CRUCIFORM-EXTRUDING REGULATORY ELEMENT CONTROLS CELL-SPECIFIC ACTIVITY OF THE TYROSINE-HYDROXYLASE GENE PROMOTER

Tyrosine hydroxylase (TH) is expressed specifically in catecholaminerg ic cells. We have identified a never regulatory sequence in the upstre am region of the bovine TH gene promoter formed by a dyad symmetry ele ment (DSE1;352/-307 bp). DSE1 supports TH promoter activity in TH-expr essing bovine adrenal medulla chromaffin (BAMC) cells and inhibits pro moter activity in non-expressing TE671 cells. DNase I footprinting of relaxed TH promoter DNA showed weak binding of nuclear BAMC cell prote ins to a short sequence in the right DSE1 arm. In BAMC cells, deletion of the right arm markedly reduced the expression of luciferase from t he TH promoter. However, deletion of the left DSE1 arm or its reversed orientation (RevL) also inactivated the TH promoter. In supercoiled T H promoter, DSE1 assumes a cruciform-like conformation i.e., it binds cruciform-specific 2D3 antibody, and S1 nuclease-cleavage and OsO4-mod ification assays have identified an imperfect cruciform extruded by th e DSE1. DNase I footprinting of supercoiled plasmid showed that crucif ormed DSE1 is targeted by nuclear proteins more efficiently than the l inear duplex isomer and that the protected site encompasses the left a rm and center of DSE1. Our results suggest that the disruption of intr astrand base-pairing preventing cruciform formation and protein,bindin g to DSE1 is responsible for its inactivation in DSE1 mutants. DSE1 cr uciform may act as a target site for activator (BAMC cells) and repres sor (TE671) proteins. Its extrusion emerges as a novel mechanism that controls cell-specific promoter activity.