Molecular characterization of BmGATA-beta, a gene involved in choriogenesis in the silkworm Bombyx mori

The chorion (or eggshell) of the silkworm Bombyx mori is synthesized by the follicular cells and consists of over 100 different proteins. HcA/B.12 is a chorion gene pair expressed during the late stages of choriogenesis, and that has been used as a model for the study of developmentally regulated gene expression. The gene BmGATAbeta encodes several transcription factors of the GATA family that bind to the promoter of HcA/B.12.;I have further characterized the BmGATAbeta gene and its transcripts. BmGATAbeta encodes at least 6 different mRNAs by alternative splicing. The 3 main isoforms differ in the organization of their DNA-binding domains: two of the transcripts (BmGATAbeta1 and BmGATAbeta2) encode GATA proteins with two zinc fingers but differ in the length of the spacer region between the zinc fingers. The third isoform encodes a GATA protein with only the C-terminal zinc finger. The other 3 isoforms lack the second exon in their N-terminal domains, and this splicing variation is found associated with all 3 types of DNA-binding domains. The promoter region of the BmGATAbeta gene and 7 kb of upstream sequence were isolated. The promoter region contains numerous potential GATA-binding sites and expression of the gene is modulated by autoregulation. The BmGATAbeta3 protein isoform, but not BmGATAbeta2 or BmGATAbeta3, is a potent activator of the BmGATAbeta gene in co-transfection assays. Antibodies were raised against various regions of the BmGATAbeta protein. BmGATAbeta is first detectable at the late stages of vitellogenesis and accumulates in the nuclei of follicular cells during choriogenesis. In contradiction to earlier research, I found that BmGATAbeta is predominantly located in the nuclei of the follicular cells at all stages of choriogenesis, and there is no substantial pool of the protein in the cytoplasm at the early stages of choriogenesis. Also, the subcellular localization of BmGATAbeta in Bm5 tissue culture cells can not be altered by various chemicals that affect the activity of kinases and phosphatases. Finally, the expression of BmGATAbeta in follicular cells could not be disrupted by the use of double-stranded RNA interference. The finding that BmGATAbeta is nuclear throughout choriogenesis suggests that it may be involved in the expression of all the chorion genes.