Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks

Abstract

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5'-GNN-3' subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5'-(GNN)6-3' family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5'-untranslated region of this gene was converted into a transcriptional repressor by fusion with Kr uppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16's minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.

Figure 1

Nucleotide sequence of the human erbB-2 promoter fragment used in these studies. Nucleotide positions −758 to −1 relative to the ATG initiation codon are shown, with known transcription factor binding sites (16) underlined. Sp1 binding sites are marked as mapped by DNase footprinting (32). The two palindromic sequences Pal I and II bound by RBPJκ (11) are marked by inverted arrows. CCAAT and TATAA sequences are boxed. The major transcription initiation sites (19) are indicated by asterisks. The zinc finger target sequence e2c is underlain with a gray box. The short stretch of sequence identity between human, rat, and mouse genes is indicated by a dashed underline. Restriction sites used for cloning of the promoter fragment are indicated. The arrowhead at position −24 denotes the 3′ end of an erbB-2 control promoter fragment lacking the zinc finger target sequence e2c.

Figure 2

ELISA analysis of zinc finger DNA-binding specificities. The indicated three-finger proteins (A) and six-finger proteins (B) were expressed in E. coli as MBP fusion proteins. Specificity of binding was analyzed by measuring the binding activity in total lysates to immobilized biotinylated hairpin oligonucleotides containing the indicated 9-bp (A) or 18-bp (B) targets. The nucleotide sequences of the six-finger nontarget oligonucleotides were as follows: e1a, 5′-GCC GAG GCG GCC GGA GTC-3′; e1b, 5′-GTT GTG GCG TTG GCG GCG-3′; b3, 5′-GCC TGA GAG GGA GCG GTG-3′; c5, 5′-GCG GAG GCA GGA GGC GGG-3′; zif-zif, 5′-GCG TGG GCG GCG TGG GCG-3′ (B). Assays were performed in duplicate and the maximal signals were normalized to 1. The open box on top of each bar represents the standard deviation.

Figure 3

Amino acid sequence alignment of six-finger proteins specific for the 18-bp target sequence e2c. Recognition helix positions −2 to 6 of each finger (F1 to F6) are underlain with dark gray boxes and labeled according to their DNA-binding specificity. Sequence identities in the framework regions are underlain with light gray boxes. Positions of the conserved cysteine and histidine residues are marked by asterisks. The corresponding positions of the SfiI recognition sites, used for cloning the zinc finger coding regions into the various expression vectors, as well as the BsrF1 and XmaI recognition sites, used for the construction of DNAs encoding six-finger proteins, are indicated.

Figure 4

Structure of zinc finger–effector domain fusion proteins. Effector domains are as labeled. White boxes, zinc fingers (ZF); black boxes, simian virus 40 nuclear localization signal; light gray boxes, hemagglutinin epitope tag; dark gray boxes, VP16 minimal activation domain.

Figure 5

Specific repression (A) or activation (B) of erbB-2 promoter activity by using zinc finger–effector domain fusion proteins. HeLa cells were cotransfected with the indicated zinc finger expression plasmids and erbB-2 promoter–luciferase reporter constructs. The erbB-2 (−1571 to −24) reporter plasmid lacks the zinc finger target sequence. Luciferase activity in total cell extracts was measured 48 h after transfection. Each bar represents the mean value (±SD) of duplicate (A) or triplicate (B) measurements.