cultured cell line, and screened for activities enhancing the basal promoter levels of the silk genes. Using these extracts a differential transcription of plural tissue‐specific genes was tried. The fibroin gene was preferentially transcribed than the sericin‐1 gene in the extracts from the posterior silk glands where the fibroin gene is specifically transcribed in vivo, while the sericin‐1 gene was dominant to the fibroin gene in the extracts from the middle silk glands where the sericin‐1 gene is specifically transcribed. Thus, these nuclear extracts offer us a biochemical clue assaying factors responsible for the differential transcription.

DNA-Binding Property of the Novel DNA-Binding Domain STPR in FMBP-1 of the Silkworm Bombyx mori

The STPR domain is a novel DNA-binding domain composed of repeats of 23 amino-acid-long peptide found in the fibroin-modulator-binding protein-1 (FMBP-1) of the silkworm Bombyx mori. Theoretical proteins having the STPR domain are highly conserved, particularly in vertebrates, but the functions are mostly unknown. In this study, the DNA-binding property of the STPR domain in FMBP-1 was examined. Use of reagents selecting the DNA groove and an oligonucleotide in which the dA:dT pairs of the probe were replaced with dI:dC pairs in mobility shift assay demonstrated that FMBP-1 approaches DNA from the major groove. Permutation electrophoresis using probes of the same length but containing the FMBP-1-binding site at different positions showed that FMBP-1 bends DNA through its binding. To induce the sharp bend of DNA, the STPR domain alone was insufficient and the long N-terminal extending region was necessary. Moreover, the basic region extending from the N-terminus of the STPR domain stabilized the DNA binding of the STPR domain. These results suggested that DNA-binding properties of the STPR domain are affected strongly by the structure of the flanking regions in the STPR domain-containing proteins.