Chalertpet Kanwalat, Pin-On Piyapat, Aporntewan Chatchawit, Patchsung Maturada, Ingrungruanglert Praewphan, Israsena Nipan, Mutirangura Apiwat
Interdisciplinary Program of Biomedical Sciences, Faculty of the Graduate School, Chulalongkorn University, Bangkok, Thailand.
Faculty of Medicine, King Mongkut's Institute of Technology Ladkrabang, Bangkok, Thailand.
Front Genet. 2019 Jul 4;10:645. doi: 10.3389/fgene.2019.00645. eCollection 2019.
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 methylated the bound DNA sequences. Therefore, we discovered that AGO4 plays a role in human RdDM.
特定基因组位置的DNA甲基化有助于多细胞生物发挥不同的功能。DNA甲基化可由RNA依赖的DNA甲基化（RdDM）调控。RdDM由内源性小RNA引导的表观基因组编辑复合物执行，该复合物会在精确的DNA位置添加一个甲基基团。在植物中，AGO4蛋白是参与RdDM的主要催化成分之一。虽然小干扰RNA或短发夹RNA已被证明能够在人类细胞中引导DNA甲基化，但人类中AGO蛋白调控的RdDM尚未得到评估。本研究旨在通过生物信息学鉴定参与人类RdDM的关键调控AGO蛋白，并通过AGO4敲低、Alu小干扰RNA转染、AGO4表达质粒转染、染色质免疫沉淀、细胞穿透肽标记的AGO4联合Alu单向导RNA转染以及甲基化分析等方法，探索其在RdDM中的功能。我们发现，首先，与AGO1 - AGO3相比，人类AGO4在全基因组范围内与DNA甲基化的关联更强。其次，内源性AGO4缺失使已知AGO4结合位点的DNA去甲基化。最后，外源性AGO4使结合的DNA序列甲基化。因此，我们发现AGO4在人类RdDM中发挥作用。
