Abstract
DNA methylation is a prominent epigenetic modification in plants and animals regulated by similar mechanisms but the process of DNA demethylation is profoundly different. Unlike vertebrates that require a series of enzymatic conversions of 5-methylcytosine (5mC) into other bases for DNA demethylation, plants utilize the DEMETER (DME) family of 5mC DNA glycosylases to catalyze a direct removal of 5mC from DNA. Here we introduced Arabidopsis DME into human HEK-293T cells to allow direct 5mC excision, and observed that direct DNA demethylation activity was successfully implemented by DME expression. In addition, DME induced diverse cellular responses such as cell proliferation inhibition, cell cycle dysregulation and S phase arrest. Microarray and methylome analyses revealed that DME upregulated a number of genes including cell cycle components, heat shock proteins, and notably, various interferon-stimulated genes. Moreover, DME-mediated DNA demethylation activated endogenous repeat elements, which are likely to form dsRNAs as viral mimics and eventually trigger interferon cascades to establish the antiviral state. This work demonstrates that plant DNA demethylase catalyzes DNA demethylation with a bypass of initial base conversion steps, and the interferon signaling plays a pivotal role to alleviate genotoxic stresses associated with DME-induced DNA demethylation in mammalian cells.
Highlights
DNA methylation has a variety of functions in many cellular processes such as transcriptional regulation, differentiation, gene imprinting and transposable element silencing[1,2,3]
In order to implement direct DNA demethylation activity in animal cells, we introduced Arabidopsis DME DNA demethylase into human embryonic kidney (HEK)-293T cells by transfection because of their reliable growth, transfection feasibility, and stable expression of exogenous genes
The green fluorescent protein (GFP)-DMEΔ fusion protein was found to be localized in the nucleus (Supplementary Fig. 1), and the whole cell extract prepared from HEK-293T cells expressing GFP-DMEΔ was able to catalyse the excision of 5mC from a double-stranded oligonucleotide substrate in vitro, whereas the extract obtained from the HEK-293T cells expressing only GFP (293T-GFP) did not process the methylated DNA substrate (Fig. 1c)
Summary
DNA methylation has a variety of functions in many cellular processes such as transcriptional regulation, differentiation, gene imprinting and transposable element silencing[1,2,3]. Active DNA demethylation is replication-independent, and DNA methylation is enzymatically removed without cell division. Plants utilize DEMETER (DME)/REPRESSOR OF SILENCING 1 (ROS1) DNA glycosylase family proteins to recognize and excise 5mC from DNA4–6. It is believed that DME induces demethylation of many transposable elements (TEs) in the central cell and vegetative cells producing small RNAs, which are likely to translocate to nearby gamete cells such as an egg www.nature.com/scientificreports/. The ten-eleven translocation (TET) family of proteins catalyse successive conversions of 5mC into 5-hydroxymethylcytosine (5hmC) and higher oxidative derivatives 5-formylcytosine (5fC) and 5-carboxycytosine (5caC) prior to base excision by a T:G mismatch-specific thymine DNA glycosylase (TDG)[13,14,15,16,17]. Active DNA demethylation in animals requires additional steps at the initial stage compared to the plant demethylation pathway
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