title: Induction of DNA methylation by artificial piRNA production in male germ cells.
abstract: Global DNA demethylation and subsequent de novo DNA methylation take place in mammalian male embryonic germ cells [1-3]. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), which are germline-specific small RNAs, have been postulated to be critically important for de novo DNA methylation of retrotransposon genes, and many proteins, including PIWI family proteins, play pivotal roles in this process [4-6]. In the embryonic mouse testis, two mouse PIWI proteins, mouse PIWI-like (MILI) and mouse PIWI2 (MIWI2), are involved in the biogenesis of piRNAs through the so-called ping-pong amplification cycle [7-10], and long single-stranded RNAs transcribed from the gene regions of piRNA clusters have been proposed to be the initial material [11-16]. However, it remains unclear whether transcription from the piRNA clusters is required for the biogenesis of piRNAs. To answer this question, we developed a novel artificial piRNA production system by simple expression of sense and antisense EGFP mRNAs in embryonic male germ cells in the piRNA biogenesis phase. EGFP expression was silenced by piRNA-dependent DNA methylation, indicating that concomitant expression of sense and antisense RNA transcripts is necessary and sufficient for piRNA production and subsequent piRNA-dependent gene silencing. In addition, we demonstrated that this artificial piRNA induction paradigm could be applied to an endogenous gene essential for spermatogenesis, DNMT3L [3, 17, 18]. This study not only provides novel insights into the molecular mechanisms of piRNA production, but also presents an innovative strategy for inducing epigenetic modification in germ cells.
authors.affiliation: Department of Pathology, Graduate School of Frontier Biosciences, Osaka University, Yamada-oka 2-2 Suita, Osaka 565-0871, Japan; CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Itou
authors.affiliation: Medical School, Osaka University, Yamada-oka 2-2 Suita, Osaka 565-0871, Japan; CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Shiromoto
authors.affiliation: Department of Pathology, Graduate School of Frontier Biosciences, Osaka University, Yamada-oka 2-2 Suita, Osaka 565-0871, Japan; CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
authors.forename: Shin-ya
authors.initials: SY
authors.lastname: Yukiho
authors.affiliation: Department of Pathology, Graduate School of Frontier Biosciences, Osaka University, Yamada-oka 2-2 Suita, Osaka 565-0871, Japan; CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
authors.forename: Chika
authors.initials: C
authors.lastname: Ishii
authors.affiliation: Department of Pathology, Graduate School of Frontier Biosciences, Osaka University, Yamada-oka 2-2 Suita, Osaka 565-0871, Japan; CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Nishimura
authors.affiliation: RIKEN BioResources Center, Tsukuba 305-0074, Ibaraki, Japan.
authors.forename: Narumi
authors.initials: N
authors.lastname: Ogonuki
authors.affiliation: RIKEN BioResources Center, Tsukuba 305-0074, Ibaraki, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Ogura
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Yamada-oka 2-2 Suita, Osaka 565-0871, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Hasuwa
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Yamada-oka 2-2 Suita, Osaka 565-0871, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Fujihara
authors.affiliation: Medical School, Osaka University, Yamada-oka 2-2 Suita, Osaka 565-0871, Japan; CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan. Electronic address: smiya@patho.med.osaka-u.ac.jp.
authors.forename: Satomi
authors.initials: S
authors.lastname: Kuramochi-Miyagawa
authors.affiliation: Department of Pathology, Graduate School of Frontier Biosciences, Osaka University, Yamada-oka 2-2 Suita, Osaka 565-0871, Japan; Medical School, Osaka University, Yamada-oka 2-2 Suita, Osaka 565-0871, Japan; CREST, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan. Electronic address: tnakano@patho.med.osaka-u.ac.jp.
authors.forename: Toru
authors.initials: T
authors.lastname: Nakano
mesh_terms: D000818:Animals; D019175:DNA Methylation / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods; D018507:Gene Expression Regulation, Developmental; D005854:Germ Cells / Q000254:growth & development / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins