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TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts
Diana L. Bernstein, … , Elena G. Ruano, Klaus H. Kaestner
Diana L. Bernstein, … , Elena G. Ruano, Klaus H. Kaestner
Published April 13, 2015
Citation Information: J Clin Invest. 2015;125(5):1998-2006. https://doi.org/10.1172/JCI77321.
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Technical Advance Genetics

TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts

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Abstract

Current strategies to alter disease-associated epigenetic modifications target ubiquitously expressed epigenetic regulators. This approach does not allow specific genes to be controlled in specific cell types; therefore, tools to selectively target epigenetic modifications in the desired cell type and strategies to more efficiently correct aberrant gene expression in disease are needed. Here, we have developed a method for directing DNA methylation to specific gene loci by conjugating catalytic domains of DNA methyltransferases (DNMTs) to engineered transcription activator–like effectors (TALEs). We demonstrated that these TALE-DNMTs direct DNA methylation specifically to the targeted gene locus in human cells. Further, we determined that minimizing direct nucleotide sequence repeats within the TALE moiety permits efficient lentivirus transduction, allowing easy targeting of primary cell types. Finally, we demonstrated that directed DNA methylation with a TALE-DNMT targeting the CDKN2A locus, which encodes the cyclin-dependent kinase inhibitor p16, decreased CDKN2A expression and increased replication of primary human fibroblasts, as intended. Moreover, overexpression of p16 in these cells reversed the proliferative phenotype, demonstrating the specificity of our epigenetic targeting. Together, our results demonstrate that TALE-DNMTs can selectively target specific genes and suggest that this strategy has potential application for the development of locus-specific epigenetic therapeutics.

Authors

Diana L. Bernstein, John E. Le Lay, Elena G. Ruano, Klaus H. Kaestner

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Figure 1

Targeted CpG methylation of the p16 (CDKN2A) locus using TALE-DNMT fusion proteins.

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Targeted CpG methylation of the p16 (CDKN2A) locus using TALE-DNMT fusio...
(A) TALE-DNMT strategy for altering the epigenetic state of the p16 (CDKN2A) promoter. Locus-specific TALEs were fused to the catalytic domain of DNA methyltransferase (p16 TALE-DNMT), or a catalytically inactive DNA methyltransferase with the point mutation E752A (p16 TALE-DNMT Mut). (B) Detailed diagram of TALE-DNMT construct and target site in the p16 (CDKN2A) locus. Black boxes indicate the 3 exons of the p16 transcript, and green boxes indicate CpG islands. The TALE-DNMT was targeted to the CpG island at the promoter just before the transcription start site. The legend on the right side of the diagram indicates which nucleotide is targeted by each of the 4 different TALE repeat monomers, which are color-coded. (C) Percent methylation of individual CpGs within the CDKN2A promoter in FACS-sorted GFP-positive populations compared with untreated HeLa cells. HeLa cells were transfected with the p16 TALE-DNMT WT or p16 TALE-DNMT mutant construct and cultured for 48 hours. Cells were then FACS-sorted for GFP to isolate transfected populations. DNA methylation was quantified by sequencing of PCR-amplified bisulfite-converted genomic DNA. Graphs reflect percent DNA methylation at each CpG and its position relative to the transcription start site (TSS) (mean ± SEM; n = 3). The diagram below the graph illustrates the region of the p16 (CDKN2A) promoter that was analyzed. Data points outlined in black are significantly elevated in the p16 TALE-DNMT population compared with the p16 TALE-DNMT mutant population (P < 0.05, multiple t tests).
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