Abstract
DNA methylation of specific genome locations contributes to the distinct functions of multicellular organisms. DNA methylation can be governed by RNA-dependent DNA methylation (RdDM). RdDM is carried out by endogenous small-RNA-guided epigenomic editing complexes that add a methyl group to a precise DNA location. In plants, the Argonaute 4 (AGO4) protein is one of the main catalytic components involved in RdDM. Although small interfering RNA or short hairpin RNA has been shown to be able to guide DNA methylation in human cells, AGO protein-regulated RdDM in humans has not yet been evaluated. This study aimed to identify a key regulatory AGO protein involved in human RdDM by bioinformatics and to explore its function in RdDM by a combination of AGO4 knockdown, Alu small interfering RNA transfection, AGO4-expressing plasmid transfection, chromatin immunoprecipitation, cell-penetrating peptide-tagged AGO4 combined Alu single-guide RNA transfection, and methylation analyses. We found that first, human AGO4 showed stronger genome-wide association with DNA methylation than AGO1–AGO3. Second, endogenous AGO4 depletion demethylated DNA of known AGO4 bound loci. Finally, exogenous AGO4 de novo methylated the bound DNA sequences. Therefore, we discovered that AGO4 plays a role in human RdDM.
Highlights
DNA methylation plays critical regulatory roles in both of prokaryotic (Adhikari and Curtis, 2016) and eukaryotic (Tang et al, 2012; Zhang et al, 2018) organisms, including mammals (Jones and Takai, 2001)
We investigated whether human Argonaute 4 (AGO4) has a plant-like RNA-directed DNA methylation (RdDM) function and hypothesized that the human AGO4 protein might be involved in an increase in DNA methylation levels that participates in RdDM
We performed several experiments to determine that human AGO4 is a crucial protein in the RdDM complex
Summary
DNA methylation plays critical regulatory roles in both of prokaryotic (Adhikari and Curtis, 2016) and eukaryotic (Tang et al, 2012; Zhang et al, 2018) organisms, including mammals (Jones and Takai, 2001). Specific methylation controls cellular processes involved in many characteristics of multicellular organisms, such as tissue-specific expression and differentiation (Khavari et al, 2010; Wan et al, 2015), genomic imprinting, X-chromosome inactivation (Chen and Li, 2006; Fedoriw et al, 2012), and silencing of transposable element transcription (Hollister and Gaut, 2009; Rerkasem et al, 2015; Anwar et al, 2017). Argonaute 4 Functions in Human RdDM and genomic stability through RNA-mediated transcriptional gene silencing (Morris, 2008; Moazed, 2009). One mechanism of RNA-mediated transcriptional gene silencing is RNA-directed DNA methylation (RdDM), which was first discovered in plants and described as noncoding RNA-mediated epigenetic modification (Wassenegger et al, 1994). We investigated whether human AGO4 has a plant-like RdDM function and hypothesized that the human AGO4 protein might be involved in an increase in DNA methylation levels that participates in RdDM
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