Fibroin gene promoter contains a cluster of homeodomain binding sites that interact with three silk gland factors

By nuclease protection and mobility shift assays, we have shown that the 5′ flanking sequence of the Bombyx mori fibroin gene, which is known to be required for a maximal level of transcription in vitro , contains five regions (A to E) that bind protein factors from the posterior silk gland extract. A silk gland-specific factor (SGF-1) and a ubiquitous factor (FBF-A1) were found to interact with the proximal A region, while the related B region can only bind SGF-1. The three distal regions (C, D and E) bind one posterior silk gland-specific factor (SGF-2) and two ubiquitous factors (SGF-3 and −4). SGF-1 might play an important role in the expression of silk protein genes because it also binds to a similar site in the sericin-1 gene and potential SGF-1 binding sites can be found in two other silk protein genes of Bombyx mori and the fibroin gene of Antheraea yamamai . The three distal regions for SGF-2, −3 and −4 contain ten base-pairs of A+T-rich repeats that resemble the consensus binding site (TCAATTAAAT) of a number of homeodomain proteins. The TAAT motif in the core of these regions is shown to be important for the binding of these three proteins and, as described elsewhere, two Drosophila homeodomain proteins, ZEN ( zerknullt ) and EVE ( even-skipped ). Interestingly, SGF-3 appears to be a Bombyx octamer binding protein and may also be involved in the regulation of the sericin-1 gene. The possibility that the fibroin gene and other silk protein genes may be a group of target genes for some members of the homeobox gene family is discussed.