title: Engineered zinc-finger transcription factors activate OCT4 (POU5F1), SOX2, KLF4, c-MYC (MYC) and miR302/367.
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.
authors.affiliation: Sigma-Aldrich Corporation, Saint Louis, MO 63103, USA.
authors.forename: Qingzhou
authors.initials: Q
authors.lastname: Ji
authors.affiliation: Sigma-Aldrich Corporation, Saint Louis, MO 63103, USA.
authors.forename: Ashley L
authors.initials: AL
authors.lastname: Fischer
authors.affiliation: Sigma-Aldrich Corporation, Saint Louis, MO 63103, USA.
authors.forename: Clyde R
authors.initials: CR
authors.lastname: Brown
authors.affiliation: Sigma-Aldrich Corporation, Saint Louis, MO 63103, USA.
authors.forename: Erik R
authors.initials: ER
authors.lastname: Eastlund
authors.affiliation: Sigma-Aldrich Corporation, Saint Louis, MO 63103, USA.
authors.forename: Tamar
authors.initials: T
authors.lastname: Dvash
authors.affiliation: Sigma-Aldrich Corporation, Saint Louis, MO 63103, USA.
authors.forename: Bonan
authors.initials: B
authors.lastname: Zhong
authors.affiliation: Sigma-Aldrich Corporation, Saint Louis, MO 63103, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Gerber
authors.affiliation: Sigma-Aldrich Corporation, Saint Louis, MO 63103, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Lyons
authors.affiliation: Sigma-Aldrich Corporation, Saint Louis, MO 63103, USA.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Knight
authors.affiliation: Sigma-Aldrich Corporation, Saint Louis, MO 63103, USA ckreader@sial.com.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Kreader
mesh_terms: D002460:Cell Line; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis / Q000235:genetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation; D016335:Zinc Fingers
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C532169:MIRN302A microRNA, human; C568081:MIRN367 microRNA, human; C489427:MYC protein, human; D035683:MicroRNAs; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D015534:Trans-Activators