Synergistic and tunable human gene activation by combinations of synthetic transcription factors

Pérez-Piñera, Pablo; Ousterout, David G.; Brunger, Jonathan M.; Farin, Alicia M.; Glass, Katherine A.; Guilak, Farshid; Crawford, Gregory E.; Hartemink, Alexander J.; Gersbach, Charles A.

doi:10.1038/nmeth.2361

Cited by 226 publications

(252 citation statements)

References 25 publications

Supporting

Mentioning

236

Contrasting

Order By: Relevance

“…Although nucleosome occlusion will prevent the binding of some TALEs, the data we present here suggest that in the absence of such occlusion, TALEs can bind to repressed chromatin at least in some cells in the population. Together with the recent report that TALEs synergise to perform their function, our data reinforce the idea that using a series of TALEs with adjacent binding sites [23,24] is likely to increase the chances of TALE proteins being effective in vivo. Southern blots to determine nucleosome positioning.…”

Section: Discussionsupporting

confidence: 87%

“…Instead, we find that there is a relatively small difference in binding to active and repressed loci, implying that TALEs should function at inactive loci. Consistent with this, TALEs were recently shown to function at such loci [23].…”

Section: Discussionmentioning

confidence: 53%

“…This, together with the recent reports that TALE proteins synergise to activate genes [23,24], begins to explain how TALEs can function on repressed chromatin substrates [23]: The binding of a TALE to the linker DNA or to the more accessible regions of the nucleosome could increase binding of a second TALE or another transcription factor closer to the nucleosome dyad, a phenomenon first described for GAL4 [7].…”

Section: Discussionmentioning

confidence: 90%

See 2 more Smart Citations

TALE proteins bind to both active and inactive chromatin

Scott

Kupinski

Kirkham

et al. 2014

Biochemical Journal

View full text Add to dashboard Cite

Transcription activator-like effector (TALE) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. Here, we systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only two-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.

show abstract

Section: Discussionsupporting

confidence: 87%

Section: Discussionmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 90%

See 1 more Smart Citation

TALE proteins bind to both active and inactive chromatin

Scott

Kupinski

Kirkham

et al. 2014

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…Like zinc fingers, TALEs are also compatible with numerous epigenetic modifiers, including the TET1 hydroxylase catalytic domain (Maeder et al 2013b) and the lysine-specific histone demethylase 1 (LSD1) (Mendenhall et al 2013) domains, which have been used for targeted CpG demethylation and histone demethylation, respectively. In particular, the ease with which a large number of TALEs can be constructed has enabled the discovery that tiling a promoter sequence with combinations of synthetic transcription factors can lead to a synergistic increase in gene expression (Maeder et al 2013b;Perez-Pinera et al 2013). And, like zinc fingers (Beerli et al 2000b;Pollock et al 2002;Magnenat et al 2008;Polstein and Gersbach 2012), TALE activators have also been successfully engineered to regulate gene expression in response to external or endogenous ) chemical stimuli, optical signals , and even proteolytic cues (Copeland et al 2016;Lonzaric et al 2016).…”

Section: Targeted Transcription Factors Tools For Modulating Gene Expmentioning

confidence: 99%

Genome-Editing Technologies: Principles and Applications

Gaj

Sirk

Shui

et al. 2016

Cold Spring Harb Perspect Biol

297

142

View full text Add to dashboard Cite

Targeted nucleases have provided researchers with the ability to manipulate virtually any genomic sequence, enabling the facile creation of isogenic cell lines and animal models for the study of human disease, and promoting exciting new possibilities for human gene therapy. Here we review three foundational technologies-clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs). We discuss the engineering advances that facilitated their development and highlight several achievements in genome engineering that were made possible by these tools. We also consider artificial transcription factors, illustrating how this technology can complement targeted nucleases for synthetic biology and gene therapy.

show abstract

“…Recent studies have demonstrated that the nucleasenull Cas9 (dCas9), which was fused with transcription activation domains (e.g. VP16 or VP64), functioned as a transcriptional activator [8]- [18] , suggesting that the CRISPR/Cas9-based system can be used to control the expression of specific genes. Since the transcriptional activity of the dCas9 activators was not high [8], [16] , however, the efficacy of the cell-fate changes was still very low (~10%) [17] .…”

mentioning

confidence: 99%

A Powerful CRISPR/Cas9‐Based Method for Targeted Transcriptional Activation

et al. 2016

View full text Add to dashboard Cite

Abstract:The targeted transcriptional activation of endogenous genes is an important tool for understanding physiological transcriptional networks, synthesizing genetic circuits, and inducing cellular phenotype changes. The CRISPR/Cas9 system has a great potential to achieve this purpose, however it has not yet succeeded to activate endogenous genes and induce cellular phenotype changes, efficiently. We herein show a powerful method for transcriptional activation by the CRISPR/Cas9-based system. Replacement of the methylated promoter with unmethylated one by the CRISPR/Cas9-based system was sufficient to activate the expression of the neural cell-specific gene OLIG2 and the embryonic stem cell-specific gene NANOG in HEK293T cells. Moreover, CRISPR/Cas9-based OLIG2 activation induced embryonic carcinoma cell line NTERA-2 to express neuronal marker βIII-tubulin. Thus, these data provide a novel method to handle genetic circuits and phenotype changes.Technologies for editing the epigenetic marks of targeted genes are required to understand complex transcriptional networks, accurately. Small molecules, such as DNA methylation inhibitors, alter the epigenetic state globally, but cannot target any specific loci. The CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat/CRISPR-associated protein 9) system has been shown to target specific genomic locus and induce the sitedirected DNA brake, when combined with a single-guide RNA (sgRNA) that contains the complementary 20 nucleotides of the target sequence and the protospacer-adjacent motif (PAM), NGG [1]-[7] . Recent studies have demonstrated that the nucleasenull Cas9 (dCas9), which was fused with transcription activation domains (e.g. VP16 or VP64), functioned as a transcriptional activator [8]-[18] , suggesting that the CRISPR/Cas9-based system can be used to control the expression of specific genes. Since the transcriptional activity of the dCas9 activators was not high [8], [16] , however, the efficacy of the cell-fate changes was still very low (~10%) [17] . In addition, it was concerned that the dCas9 activators may trigger non-targeted transcriptional activation with unknown transcription regulators and their transcriptional activity may not be a physiological level. It was, therefore, required to establish new system that activates the target gene expression at physiological level and induces cell-fate changes.In order to achieve this, we have focused on the microhomology-mediated end-joining (MMEJ)-dependent integration of donor DNA using CRISPR/Cas9 [19], [20] . MMEJ is a DNA double-stranded break (DSB) repair mechanism that utilizes microhomologous sequences (5-25 bp) for error-prone end-joining [21] . Using the MMEJ-combined CRISPR/Cas9 system, here we show a powerful method that activates the silenced gene by replacing its methylated promoter with unmethylated one. This system can be used for the analysis of transcriptional networks, cell-fates and genetic circuits in various types of cells.We designed a CRISPR/Cas9-based epigenome edit...

show abstract

Synergistic and tunable human gene activation by combinations of synthetic transcription factors

Cited by 226 publications

References 25 publications

TALE proteins bind to both active and inactive chromatin

TALE proteins bind to both active and inactive chromatin

Genome-Editing Technologies: Principles and Applications

A Powerful CRISPR/Cas9‐Based Method for Targeted Transcriptional Activation

Contact Info

Product

Resources

About