Development of zinc finger domains for recognition of the 5'-ANN-3' family of DNA sequences and their use in the construction of artificial transcription factors.
In previous studies we have developed Cys(2)-His(2) zinc finger domains that specifically recognized each of the 16 5'-GNN-3' DNA target sequences and could be used to assemble six-finger proteins that bind 18-base pair DNA sequences (Beerli, R. R., Dreier, B., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1495--1500). Such proteins provide the basis for the construction of artificial transcription factors to study gene/function relationships in the post-genomic era. Central to the universal application of this approach is the development of zinc finger domains that specifically recognize each of the 64 possible DNA triplets. Here we describe the construction of a novel phage display library that enables the selection of zinc finger domains recognizing the 5'-ANN-3' family of DNA sequences. Library selections provided domains that in most cases showed binding specificity for the 3-base pair target site that they were selected to bind. These zinc finger domains were used to construct 6-finger proteins that specifically bound their 18-base pair target site with affinities in the pm to low nm range. When fused to regulatory domains, these proteins containing various numbers of 5'-ANN-3' domains were capable of specific transcriptional regulation of a reporter gene and the endogenous human ERBB-2 and ERBB-3 genes. These results suggest that modular DNA recognition by zinc finger domains is not limited to the 5'-GNN-3' family of DNA sequences and can be extended to the 5'-ANN-3' family. The domains characterized in this work provide for the rapid construction of artificial transcription factors, thereby greatly increasing the number of sequences and genes that can be targeted by DNA-binding proteins built from pre-defined zinc finger domains.
Dreier
B
B
Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037.
Beerli
R R
RR
Segal
D J
DJ
Flippin
J D
JD
Barbas
C F
CF
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D066246:ErbB Receptors / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018815:Genes, erbB; D018773:Genes, erbB-1; D018734:Genes, erbB-2; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D020893:Receptor, ErbB-3 / Q000235:genetics; D014157:Transcription Factors / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D016335:Zinc Fingers
D004247:DNA; D017931:DNA Primers; D066246:ErbB Receptors; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; D014157:Transcription Factors