The use of zinc finger peptides to study the role of specific factor binding sites in the chromatin environment

doi:10.1016/S1046-2023(02)00009-9

Methods

Volume 26, Issue 1, 2 January 2002, Pages 76-83

https://doi.org/10.1016/S1046-2023(02)00009-9 Get rights and content

Abstract

The once ambitious goal of creating custom DNA-binding factors has been achieved. Advances in construction methodology now enable any laboratory to create site-specific binding proteins to nearly any sequence. Using predefined zinc finger modules, new proteins can be constructed in days with minimal cost and using only standard polymerase chain reaction techniques. The existing spectrum of modules can be rearranged to produce more than one billion different proteins that bind with high affinity and specificity. Artificial transcription factors based on modified zinc finger domains have recently been shown by several groups to be capable of activating or repressing transcription of a handful of endogenous genes in the chromatin environment of plant and animal cells. These proteins can also be used in a number of ways to compete with endogenous factors for specific binding sites in vivo. Zinc finger peptides are therefore useful tools in the study of gene regulation and signal transduction. A detailed description of the construction method is presented, along with a full discussion of potential caveats and future expectations.

Introduction

The ability to recognize and bind specific DNA sequences within the chromatin environment of the nucleus has wide-ranging potential application in the areas of experimental and applied biology. Several promising approaches have been developed recently, including triple-helix-forming oligonucleotides (TFO), peptide nucleic acids (PNAs), polyamides, and modified DNA-binding proteins [1], [2], [3], [4], [5]. All have been shown to function in the chromatin environment to varying degrees, and all are commercially available (e.g., TFO, Operon Technologies, Inc., Alameda, CA; PNA, Pantheco A/S, Hørsholm, Denmark; polyamides, GeneSoft, Inc., South San Francisco, CA; modified zinc finger proteins, Sangamo BioSciences, Inc., Richmond, CA). Advances in these technologies have generally been driven by the goal of creating novel therapeutics. For example, polyamides have been used to inhibit HIV-1 replication in cell culture, and artificial transcription factors based on modified zinc-finger proteins have been shown to regulate the therapeutically relevant endogenous genes erbB-2, erbB-3, epo, and vegf-a [6], [7], [8], [9], [10]. However, the therapeutic potential of these compounds often overshadows their use as tools for investigation of gene expression and signal transduction.

In principle, custom DNA-binding compounds can be applied to the study of gene regulation in several ways. For example, they can be used to study the function of putative transcription factors or other components of signal transduction pathways. The primary methods for understanding gene function involve enhancing or abolishing expression of the gene, followed by observation of phenotypic effects. Applying the above technologies to produce a “functional” gene knockout requires only a fraction of the effort required to produce a genetic knockout, and the techniques do not require alterations to the cellular factors or their DNA binding sites. Modified DNA-binding proteins offer a distinct advantage in this area because they can be easily engineered to be transcriptional activators as well as repressors [7], [11]. Chemically regulated, zinc finger-based transcription factors enable conditional expression of the gene of interest [7], [12].

This article focuses on a different use of DNA-binding compounds, as in vivo competitors to endogenous binding factors for specific binding sites. Theoretically, a compound can be designed to bind the same target site as a cellular binding factor. In this case, the compound would be expected to compete for all of the factor's binding sites (Fig. 1A) or, if the factor had degenerate binding specificity, for a subset of the binding sites. Alternatively, the compound could be targeted in such a way that it recognizes the binding site for the protein of interest as well as unique sequence flanking the binding site (Fig. 1B). In this way, the compound can be used to examine the binding of an endogenous factor at one particular site. Such a tool would be useful to understand if the binding of a factor at a particular site was physiologically relevant. If several binding sites for the same factor were present, such a tool could help to determine if all the sites were required, and if eliminating certain sites produced more dramatic effects than eliminating others (Fig. 1C). If binding sites for different factors were arranged spatially in a promoter, the compound could disrupt binding at one site without affecting the others (Fig. 1D). Although such studies could be performed in vitro using modified DNA fragments, the compound should allow exploration within the more physiologically relevant environment of living cells and on native, unmodified, chromatin DNA.

