Estrogen receptor-KRAB chimeras are potent ligand-dependent repressors of estrogen-regulated gene expression

As an approach to targeted repression of genes of interest, we describe the development of human estrogen receptor (ER) alpha-KRAB repressor domain ch imeras that are potent ligand-dependent repressors of the transcription of estrogen response element (ERE)-containing promoters and analyze their mech anisms of action. Repression by the KRAB domain was dominant over transacti vation mediated by ER AF1 and AF2. An ERE and an ER ligand (estrogen or ant iestrogen) were required for repression. Studies with several promoters and cell lines demonstrated that the presence of EREs, rather than the capacit y for estrogen induction, determines the potential for repression of a gene by the KRAB-ER alpha-KRAB (HERK) chimera. A single consensus ERE was suffi cient for repression, but the KERK chimera was unable to suppress transcrip tion from the imperfect ERE in the native pS2 promoter. We recently reporte d mutations that enhance binding of a steroid receptor DNA-binding domain t o the ERE. Introducing these mutations into wild-type ER enhanced transacti vation from the pS2 ERE. Insertion of these mutations into KERK created the novel repressor KERK-3M, which is a potent repressor of both ER-induced an d basal transcription on a promoter containing the pS2 ERE. These modified ER-KRAB chimeras should prove useful as new tools for the functional analys is and repression of ER-regulated genes.