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.

Figure 4. Alterations in p16 levels due to p16 TALE-DNMT result in increased proliferation in primary human cells.

(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.

Figure 3. Targeted CpG methylation at the p16 (CDKN2A) locus results in decreased gene expression in primary human cells.

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.

Figure 2. Minimizing direct repeats permits lentiviral expression of TALE fusion proteins.

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).

Figure 1. Targeted CpG methylation of the p16 (CDKN2A) locus using TALE-DNMT fusion proteins.

(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).