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Nucleic Acids Res. 2014 Apr;42(7):4375-90. doi: 10.1093/nar/gku109. Epub 2014 Feb 5.

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

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1
Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China, SBS Core Laboratory, CUHK Shenzhen Research Institute, Shenzhen, China, Bone Marrow Transplantation Centre, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, China, Department of Bioinformatics and Genomics, The University of North Carolina at Charlotte, Charlotte, NC 28223, USA, Advanced Biomedical Computing Center, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, China and Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR, China.

Abstract

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

PMID:
24500196
PMCID:
PMC3985678
DOI:
10.1093/nar/gku109
[Indexed for MEDLINE]
Free PMC Article
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