Journal: Frontiers in Genetics
Article Title: Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells
doi: 10.3389/fgene.2019.00645
Figure Lengend Snippet: Association of the AGO4-binding genes and methylation levels. (A) Scheme showing that tetracycline-treated cells switch off AGO4 protein expression (Tet+) from day 1 to 12 and that 5’-azacytidine treatment from day 7 to 9 inhibits DNA methyltransferase (5-AC). (B) Detection of AGO4 mRNA in Tet- (AGO4 expression) and Tet+ (No AGO4 expression) cells; AGO4 was significantly expressed in the absence of Tet. (C) Confirmation of AGO4-binding genes showed that AGO4 only bounds to the selected gene from bioinformatics data as exemplified by C16ORF89 . (D) However, AGO4 did not bind to a gene lacking the AGO4-binding site, MSN . (E) The AGO4 methylation level of the gene, which contains AGO4-binding sites, was increased due to the presence of the AGO4 protein (Tet-). This increased methylation level was not associated with the presence of DNA methyltransferase (5-AC+). (F) These results were not observed for a gene lacking AGO4-binding sites, MSN . (G to H) Recovery of DNA methylation levels is found in cells expressing the AGO4 protein (Tet-) with 5’-azacytidine withdrawal in both of AGO4-binding gene, C16ORF89 and non-AGO4-binding gene, MSN ; AGO4 is inferred to methylate previously demethylated loci. The above data are the representative dataset from three independent experiments presented as the mean ± SEM. Statistical analyses were performed using a paired-sample t -test, where p < 0.05, p < 0.01, and p < 0.005 are represented as *, **, and ***, respectively, where Tet+ means tetracycline treatment, 5-AC+ means azacytidine treatment, and Tet- and 5-AC- mean untreated groups (I). Schematic overview of the role of AGO4 in de novo methylation. After DNA replication, DNA methylation is maintained by DNMT1 by the recognition of hemimethylated CpG on the parental strand as a template and the addition of newly methylated CpG to the daughter strand. DNMT1 is inhibited by the action of 5-AC, and the methylation level should be decreased by half under 5-AC treatment. In our study, when tetracycline was added, AGO4 shRNA was manipulated, resulting in AGO4 expression repression. Thus, without tetracycline, AGO4 is upregulated and binds to DNA loci, and DNA methylation is maintained, although 5-AC is added, suggesting that AGO4 involved in de novo methylation
Article Snippet: Overexpression of AGO4 was performed by the using pIRESneo-FLAG/HA AGO4 plasmid (Addgene, MA, USA).
Techniques: Binding Assay, Methylation, Expressing, DNA Methylation Assay, shRNA
![Viral titers and viral RNA of (A) influenza-A-infected (PR/8/1934), (B) encephalomyocarditis (EMCV)-infected, or (C) vesicular stomatitis virus (VSV)-infected (indicated multiplicities of infection [MOIs] for 16 h) bone-marrow-derived macrophages (BMDMs) from Ago1 +/+ (black) and Ago1 −/− (yellow), (D–F) Ago3 +/+ (black) and Ago3 −/− (green), or (G–I) <t>Ago4</t> +/+ (black) and Ago4 −/− (red) littermate mice. Representative plaque assay images are shown. Influenza Ns1, EMCV 2A-2B , or VSV G RNA levels were quantified by qPCR relative to TATA box protein ( Tbp ). All data are from two or three biological replicates performed in triplicate and combined. Errors bars represent SEM. *p < 0.05, **p < 0.01, and ***p < 0.001, as measured by two-way ANOVA with Bonferroni multiple comparison test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9342/pmc07039342/pmc07039342__nihms-1560134-f0002.jpg)
![Knockout of DUSP11 decreases AGO association with 5′ triphosphorylated BLV 5p miRNAs. ( A ) Immunoblot analysis of input lysate (0.1%) and RIP (5%) of endogenous AGO proteins (∼95 kDa) using pan-AGO antibody (2A8) in parental (wild-type [WT]) and DUSP11 knockout (D11-KO) HEK293T cells transfected with either pBLV-B2 or pBLV-B5 expression vectors. Asterisks indicate IgG light and heavy chains. ( B ) Northern blot analysis of input RNA (2.5%) and RNA recovered from RIP samples (50%) in A . The membrane was first probed with 5p probes, stripped, and reprobed with the 3p probes. ( C ) RIP–Northern blot analysis of BLV B2 and B5 miRNAs associated with individual AGO proteins. Parental (wild-type) and DUSP11 knockout HEK293T cells were cotransfected with pBLV-B2 and pBLV-B5 and each of the indicated AGO-Flag expression vectors. The individual AGOs were precipitated using the anti-Flag M2 antibody. Total input RNA (5% for AGOs1–3 and 10% for <t>AGO4)</t> and RIP samples (95%) were subjected to Northern blot analysis. Blots were first probed for BLV-miR-B5-5p, sequentially stripped, and reprobed for BLV-miR-B5-3p, BLV-miR-B2-5p, and BLV-miR-B2-3p. ( D ) RIP–Northern blot analysis of parental and DUSP11 knockout HEK293T cells cotransfected with pAGO1-Flag and the BLV-B5 pre-miRNA mimic pretreated with (+; 5′-p-B5) or without (−; 5′-ppp-B5) RNA 5′ polyphosphatase. RIP was performed using the anti-Flag M2 antibody 48 h after transfection of mimics and pAGO1-Flag. RNA recovered from RIP-Flag was subjected to Northern blot analysis. The membrane was first probed for the 5p miRNA arm, stripped, and reprobed for the 3p arm. Immunoblot analysis of the RIP-Flag samples is shown below the Northern blot to show the immunoprecipitation efficiency of AGO1-Flag between the RNA 5′ polyphosphatase-treated (+) and untreated (−) samples. ( E ) Similar to D except using the BLV-B5 duplexed miRNA mimics in which the 5p arm was pretreated with (+) or without (−) RNA 5′ polyphosphatase. All of the duplexes contained a 3p miRNA mimic that was treated with RNA 5′ polyphosphatase to mimic the 5′ monophosphate generated by Dicer processing.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6614/pmc05066614/pmc05066614__2076f05.jpg)
