TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts
Diana L. Bernstein, John E. Le Lay, Elena G. Ruano, Klaus H. Kaestner
Diana L. Bernstein, John E. Le Lay, Elena G. Ruano, Klaus H. Kaestner
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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 2

Minimizing direct repeats permits lentiviral expression of TALE fusion proteins.

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Minimizing direct repeats permits lentiviral expression of TALE fusion p...
HeLa cells were infected with p16 jumbled TALE-DNMT, p16 jumbled TALE-DNMT mutant, or GFP control lentiviruses and harvested after 4 days. (A) Western blot of HeLa cells infected with p16 jTALE-DNMT or p16 jTALE-DNMT mutant lentivirus showing production of the full-length protein. (B) PCR amplification of the full-length TALE repeat moiety from genomic DNA (gDNA), demonstrating integration of the intact construct into the host genome, and from cDNA, demonstrating transcription of full-length mRNA, in infected HeLa cells. Amplification of plasmid DNA is shown as a reference. jTALE, jumbled TALE; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. (C) Percent DNA methylation of the p16 (CDKN2A) locus in HeLa cells infected with p16 jTALE-DNMT WT and p16 jTALE-DNMT mutant lentivirus (mean ± SEM; n = 3). Data points outlined in black indicate CpGs in which DNA methylation is significantly elevated in p16 jTALE-DNMT WT–infected cells compared with p16 jTALE-DNMT mutant–infected cells (P < 0.05, multiple t tests).