An induction gene trap for identifying a homeoprotein-regulated locus

An important issue in developmental biology is the identification of homeop rotein target genes. We have developed a strategy based on the internalizat ion and nuclear addressing of exogenous homeodomains, using an engrailed ho meodomain (EnHD) to screen an embryonic stem (ES) cell gene trap library. E ight integrated gene trap loci responded to EnHD. One is within the bullous pemphigoid antigen 1 (BPAG1) locus, in a region that interrupts two neural isoforms. By combining in vivo electroporation with organotypic cultures, we show that an already identified BPAG1 enhancer/promoter is differentiall y regulated by homeoproteins Hoxc-8 and Engrailed in the embryonic spinal c ord and mesencephalon. This strategy can therefore be used for identifying and mutating homeoprotein targets. Because homeodomain third helices can in ternalize proteins, peptides, phosphopeptides, and antisense oligonucleotid es, this strategy should be applicable to other intracellular targets for c haracterizing genetic networks involved in a large number of physiopatholog ical states.