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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.

SUBMITTER: Bernstein DL

PROVIDER: S-EPMC4463192 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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

Bernstein Diana L DL Le Lay John E JE Ruano Elena G EG Kaestner Klaus H KH

The Journal of clinical investigation 20150413 5

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 ...[more]

PMID: 25866970

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Project description:Human norovirus (HuNoV) is the main cause of gastroenteritis worldwide, yet no therapeutics are currently available. Here, we utilize a human norovirus replicon in human gastric tumor (HGT) cells to identify host factors involved in promoting or inhibiting HuNoV replication. We observed that an interferon (IFN)-cured population of replicon-harboring HGT cells (HGT-Cured) was enhanced in their ability to replicate transfected HuNoV RNA compared to parental HGT cells, suggesting that differential gene expression in HGT-Cured cells created an environment favoring norovirus replication. Microarrays were used to identify genes differentially regulated in HGT-NV and HGT-Cured compared to parental cells. We found that IFN lambda receptor (IFNLR1) expression was highly reduced in HGT-NV and HGT-Cured cells. While all three cell lines responded to exogenous IFN-β by inducing interferon-stimulated genes, HGT-NV and HGT-Cured cells failed to respond to exogenous IFN-λ. Methylation-sensitive PCR showed that an increased methylation of the IFNLR1 promoter and inhibition of DNA methyltransferase activity partially reactivated IFNLR1 expression in HGT-NV and HGT-Cured cells, indicating that host adaptation occurred via epigenetic reprogramming. Moreover, IFNLR1 ectopic expression rescued response to IFN-λ and restricted HuNoV replication in HGT-NV cells. We conclude that type III IFN is important in inhibiting HuNoV replication in vitro and that the loss of IFNLR1 enhances replication of HuNoV. This study unravels for the first time epigenetic reprogramming of the interferon lambda receptor as a new mechanism of cellular adaptation during long-term RNA virus replication and shows that an endogenous level of interferon lambda signaling is able to control human norovirus replication.IMPORTANCE Noroviruses are one of the most widespread causes of gastroenteritis, yet no suitable therapeutics are available for their control. Moreover, to date, knowledge of the precise cellular processes that control the replication of the human norovirus remains ill defined. Recent work has highlighted the importance of type III interferon (IFN) responses in the restriction of viruses that infect the intestine. Here, we analyzed the adaptive changes required to support long-term replication of noroviruses in cell culture and found that the receptor for type III IFN is decreased in its expression. We confirmed that this decreased expression was driven by epigenetic modifications and that cells lacking the type III IFN receptor are more permissive for norovirus replication. This work provides new insights into key host-virus interactions required for the control of noroviruses and opens potential novel avenues for their therapeutic control.

Dataset Information

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

Publications

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

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