Nuclear RNA interference provides a unique approach to the study of RNA-mediated transgenerational epigenetic inheritance. A paradox in the field is that expression of target loci is necessary for the initiation and maintenance of their silencing. How expression and repression are coordinated during animal development is poorly understood. To resolve this gap, we took imaging, deep-sequencing and genetic approaches towards delineating the developmental regulation and subcellular localization of RNA transcripts of two representative endogenous targets, the LTR retrotransposons Cer3 and Cer8. By examining wild-type worms and a collection of mutant strains, we found that the expression and silencing cycle of Cer3 and Cer8 is coupled with embryonic and germline development. Strikingly, endogenous targets exhibit a hallmark of nuclear enrichment of their RNA transcripts. In addition, germline and somatic repressions of Cer3 have different genetic requirements for three heterochromatin enzymes, MET-2, SET-25 and SET-32, in conjunction with the nuclear Argonaute protein HRDE-1. These results provide the first comprehensive cellular and developmental characterization of nuclear RNAi activities throughout the animal reproductive cycle.
Small RNA-guided chromatin silencing, also referred to as nuclear RNAi, plays an essential role in genome surveillance in eukaryotes and provides a unique paradigm to explore the complexity in RNA-mediated chromatin regulation and transgenerational epigenetics. A well-recognized paradox in this research area is that transcription of the target loci is necessary for the initiation and maintenance of the silencing at the same loci. How the two opposing activities (transcriptional activation and repression) are coordinated during animal development is poorly understood. To resolve this gap, we took single-molecule RNA imaging, deep-sequencing, and genetic approaches towards delineating the developmental regulation and subcellular localization of RNA transcripts of two exemplary endogenous germline nuclear RNAi targets in C. elegans, Cer3 and Cer8 LTR retrotransposons.By examining the wild type and a collection of mutant strains, we found that transcription and silencing cycle of Cer3 and Cer8 is tightly coupled with the early embryogenesis and germline mitotic and meiotic cell cycles. Strikingly, Cer3 and Cer8 transcripts are exclusively localized in the nuclei of germ cells in both wild type and germline nuclear RNAi-defective mutant animals. RNA-sequencing analysis found that this nuclear enrichment feature is a general feature for the endogenous targets of the germline nuclear RNAi pathway. In addition, the germline and somatic repressions of Cer3 have different genetic requirement for the three H3K9 histone methyltransferases, MET-2, SET-25, and SET-32, in conjunction with the nuclear Argonaute protein WAGO-9/HRDE-1. These results provide a first comprehensive cellular and developmental characterization of the nuclear RNAi-targeted endogenous targets throughout animal reproductive cycle. Altogether, these results support a model in which (1) both the transcriptional activation and repression steps of the germline nuclear RNAi pathway are tightly coupled with animal development, (2) the endogenous targets exhibit a hallmark of nuclear enrichment of their transcripts, and (3) different heterochromatin enzymes play distinct roles in somatic and germline silencing of the endogenous targets.
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