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Transgenerational Epigenetic Inheritance Is Negatively Regulated by the HERI-1 Chromodomain Protein

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Transgenerational Epigenetic Inheritance Is Negatively Regulated by the HERI-1 Chromodomain Protein | INVESTIGATION Transgenerational Epigenetic Inheritance Is Negatively Regulated by the HERI-1 Chromodomain Protein Roberto Perales,* Daniel Pagano,* Gang Wan,* Brandon D. Fields,*,† Arneet L. Saltzman,‡ and Scott G. Kennedy*,1 *Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, †Laboratory of Genetics, University of Wisconsin–Madison, Wisconsin 53706, and ‡Department of Cell and Systems Biology, University of Toronto, Ontario M5S 3G5, Canada ORCID ID: 0000-0002-3964-9734 (R.P.) ABSTRACT Transgenerational epigenetic inheritance (TEI) is the inheritance of epigenetic information for two or more generations. In most cases, TEI is limited to a small number of generations (two to three). The short-term nature of TEI could be set by innate biochemical limitations to TEI or by genetically encoded systems that actively limit TEI. In Caenorhabditis elegans, double-stranded RNA (dsRNA)-mediated gene silencing [RNAi (RNA interference)] can be inherited (termed RNAi inheritance or RNA-directed TEI). To identify systems that might actively limit RNA-directed TEI, we conducted a forward genetic screen for factors whose mutation enhanced RNAi inheritance. This screen identified the gene heritable enhancer of RNAi (heri-1), whose mutation causes RNAi inheritance to last longer (. 20 generations) than normal. heri-1 encodes a protein with a chromodomain, and a kinase homology domain that is expressed in germ cells and localizes to nuclei. In C. elegans, a nuclear branch of the RNAi pathway [termed the nuclear RNAi or NRDE (nuclear RNA defective) pathway] promotes RNAi inheritance. We find that heri-1(2) animals have defects in spermatogenesis that are suppressible by mutations in the nuclear RNAi Argonaute (Ago) HRDE-1, suggesting that HERI-1 might normally act in sperm progenitor cells to limit nuclear RNAi and/or RNAi inheritance. Consistent with this idea, we find that the NRDE nuclear RNAi pathway is hyperresponsive to experimental RNAi treatments in heri-1 mutant animals. Interestingly, HERI-1 binds to genes targeted by RNAi, suggesting that HERI-1 may have a direct role in limiting nuclear RNAi and, therefore, RNAi inheritance. Finally, the recruitment of HERI-1 to chromatin depends upon the same factors that drive cotranscriptional gene silencing, suggesting that the generational perdurance of RNAi inheritance in C. elegans may be set by competing pro- and antisilencing outputs of the nuclear RNAi machinery. KEYWORDS transgenerational epigenetic inheritance; RNAi; chromatin; small RNAs HE inheritance of epigenetic information for two or more et al. 2012), and the inheritance of acquired traits in mice Tgenerations is referred to as transgenerational epigenetic (Carone et al. 2010; Walker and Gore 2011; Radford et al. inheritance (TEI) (Heard and Martienssen 2014). Many ex- 2012; Castel and Martienssen 2013; Padmanabhan et al. amples of TEI have now been documented including, but 2013; Somer and Thummel 2014; Holoch and Moazed 2015; not limited to, paramutation in plants (Arteaga-Vazquez and Martienssen and Moazed 2015; Rankin 2015). In many cases, Chandler 2010), protein-based inheritance in yeast (Shorter TEI is limited to a small number of generations (e.g.,twoto and Lindquist 2005), double-stranded RNA (dsRNA)-mediated three) (D’Urso and Brickner 2014; Heard and Martienssen gene silencing [RNA interference (RNAi)] in Caenorhabditis 2014). In other cases, such as piwi-interacting RNAs (piRNA)- elegans (Vastenhouw et al. 2006; Ashe et al. 2012; Buckley mediated gene silencing in C. elegans and paramutation in plants, TEI can be long-lasting (. 10 generations). Molecular Copyright © 2018 by the Genetics Society of America mechanisms that set the generational duration of TEI are doi: https://doi.org/10.1534/genetics.118.301456 largely a mystery. Manuscript received August 2, 2018; accepted for publication October 23, 2018; Recently, small noncoding RNAs have emerged as impor- published Early Online November 2, 2018. Supplemental material available at Figshare: https://doi.org/10.25386/genetics. tant mediators of epigenetic inheritance in eukaryotes. For 7229294. example, in plants, the RNA-dependent RNA Polymerase 1Corresponding author: Department of Genetics, Harvard Medical School, 77 Ave. Louis Pasteur, New Research Bldg. 266, Boston, MA 02115. E-mail: kennedy@ (RdRP) mop1 produces small interfering (si)RNAs thought genetics.med.harvard.edu to mediate paramutation (Alleman et al. 2006). In the yeast Genetics, Vol. 210, 1287–1299 December 2018 1287 Schizosaccharomyces pombe, siRNAs help direct and maintain H3K27me3), as well as inhibition of RNAP II during the elon- stable, and in some cases heritable, heterochromatic states at gation step of transcription via an unknown mechanism pericentromeres (Volpe et al. 2002; Martienssen et al. 2005; (Guang et al. 2008, 2010; Burkhart et al. 2011; Buckley et al. Ragunathan et al. 2015). In Drosophila, maternally inherited 2012; Mao et al. 2015). While it is not yet clear why nuclear