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Nucleic Acids Research Advance Access originally published online on May 3, 2007
Nucleic Acids Research 2007 35(10):3465-3477; doi:10.1093/nar/gkm241
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Nucleic Acids Research, 2007, Vol. 35, No. 10 3465-3477
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Rational design of an estrogen receptor mutant with altered DNA-binding specificity

Denis Nguyen1, Martine Bail1,2, Genevieve Pesant1, Virginie N. Dupont1,2, Étienne Rouault1, Julie Deschênes1,2, Walter Rocha1,2, Geneviève Melançon1, Sergey V. Steinberg1 and Sylvie Mader1,2,*

1Biochemistry Department and 2Institute for Research in Immunology and Cancer, Université de Montréal, C.P. 6128 Succursale Centre Ville, Montréal QC H3C 3J7, Canada

*To whom correspondence should be addressed. Tel: +1 514 343 7166; Fax: +1 514 343 6843; Email: sylvie.mader{at}umontreal.ca

Received November 1, 2006. Revised March 24, 2007. Accepted April 2, 2007.

Although artificial C2-H2 zinc fingers can be designed to recognize specific DNA sequences, it remains unclear to which extent nuclear receptor C4 zinc fingers can be tailored to bind novel DNA elements. Steroid receptors bind as dimers to palindromic response elements differing in the two central base pairs of repeated motifs. Predictions based on one amino acid—one base-pair relationships may not apply to estrogen receptors (ERs), which recognize the two central base pairs of estrogen response elements (EREs) via two charged amino acids, each contacting two bases on opposite DNA strands. Mutagenesis of these residues, E203 and K210 in ER{alpha}, indicated that both contribute to ERE binding. Removal of the electric charge and steric constraints associated with K210 was required for full loss of parental DNA-binding specificity and recognition of novel sequences by E203 mutants. Although some of the new binding profiles did not match predictions, the double mutation E203R-K210A generated as predicted a mutant ER that was transcriptionally active on palindromes of PuGCTCA motifs, but not on consensus EREs. This study demonstrates the feasibility of designing C4 zinc finger mutants with novel DNA-binding specificity, but also uncovers limitations of this approach.


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V. Bourdeau, J. Deschenes, D. Laperriere, M. Aid, J. H. White, and S. Mader
Mechanisms of primary and secondary estrogen target gene regulation in breast cancer cells
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[Abstract] [Full Text] [PDF]


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