Selective binding of Drosophila BR-C isoforms to a distal regulatory element in the hsp23 promoter

The Broad-Complex (BR-C) gene plays a key role in the ecdysone regulatory hierarchy. Together with other early ecdysone-inducible genes BR-C transmits the hormonal signal to a set of secondary response genes in a tissue-specific manner. Among its targets is the hsp23 gene. Previously we showed that expression of the hsp23 gene in late third instar is BR-C-dependent, and accompanied by the appearance of a BR-C-dependent DNase I hypersensitive site at position -1400 (DHS-1400). BR-C encodes a family of transcription factors, and we show here that at least three BR-C protein isoforms--Z1, Z2, and Z3--bind to the sequences around DHS-1400 in vitro. A DNase I footprinting assay reveals five protected regions, designated site 1 to site 5, each of which specifically associates with one or several BR-C protein isoforms. We also show that a 100 bp region overlapping site 5, which binds all three isoforms in vitro, is required for hsp23 activity in vivo. The deletion of binding site 5 in a reporter gene construct reproduced the effect of the npr class mutations, that is, hsp23 is no longer expressed in any tissue tested except brain. Thus, BR-C regulates hsp23 expression via direct interaction of the predominant isoform with the distal regulatory element.