piRNAs direct heritable silencing of transposable elements RNAi is needed for RNAi inheritance, it is known that nuclear (Le Thomas et al. 2014). Finally, in mice, the effects of stress RNAi is needed for transgenerational siRNA expression, sug- and metabolic disease are reported to pass from parent to gesting that cotranscriptional gene silencing and cytoplasmic progeny, and microRNAs and short tRNA fragments have siRNA amplification systems may be connected in some way. been implicated in mediating this inheritance (Carone et al. If RdRP enzymes can amplify small RNA populations each 2010; Gapp et al. 2014; Sharma et al. 2016). Thus, small generation during RNAi inheritance, then why does RNAi regulatory RNAs are good candidates for being the informa- inheritance not last forever? There could be fundamental tional vectors of TEI in eukaryotes (termed RNA-directed biochemical limitations that prevent RNAi inheritance from TEI). Small noncoding RNAs are also linked to TEI in lasting forever, or C. elegans might possess systems that ac- C. elegans. For instance, piRNAs can trigger transgenerational tively prevent long-term inheritance. Recently,loss-of-function gene silencing via a process termed RNA-induced epigenetic mutations in met-2,whichisoneoftwoC. elegans H3K9 silencing (Ashe et al. 2012; Shirayama et al. 2012). Addition- methyltransferases responsible for depositing the majority ally, dsRNA-mediated gene silencing (RNAi) is heritable in of H3K9me2 found in C. elegans, were shown to cause RNA- C. elegans; the progeny of animals treated with dsRNAs retain directed TEI to last longer than normal (Andersen and Horvitz the ability to silence RNAi-targeted genes for many genera- 2007; Checchi and Engebrecht 2011; Towbin et al. 2012; tions, even after the removal of dsRNA triggers (termed RNAi Lev et al. 2017). The reasons why loss of MET-2 enhances inheritance) (Vastenhouw et al. 2006; Alcazar et al. 2008). RNAi inheritance are not known, but may involve global al- During RNAi inheritance in C. elegans, dsRNAs are processed terations in gene expression that indirectly promote RNAi by Dicer into “primary” siRNAs, which are bound by Argo- inheritance (Lev et al. 2017). To further our understanding naute (AGO) proteins to regulate complementary cellular of why RNAi inheritance does not last forever, we conducted RNAs (Meister 2013). AGO-bound primary siRNAs recruit, a forward genetic screen to identify additional factors that by an unknown mechanism, RdRPs to homologous mRNA limit the duration of RNAi inheritance. Our screen identified templates to produce amplified pools of “secondary siRNAs” the gene-heritable enhancer of RNAi 1 (heri-1). We find that (Sijen et al. 2001). Repeated RdRP-based siRNA amplifica- HERI-1 inhibits nuclear RNAi-based cotranscriptional gene tion in germ cells each generation is likely responsible for silencing and that this ability is likely the reason why loss driving RNAi inheritance in C. elegans (Ashe et al. 2012; of HERI-1 promotes RNAi inheritance. Interestingly, HERI-1 Buckley et al. 2012; Sapetschnig et al. 2015). is physically recruited to genes undergoing nuclear RNAi, Forward genetic screens have identified factors that are suggesting that HERI-1 may be a direct and dedicated regu- required for RNAi inheritance in C. elegans (Ashe et al. 2012; lator of nuclear RNAi, and therefore TEI. Surprisingly, the Buckley et al. 2012; Spracklin et al. 2017; Wan et al. 2018). recruitment of this negative regulator of nuclear RNAi to The factors fall into two general categories. The first category genes is itself dependent upon nuclear RNAi. To explain these includes factors that localize to cytoplasmic liquid-like con- results, we propose that nuclear RNAi has both pro- and densates such as the P granule, the Z granule, or Mutator foci. antisilencing functions, and that the generational perdurance These factors likely promote RNAi inheritance by acting of RNAi inheritance is set by the relative contributions of with RdRPs to amplify siRNA populations each generation these two opposing outputs. (Spracklin et al. 2017; Wan et al. 2018). The second category of factors are members of a nucleus-specific branch of the Materials and Methods RNAi pathway [the nuclear RNAi or NRDE (nuclear RNA Strains defective) pathway] (Ashe et al. 2012; Buckley et al. 2012; Shirayama et al. 2012; Spracklin et al. 2017; Wan et al. 2018). For a complete list of strains used in this study, see Supple- According to current models of nuclear RNAi, AGOs bind and mental Material, Table S1. All strains were maintained using escort siRNAs to nuclei, where these ribonucleoprotein com- standard laboratory conditions. plexes locate RNA Polymerase II (RNAP II)-dependent nascent heri screen transcripts based on complementarity to trigger cotranscrip- tional gene silencing (termed nuclear RNAi) (Guang et al. Larval stage 4 (L4) worms carrying both the oma-1(zu405) 2008, 2010; Buckley et al. 2012). HRDE-1 and NRDE-3 are allele and the pie-1::gfp::h2b transgene were mutagenized two tissue-specific nuclear AGOs that drive nuclear RNAi in with ethyl methanesulfonate (EMS) at a concentration of germ cells and somatic cells, respectively (Guang et al. 2008; 47 mM for 4 hr at room temperature.
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