Engineered zinc-finger transcription factors activate OCT4 (POU5F1),SOX2,KLF4,c-MYC (MYC) and miR302/367.

Qingzhou Ji,Erik R Eastlund,Scott W Knight,Clyde R Brown,Ashley L Fischer,Mark A Gerber,Carol A Kreader,Bonan Zhong,Tamar Dvash,Ian Lyons

doi:10.1093/nar/gku243

Abstract

Artificial transcription factors are powerful tools for regulating gene expression. Here we report results with engineered zinc-finger transcription factors (ZF-TFs) targeting four protein-coding genes, OCT4, SOX2, KLF4 and c-MYC, and one noncoding ribonucleic acid (RNA) gene, the microRNA (miRNA) miR302/367 cluster. We designed over 300 ZF-TFs whose targets lie within 1 kb of the transcriptional start sites (TSSs), screened them for increased messenger RNA or miRNA levels in transfected cells, and identified potent ZF-TF activators for each gene. Furthermore, we demonstrate that selected ZF-TFs function with alternative activation domains and in multiple cell lines. For OCT4, we expanded the target range to −2.5 kb and +500 bp relative to the TSS and identified additional active ZF-TFs, including three highly active ZF-TFs targeting distal enhancer, proximal enhancer and downstream from the proximal promoter. Chromatin immunoprecipitation (FLAG-ChIP) results indicate that several inactive ZF-TFs targeting within the same regulatory region bind as well as the most active ZF-TFs, suggesting that efficient binding within one of these regulatory regions may be necessary but not sufficient for activation. These results further our understanding of ZF-TF design principles and corroborate the use of ZF-TFs targeting enhancers and downstream from the TSS for transcriptional activation.

Highlights

Various classes of naturally occurring deoxyribonucleic acid (DNA)-binding molecules, including zinc-finger (ZF) proteins [1,2], triplex forming oligos [3], meganucleases [4] and transcription activator-like effectors (TALEs) [5,6,7], as well as the CRISPR cas9 protein-guide ribonucleic acid (RNA) system [8,9], have been engineered to bind sequencespecific endogenous target sites
Each six or five-finger ZF coding sequence assembly was cloned between an N-terminal nuclear localization signal and a C-terminal NF-B p65 activation domain to generate a series of zinc-finger transcription factors (ZF-transcription factors (TFs)) expression constructs for each gene
Previous research on ZF- and TALE-TFs has primarily focused on characterizing a limited number of artificial TFs, often targeted to DNase-hypersensitive or known TFbinding regions [6,12,13,14,15,16,17,19]

Summary

Introduction

Various classes of naturally occurring deoxyribonucleic acid (DNA)-binding molecules, including zinc-finger (ZF) proteins [1,2], triplex forming oligos [3], meganucleases [4] and transcription activator-like effectors (TALEs) [5,6,7], as well as the CRISPR cas protein-guide ribonucleic acid (RNA) system [8,9], have been engineered to bind sequencespecific endogenous target sites Such sequence-specific binding proteins, when coupled to various functional domains, are powerful tools for modifying the genome and regulating gene expression. ZF and other DNA-binding proteins have been fused with the endonuclease domain from FokI to allow site-specific cutting, followed by insertion and/or deletion [10,11] These proteins have been fused to transcriptional activation or repression domains, such as VP16 from the herpes simplex virus or a Krueppel-associated box (KRAB) domain. These engineered transcription factors (TFs) effectively up or downregulate target gene expression when delivered into cells [12,13,14,15,16,17,18]

Methods

Results

Conclusion