Section snippets

Custom zinc finger proteins as tools

This article describes the construction and use of modified zinc finger DNA-binding proteins as site-specific tools. The use of zinc finger proteins for the purposes described above has been extensively investigated. Several studies evaluated the potential of repressing transcription by interfering with the assembly of the transcription machinery. In one study, binding sites for the murine transcription factor Zif268 were placed in an artificial promoter at various distances from the TATA box

Building custom zinc finger proteins

Among the many naturally occurring DNA-binding proteins, the Cys₂–His₂ zinc finger domain has emerged as the scaffold of choice for the design of novel sequence-specific DNA-binding proteins [5]. Each 30-amino-acid residue domain or finger forms a stable ββα fold. The N terminus of the α helix typically recognizes a 3-bp subsite. Recognition of extended sequences is achieved by linking the domains into tandem arrays. The versatility of zinc finger proteins in DNA recognition is perhaps best

Step 1: Select target sites

If a protein could be constructed to bind any desired sequence, the investigator would have infinite flexibility in choosing a target site. However, given the current state of the technology, there are several constraints. The primary constraint is that the site must be targetable by the available modules (Table 1). It is therefore helpful to begin by identifying a general area to be targeted, then searching the area for sequences with contiguous blocks of three or more of the triplets

Concluding remarks

This article describes methods for the construction of custom DNA-binding proteins that can be used to compete with endogenous binding factors in a chromatin environment. The ability to create modified zinc finger proteins is now readily accessible. Any molecular biologist can create these proteins using generic materials and techniques, without the need for collaboration with specialized laboratories. More than one billion different zinc finger proteins can be rapidly assembled from predefined

Acknowledgments

I thank Professor Carlos F. Barbas III and Dr. Pilar Blancafort for comments and discussion on this manuscript. I also thank the members of the Barbas laboratory for many contributions to this area.

References (38)

B.P. Casey et al.
Prog. Nucleic Acid Res. Mol. Biol.
(2001)
P.E. Nielsen
Meth. Enzymol.
(2001)
D.J. Segal et al.
Curr. Opin. Chem. Biol.
(2000)
B. Dreier et al.
J. Biol. Chem.
(2001)
P.Q. Liu et al.
J. Biol. Chem.
(2001)
L. Zhang et al.
J. Biol. Chem.
(2000)
D.J. Segal et al.
Curr. Opin. Biotechnol.
(2001)
R.R. Beerli et al.
J. Biol. Chem.
(2000)
J.S. Kim et al.
J. Biol. Chem.
(1997)
J.S. Kang et al.
J. Biol. Chem.
(2000)

M.H. Kuo et al.

Methods

(1999)

C.O. Pabo et al.

J. Mol. Biol.

(2000)

S.A. Wolfe et al.

Structure

(2001)

S.A. Wolfe et al.

J. Mol. Biol.

(1999)

B. Dreier et al.

J. Mol. Biol.

(2000)

M. Isalan et al.

Meth. Enzymol.

(2001)

N. Corbi et al.

Biochem. Biophys. Res. Commun.

(1998)

M. Elrod-Erickson et al.

Structure

(1996)

A.F. Faruqi et al.

Mol. Cell. Biol.

(1996)

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The use of zinc finger peptides to study the role of specific factor binding sites in the chromatin environment

Abstract

Introduction

Section snippets

Custom zinc finger proteins as tools

Building custom zinc finger proteins

Step 1: Select target sites

Concluding remarks

Acknowledgments

Prog. Nucleic Acid Res. Mol. Biol.

Meth. Enzymol.

Curr. Opin. Chem. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Biotechnol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Methods

J. Mol. Biol.

Structure

J. Mol. Biol.

J. Mol. Biol.

Meth. Enzymol.

Biochem. Biophys. Res. Commun.

Structure

Mol. Cell. Biol.