Estrogen Receptor-KRAB Chimeras Are Potent Ligand-dependent Repressors of Estrogen-regulated Gene Expression

Georgius De Haan,Sudsanguan Chusacultanachai,Chengjian Mao,Benita S Katzenellenbogen,David J Shapiro

doi:10.1074/jbc.275.18.13493

Georgius De Haan, Sudsanguan Chusacultanachai + Show 3 more

Open Access

https://doi.org/10.1074/jbc.275.18.13493

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Abstract

As an approach to targeted repression of genes of interest, we describe the development of human estrogen receptor (ER) alpha-KRAB repressor domain chimeras that are potent ligand-dependent repressors of the transcription of estrogen response element (ERE)-containing promoters and analyze their mechanisms of action. Repression by the KRAB domain was dominant over transactivation mediated by ER AF1 and AF2. An ERE and an ER ligand (estrogen or antiestrogen) were required for repression. Studies with several promoters and cell lines demonstrated that the presence of EREs, rather than the capacity for estrogen induction, determines the potential for repression of a gene by the KRAB-ERalpha-KRAB (KERK) chimera. A single consensus ERE was sufficient for repression, but the KERK chimera was unable to suppress transcription from the imperfect ERE in the native pS2 promoter. We recently reported mutations that enhance binding of a steroid receptor DNA-binding domain to the ERE. Introducing these mutations into wild-type ER enhanced transactivation 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 and basal transcription on a promoter containing the pS2 ERE. These modified ER-KRAB chimeras should prove useful as new tools for the functional analysis and repression of ER-regulated genes.

Highlights

Generating ligand-regulated activators or repressors targeted to DNA sequences in any gene of interest represents a challenging long-term goal of protein engineering
The ability of KERK to repress transcription of a reporter gene containing the SV40 promoter and enhancer (SV40PE) and four consensus estrogen response element (ERE) was tested. This (ERE)4-pGL3-SV40PE reporter plasmid exhibits substantial intrinsic activity, referred to as basal transcription, which is further enhanced by ligand-activated estrogen receptor (ER)
The basal promoter activity of the (ERE)4-pGL3SV40PE reporter plasmid in the absence of estrogen receptor was set at 100%

Summary

Introduction

Generating ligand-regulated activators or repressors targeted to DNA sequences in any gene of interest represents a challenging long-term goal of protein engineering. To create ligand-dependent repressors targeted to ERE-containing genes, we constructed chimeras of ER␣ and the KRAB (Kruppel-associated box) transcription repression domain (6 –9) of the KOX1 protein ( named ZNF10) [7, 8]. This novel ER-KRAB chimera (KERK-3M) is a potent repressor of both basal and estrogen-induced activities of genes containing the consensus ERE or the imperfect pS2 ERE.

Results

Conclusion