Regulation of an Endogenous Locus Using a Panel of Designed Zinc Finger Proteins Targeted to Accessible Chromatin Regions

Abstract

We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.

Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches

“…15 Most TF ZF s have been constructed for constitutive regulation of natural promoters driving reporter genes. 11,16,17 TF ZF s have been expressed from constitutive promoters transiently, retrovirally or by generating integrated stable cell lines [18][19][20][21] and have also been expressed in vivo. 22 To effectively study gene function and develop gene therapies, tightly controlled and highly inducible regulation of the target gene is desirable through direct control of the TF ZF with a cell-penetrating drug.…”

Targeted regulation of imprinted genes by synthetic zinc-finger transcription factors

“…12 From the large set of molecules able to interact with this DNA region, five different synthetic IGF2-ZFPs were chosen with the following criteria: a binding site within 7500 bp of one or more of the four IGF2 promoters (Figure 1a), and an apparent dissociation constant of o100 pM, as determined by EMSA. 19 The consensus-binding sites and apparent dissociation constants for these five proteins are given in the Materials and methods. To specify gene activation, these DNA binding moieties were fused to the VP16 activation domain.…”

“…The proteins described herein bind 9-bp recognition sites, which given a random genome, would be predicted to bind to many thousands of locations. However, the restriction on promiscuous binding enforced by the chromatin architecture of the genome, 18,19 the requirement for correct location of binding sites within regulatory regions such as promoters or enhancers, and the short functional range of the targeted effecter domains may result in sufficient specificity of function. That said, the modular nature of the zinc-finger binding domain makes possible the construction of multifinger transcription factors with four, or more, such domains 20,21,49 providing increased specificity such that a single gene is regulated genomewide.…”

“…We, and others, have constructed libraries of such rationally designed and selected zinc-fingers that bind to a vast range of different subsites, [12][13][14][15][16] and can use these for the assembly of synthetic ZFPs that recognize desired target sites within reporter genes. 17 Furthermore, these approaches have been successfully applied to the regulation of endogenous genes both in the activation of genes, for example EPO1, 18 VEGF-A, 19 erbB-2 and erbB-3, 13,20 and in the repression of gene expression, for example MDR1, 21,22 erbB-2 and erbB-3, 13,20 and PPARg. 23 Here we show that an engineered ZFP can both positively and negatively regulate the expression of the IGF2-H19 genes.…”

Persistent mismatch repair deficiency following targeted correction of hMLH1

“…12 Newer technologies, such as zinc-finger nucleases (ZFNs) for targeted gene correction in human cells; have shown great promise in overcoming some of these obstacles. 13,14 What is not evident, however, are the potential efficacy problems that targeted gene therapy may encounter in the ever-changing genetic environment of the tumor.…”