Transforming growth factor-beta 3 (TGF-beta 3) has a specific role in vivo in the patterning of embryonic and tissue-specific gene expression. We have cloned and sequenced the mouse TGF-beta 3 5'-flanking region to study the transcriptional regulation of this gene. Promoter fragments were cloned into a promoterless luciferase reporter plasmid to study functional activity in a human skin melanoma cell line A375 (A375). Sequential 5'-deletion encompassing DNA sequences from -2297 to -1003 bp exhibited high promoter activity in A375 cells, whereas the promoter activity decreased to minimal in the -742 to 104 bp regions, suggesting both positive and negative transcriptional regulation in the TGF-beta 3 promoter. The fragment containing 1.8 kb had the highest luciferase activity. Characterization of this 1.8 kb 5'-flanking region upstream of the translation start site showed a putative hunchback-binding site consensus sequence. The electrophoretic mobility shift assay (EMSA) and transient transfection experiments showed that the putative hunchback-binding site is functional and regulated TGF-beta 3 promoter transcriptional activity. The DNA-complex including the hunchback sequence binding protein (HbSBP) was important for suppression of the promoter activity in A375 cells. Mutation of the hunchback consensus sequence resulted in up to 2-fold higher promoter activity than the wild type construct. There was an absence of HbSBP in other cell lines tested including 3T3 fibroblast and B-16 mouse skin melanoma as determined by EMSA and Western blot analysis. HbSBP may function as a TGF-beta 3 gene transcriptional regulator and may be expressed in a cell type-specific manner. (c) 2006 Elsevier Inc. All rights reserved.