Argonaute 4 promotes genomic methylation

Abstract

RNA-directed DNA methylation (RdDM) is the major small RNA-mediated epigenetic pathway in plants. Similar to genome and epigenome editing with the CRISPR-Cas9 system, RdDM is composed of a regulatory protein and a “guide” RNA (gRNA), which recruits the complex to specific genomic locations containing DNA sequences homologous to the RNA sequence. In humans, we found that Argonaute-4 (AGO4) colocalization has a strong correlation to promoter methylation of AGO4-binding genes. Here, we engineered cell-penetrating tagged oligoarginines (R9) tagged AGO4 (R9-AGO4) or xentry tagged AGO4 (X-AGO4) loaded with single guided-RNA (sgRNA) as a tool to specifically add methylation at genome locations where sgRNA is homologous to. We uncovered that only R9-AGO4-sgRNA complex can methylate both repetitive sequences (Alu and LINE-1) and unique copy gene (EML2 and CCNA1) while X-AGO4-sgRNA can penetrate into cells, but did not provide methylation change. The induction of methylation by R9-AGO4-LINE-1 or R9-AGO4-CCNA1 also contributes to down-regulation of their own gene expression. Moreover, R9-AGO4-LINE-1 can delay proliferation rate of transfected cells compared with their control counterparts. Therefore, our study is the first time reported that the engineered R9-AGO4 protein can be transfected to cells with sgRNA and can be used as an alternative tool for epigenomic editing at any specific genes or DNA sequences in humans.