Cellular response against expression of Arabidopsis DEMETER DNA demethylase in animal cells

Abstract

Epigenetics refers to heritable changes of phenotype or gene expression without the modification of DNA sequence. DNA methylation, a prominent epigenetic marks, is associated with gene expression, X chromosome inactivation, gene imprinting and developmental process. DNA methylation occurs by DNA methyltransferase to add methyl group at the 5th carbon of a cytosine to produce 5-methylcytosine (5mC), which is achieved by a similar mechanism in both the plants and animals. However, DNA demethylation pathways are fundamentally different in plant and animal. In animals, DNA demethylation is achieved by several enzymatic pathways, where TET1 catalyze the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and successive oxidation of 5hmC to 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), and deamination by AID/APOBEC. More efficient DNA demethylation is accomplished in plants by the DEMETER 5-methylcytosine DNA glycosylase, which mediates direct excision of 5mC from double strand DNA. In order to see if this plant DME is working properly in animal systems, Arabidopsis DME gene was introduced into human HEK-293T cells. DME was a successfully implemented to remove 5mC and it led to dysregulation of cell proliferation, cell cycle arrest at S phase, and severe DNA damage. Gene expression profile showed that diverse genes were differentially expressed in DME expressing cells such as cell cycle component genes, heat shock proteins, and interferon stimulated genes (ISGs). Interestingly, DME expressing cells seem to induce antiviral response through the dsRNA generated from activation of retrotransposons. Furthermore, DME could be a novel candidate for epigenome editing via the successful excision of 5mC in animal cells. DME appears to activate specific endogenous gene expression in combination with TALE module and nuclease deficient CRISPR/Cas9 and, which induced DNA demethylation at the specific locus. These works demonstrated that plant DNA demethylase catalyzed DNA demethylation and the interferon signaling played a crucial role to relieve genotoxic stresses in this DME-induced DNA demethylation processes in mammalian cells. Furthermore, DME can be utilized for specific gene regulation in DNA methylation associated disease and may shed light on the role of DNA methylation in human disease.