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 May 1, 2015; First 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

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 3

Targeted CpG methylation at the p16 (CDKN2A) locus results in decreased gene expression in primary human cells.

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Targeted CpG methylation at the p16 (CDKN2A) locus results in decreased ...
Primary human fibroblasts were transduced with p16 jTALE-DNMT WT or p16 jTALE-DNMT mutant lentiviruses and incubated for 4 days. (A) Percent DNA methylation of CpGs within the p16 (CDKN2A) promoter region. Graphs reflect percent DNA methylation at each CpG (mean ± SEM; n = 3) and position relative to the transcription start site. Data points outlined in black are significantly elevated in the p16 jTALE-DNMT population compared with the p16 jTALE-DNMT mutant population (P < 0.05, multiple t tests). (B) p16 transcript expression in fibroblasts treated with p16 jTALE-DNMT WT or mutant lentiviruses relative to the mutant negative control. Expression levels were normalized to HPRT1 mRNA levels (mean ± SEM; n = 3). *P < 0.05, 2-tailed t test. (C) Average percent DNA methylation of CpGs at each CpG island within the p16 (CDKN2A) locus and at β-actin (ACTB), a housekeeping gene located on a different chromosome. The diagram below the graph illustrates the position of CpG islands at the p16 (CDKN2A) locus (mean ± SEM; n = 3). *P < 0.05, **P < 0.01, 2-tailed t test. (D) Average percent methylation at genes adjacent to p16 (CDKN2A), as described in C. The diagram above the graph indicates the position of each gene relative to p16 (CDKN2A). (E) mRNA expression of genes adjacent to p16 (CDKN2A) in lentivirally transduced human fibroblasts, determined as described in B.