Chemically regulated zinc finger transcription factors

Ligand-dependent transcriptional regulators were generated by fusion of des igned Cys(2)-His(2) zinc finger proteins and steroid hormone receptor ligan d binding domains. To produce novel DNA binding domains, three-finger prote ins binding specific 9-base pair sequences were constructed from modular bu ilding blocks. Fusion of these zinc finger proteins to a transcriptional ac tivation domain and to modified ligand binding domains derived from either the estrogen or progesterone receptors yielded potent ligand-dependent tran scriptional regulators. Together with optimized minimal promoters, these re gulators provide 4-hydroxytamoxifen- or RU486-inducible expression systems with induction ratios of up to 3 orders of magnitude. These inducible expre ssion systems are functionally independent, and each can be selectively swi tched on within the same cell. The potential use of zinc finger-steroid rec eptor fusion proteins for the regulation of natural promoters was also expl ored. A gene-specific six-finger protein binding an 18-base pair target seq uence was converted into a ligand-dependent regulator by fusion with either two estrogen receptor ligand binding domains or one ecdysone receptor and one retinoid X receptor ligand binding domain. These single-chain receptor proteins undergo an intramolecular rearrangement, rather than intermolecula r dimerization and are functional as monomers, Thus, the ability to enginee r DNA binding specificities of zinc finger proteins enables the constructio n of ligand dependent transcriptional regulators with potential for the reg ulation of virtually any desired artificial or natural promoter. It is anti cipated that the novel chemically regulated gene switches described herein will find many applications in applied and basic research, where the specif ic modulation of gene expression can be exploited.