Chi-chung Hui

Developmental expression of the Bombyx Antennapedia homologue and homeotic changes in the Nc mutant

Background: Abundant availability of homeotic mutants in Bombyx mori provides us with clues to study the body plan of the silkworm. Previous studies indicated that the ECa/ECa and EN/EN embryos, which reveal drastic morphological changes (Itikawa 1943, 1952) lack the homologue of abd‐A and the homologues of abd‐A and Ubx, respectively (Ueno et al. 1992). It will be interesting to characterize a mutant named Nc, the locus of which was mapped about 1.4 cm apart from the E loci (Itikawa 1944, 1952).

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.

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.

Developmental Regulation of Silk Gene Expression in Bombyx mori

Our major concern has been understanding how cells are specialized and express a set of genes during development. Along this line, our target has been a dissection of silk gene regulation in the silk gland of Bombyx mori (Suzuki, 1977; Suzuki et al., 1987). The development of the silk gland originates as an invagination of the ectoderm in the labial segment of stage 19 embryos, and completes morphologically by stage 25 (Nunome, 1937). Transcription of the fibroin (heavy chain) gene (Suzuki and Brown, 1972; Suzuki et al., 1972; Ohshima and Suzuki, 1977; Tsujimoto and Suzuki, 1979a, b) begins at around stage 25 of the embryos (Ohta et al., 1988). After this first activation the fibroin gene is repeatedly switched on and off in the posterior silk gland cells during larval development (Suzuki and Suzuki, 1974; Suzuki and Giza, 1976; Maekawa and Suzuki, 1980). When fibroin gene transcription in the posterior silk glands of the fifth instar larvae was analyzed by nuclear run-on assays, it was found that the transcription is restricted to the anterior region at the beginning of the instar and spreads toward the posterior region as the stage proceeds (Obara and Suzuki, 1988). Transcription of the fibroin light chain (or P25) gene was found to occur in parallel with that of the fibroin gene during the fourth molting stage and the fifth larval instar (Couble et al., 1983; Kimura et al., 1985).

Homeodomain Binding Sites in the Promoter Region of Silk Protein Genes1

Several 10 bp AT‐rich repeats containing a core sequence TTAATT or its complement, AATTAA, in the distal part of the Bombyx mori fibroin gene promoter are known to bind a group of silk gland factors and to be required for a maximal level of transcription in a posterior silk gland extract. These repeats are similar to a consensus sequence (TCAATTAAAT) of the binding sites deduced for a group of Drosophila homeodomain proteins. By DNasel footprinting assay, two of these proteins, ZEN (zerknüllt) and EVE (even‐skipped), were shown to bind these sites with high affinity. The “TTAATT” core sequence was found to be important for ZEN and EVE binding by an electrophoretic mobility shift assay. Though these homeodomain proteins apparently recognize the same AT‐rich consensus sequence, we have identified a mutant sequence that enhances ZEN but not EVE binding. Alteration of sequence flanking the repeats was also found to affect the binding of these proteins. The binding of EVE and ZEN was shown to be facilitated by multiple repeats in the binding sites. Similar AT‐rich repeats can be found in the promoter region of other silk protein genes and, as shown here, two regions in the sericin‐1 gene promoter also bind these homeodomain proteins. The interaction of some homeodomain proteins and these homeodomain binding sites might be important for the developmental regulation of a group of silk protein genes.