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

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.

[1]  Jun Ma,et al.  GAL4-VP16 is an unusually potent transcriptional activator , 1988, Nature.

[2]  J. Bonventre,et al.  The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[3]  B. Howard,et al.  The Transcriptional Coactivators p300 and CBP Are Histone Acetyltransferases , 1996, Cell.

[4]  H. Schöler,et al.  Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. , 1996, Development.

[5]  M. Hagmann,et al.  The VP16 paradox: herpes simplex virus VP16 contains a long-range activation domain but within the natural multiprotein complex activates only from promoter-proximal positions , 1997, Journal of virology.

[6]  H. Schöler,et al.  Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4 , 1998, Cell.

[7]  F. Yao,et al.  Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. , 1998, Human gene therapy.

[8]  J. Miyazaki,et al.  Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells , 2000, Nature Genetics.

[9]  N. L. La Thangue,et al.  p300/CBP proteins: HATs for transcriptional bridges and scaffolds. , 2001, Journal of cell science.

[10]  H. Schöler,et al.  Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences , 2001, Mammalian Genome.

[11]  Pilar Blancafort,et al.  Scanning the human genome with combinatorial transcription factor libraries , 2003, Nature Biotechnology.

[12]  M. Wiznerowicz,et al.  Conditional Suppression of Cellular Genes: Lentivirus Vector-Mediated Drug-Inducible RNA Interference , 2003, Journal of Virology.

[13]  Roderick T. Hori,et al.  TFIIB-facilitated recruitment of preinitiation complexes by a TAF-independent mechanism. , 2004, Nucleic acids research.

[14]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[15]  H. Cedar,et al.  G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis , 2006, Nature Cell Biology.

[16]  T. Ichisaka,et al.  Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors , 2007, Cell.

[17]  T. Swigut,et al.  H3K27 Demethylases, at Long Last , 2007, Cell.

[18]  P. Collas,et al.  Epigenetic reprogramming of OCT4 and NANOG regulatory regions by embryonal carcinoma cell extract. , 2007, Molecular biology of the cell.

[19]  Melissa Hardy,et al.  The Tol2kit: A multisite gateway‐based construction kit for Tol2 transposon transgenesis constructs , 2007, Developmental dynamics : an official publication of the American Association of Anatomists.

[20]  Dong Qi,et al.  A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis. , 2008, Journal of virological methods.

[21]  N. D. Clarke,et al.  Integration of External Signaling Pathways with the Core Transcriptional Network in Embryonic Stem Cells , 2008, Cell.

[22]  Ronnie J Winfrey,et al.  Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. , 2008, Molecular cell.

[23]  R. Jaenisch,et al.  A drug-inducible system for direct reprogramming of human somatic cells to pluripotency. , 2008, Cell stem cell.

[24]  Matthew J. Moscou,et al.  A Simple Cipher Governs DNA Recognition by TAL Effectors , 2009, Science.

[25]  Jens Boch,et al.  Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors , 2009, Science.

[26]  Thomas Lufkin,et al.  Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb , 2009, Nature Cell Biology.

[27]  Marcos J. Araúzo-Bravo,et al.  Oct4-Induced Pluripotency in Adult Neural Stem Cells , 2009, Cell.

[28]  F. Tang,et al.  Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells , 2009, Nature.

[29]  N. D. Clarke,et al.  A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity , 2010, Nature.

[30]  Erin L. Doyle,et al.  Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting , 2011, Nucleic acids research.

[31]  R. Barrangou,et al.  CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. , 2011, Annual review of genetics.

[32]  G. Church,et al.  Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. , 2011, Nature biotechnology.

[33]  R. Jaenisch,et al.  De novo DNA methylation by Dnmt3a and Dnmt3b is dispensable for nuclear reprogramming of somatic cells to a pluripotent state. , 2011, Genes & development.

[34]  J. Rousseau,et al.  Transcription activator-like effector proteins induce the expression of the frataxin gene. , 2012, Human gene therapy.

[35]  L. Bleris,et al.  Transcription activator-like effector hybrids for conditional control and rewiring of chromosomal transgene expression , 2012, Scientific Reports.

[36]  Ruhong Zhou,et al.  Comprehensive Interrogation of Natural TALE DNA Binding Modules and Transcriptional Repressor Domains , 2012, Nature Communications.

[37]  Daniel F. Voytas,et al.  Targeting G with TAL Effectors: A Comparison of Activities of TALENs Constructed with NN and NK Repeat Variable Di-Residues , 2012, PloS one.

[38]  H. Leonhardt,et al.  Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers , 2012, Nucleic acids research.

[39]  I. Dawid,et al.  Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs) , 2012, Proceedings of the National Academy of Sciences.

[40]  S. Ramaswamy,et al.  A Molecular Roadmap of Reprogramming Somatic Cells into iPS Cells , 2012, Cell.

[41]  Eric S. Lander,et al.  Chromatin modifying enzymes as modulators of reprogramming , 2012, Nature.

[42]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[43]  Xinqi Gong,et al.  Recognition of methylated DNA by TAL effectors , 2012, Cell Research.

[44]  Sandy L. Klemm,et al.  Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase , 2012, Cell.

[45]  Fayza Daboussi,et al.  Overcoming Transcription Activator-like Effector (TALE) DNA Binding Domain Sensitivity to Cytosine Methylation*♦ , 2012, The Journal of Biological Chemistry.

[46]  J. Doudna,et al.  RNA-guided genetic silencing systems in bacteria and archaea , 2012, Nature.

[47]  Rudolf Jaenisch,et al.  One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering , 2013, Cell.

[48]  Morgan L. Maeder,et al.  CRISPR RNA-guided activation of endogenous human genes , 2013, Nature Methods.

[49]  Christopher M. Vockley,et al.  RNA-guided gene activation by CRISPR-Cas9-based transcription factors , 2013, Nature Methods.

[50]  Luke A. Gilbert,et al.  CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes , 2013, Cell.

[51]  J. C. Tsang,et al.  Reprogramming to Pluripotency Using Designer TALE Transcription Factors Targeting Enhancers , 2013, Stem cell reports.

[52]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[53]  J. Keith Joung,et al.  Robust, synergistic regulation of human gene expression using TALE activators , 2013, Nature Methods.

[54]  Jeffry D. Sander,et al.  Efficient In Vivo Genome Editing Using RNA-Guided Nucleases , 2013, Nature Biotechnology.

[55]  Yarden Katz,et al.  Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system , 2013, Cell Research.

[56]  Farshid Guilak,et al.  Synergistic and tunable human gene activation by combinations of synthetic transcription factors , 2013, Nature Methods.

[57]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.

[58]  R. Janknecht,et al.  KDM4/JMJD2 histone demethylases: epigenetic regulators in cancer cells. , 2013, Cancer research.

[59]  Luke A. Gilbert,et al.  Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression , 2013, Cell.

[60]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[61]  Feng Zhang,et al.  Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system , 2013, Nucleic acids research.

[62]  G. Hong,et al.  Nucleic Acids Research , 2015, Nucleic Acids Research.