Abstract:
Argonaute proteins (AGOs) are evolutionarily conserved and ubiquitously expressed in all higher eukaryotes and have important function in several biological processes, such as cell differentiation and transposon silencing. The commonly known functions of AGOs are key in the gene-silencing pathways guided by small RNAs. There are many human AGOs; however, redundancy and the distinctive roles of different AGOs have not been well characterized. Here, I evaluated the expression of genes containing LINE-1 in AGO1-4-knocked down HEK293 cells using bioinformatics approach: Connection Up- or Down- Regulation Expression Analysis of Microarrays X (CU-DREAM-X). Furthermore, I measured the methylation levels of AGO1-4-bound LINE-1s and specific genes using Chromatin immunoprecipitation (ChIP) and quantitative combined bisulfite restriction analysis of methylation pattern (Q-COBRA-MP) and Pyrosequencing, respectively. The results showed that genes containing LINE-1s in AGO1, -2, -3 or -4-knocked down HEK293 cells prevented up-regulation, increased up-regulation, unchanged regulation and prevented up-regulation, respectively. Interestingly, AGO1-4 bound to LINE-1 differently in term of the methylation level. Although the methylation level of AGO1 and -4-bound LINE-1s was not different from the genome, AGO2- and AGO3-bound LINE-1s were hypomethylated. Finally, in case of specific genes, AGO4 bound to genes was reduced after Si-AGO4 HEK293 cell lines were treated with tetracycline for 7 days. Then, methylation levels of AGO4 binding genes were reduced after Si-AGO4 HEK293 cell lines were treated tetracycline together with 5-aza-deoxycytidine for 9 days and were recovered after Si-AGO4 HEK 293 cell lines were cultivated continuously for 3 days without tetracycline and 5-aza-deoxycytidine. Our experiments demonstrate the distinctive epigenetic roles of AGO1-4 in regulating genes containing LINE-1s and evaluate AGO4 mediate small-RNA-guided de novo methylation in human cells. It is interesting to further explore the underlining mechanism of these various functions.
