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

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 4

Alterations in p16 levels due to p16 TALE-DNMT result in increased proliferation in primary human cells.

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Alterations in p16 levels due to p16 TALE-DNMT result in increased proli...
(A) Representative immunofluorescence image of EdU incorporation in human fibroblasts infected with WT or mutant p16 jTALE-DNMT lentivirus. After 72 hours of infection, cells were incubated with EdU for 1 hour and stained for EdU. Cell nuclei are stained blue (DAPI), and EdU-positive nuclei are stained red. Images were taken at ×10 magnification (n = 7). (B) Percent EdU incorporation of cells infected with p16 jTALE-DNMT WT or mutant lentivirus, with (n = 4) or without (n = 7) coinfection of CMV-p16 lentivirus. Three random images were counted for each biological replicate (mean ± SEM). ***P < 0.001; NS, not significant; 2-tailed t test. (C) Population doubling time of human fibroblasts infected with p16 jTALE-DNMT WT or mutant lentivirus (mean ± SEM; n = 4). *P < 0.05, 2-tailed t test. (D) Transcript levels of cell cycle regulators in human fibroblasts transduced with WT p16 jTALE-DNMT lentivirus relative to mutant lentivirus. Expression was normalized to HPRT1 mRNA levels (mean ± SEM; n = 3). Two-tailed t test. (E) Average percent DNA methylation of CpGs at the nearest CpG island of cell cycle regulators evaluated in D. Average DNA methylation was measured by PCR amplification of bisulfite-converted genomic DNA followed by high-throughput sequencing (mean ± SEM; n = 3). **P < 0.01, ***P < 0.001, 2-tailed t test. CKI, cyclin-dependent kinase inhibitor.