RNA-mediated transgenerational inheritance in ciliates and plants
- 911 Downloads
In the age of next-generation sequencing (NGS) and with the availability of whole sequenced genomes and epigenomes, some attention has shifted from purely sequence-based studies to those of heritable epigenetic modifications. Transgenerational inheritance can be defined as heritable changes to the state of DNA that may be passed on to subsequent generations without alterations to the underlying DNA sequence. Although this phenomenon has been extensively studied in many systems, studies of transgenerational inheritance in mammals and other higher-level eukaryotes may be complicated by the fact that many epigenetic marks are reprogrammed during sexual reproduction. This, by definition, may obscure our interpretation of what is in fact truly transgenerational. Therefore, in this mini review, we discuss what is currently known in the field about transgenerational epigenetic inheritance in ciliates and plants, with a particular emphasis on RNA-mediated processes and changes in chromatin states.
KeywordsTransgenerational inheritance Non-Mendelian inheritance Small RNA Epigenetics Ciliates Plants
In recent years, there has been much focus on the subject of epigenetic inheritance and how heritable changes in chromatin states may be transmitted to subsequent generations. This includes changes in gene activity and gene expression levels, without altering the underlying DNA sequence, that may be passed on to generations to come. Transgenerational inheritance may involve DNA methylation or other chromatin-based mechanisms, but can also involve RNA-mediated DNA methylation and RNA-mediated DNA excision/elimination in some of the more extreme examples. Indeed, small non-coding RNAs have been implicated in many of these processes and likely mediate transgenerational inheritance across eukaryotic species, since they can induce changes in chromatin dynamics and guide histone modifications. While these phenomena have been described in mammals and extensive work has been performed to elucidate mechanisms, this review will focus primarily on RNA-mediated transgenerational inheritance in ciliated protozoans and plants.
Ciliates are large, unicellular protists that can be found ubiquitously across the globe in both marine and freshwater environments. Ciliates exhibit a special case of germline-soma specialization called nuclear dimorphism and are thus unique systems to study RNA-mediated transgenerational inheritance. Ciliates contain two completely separate caches of genetic information: the micronucleus (MIC), considered the germline nucleus, is transcriptionally silent and is used to propagate genetic information from one generation to the next and the macronucleus (MAC), considered the somatic nucleus, is used for vegetative growth of the cells (Prescott 1994). The micronuclear genome resembles that of a canonical eukaryotic genome, with many genes organized along long chromosomes. The micronuclear genome contains a large amount of “junk” DNA including transposable elements (TEs) and repetitive elements such as minisatellites, while micronuclear genes themselves are often interrupted by multiple short transposon-derived stretches of non-coding DNA called internally eliminated sequences (IESs) (Arnaiz et al. 2012; Chen et al. 2014; Guerin et al. 2017; Hamilton et al. 2016). The macronuclear genome, on the other hand, is devoid of all of this “junk” DNA and all the transcription necessary for vegetative growth occurs here (Aury et al. 2006; Duret et al. 2008; Eisen et al. 2006; Fang et al. 2012; Swart et al. 2013). During the ciliate sexual life cycle, the parental macronucleus provides genetic information in the form of transported sRNAs for the formation of a new macronucleus, which is derived from a newly formed, micronuclear precursor. During this micronucleus to macronucleus transition, the micronuclear genome is modified drastically through various processing events, including the polytenization of chromosomes and removal of repetitive DNA sequences, and IESs must be precisely removed to create functional macronuclear genes. It has been shown that small RNAs (sRNAs) are involved in the epigenetic transmission of information from parental nuclei to the developing macronucleus, leading to large-scale genomic rearrangements, altered chromatin states and ultimately the complete removal of specific DNA sequences.
In the stichotrich Oxytricha trifallax, approximately 20% of micronuclear genes exist in a non-linear, “scrambled” order that must be connected upon IES removal during macronuclear development (Chen et al. 2014). This means that in addition to targeting specific IES regions for elimination from the genome, the cells must also sort and reorder the remaining macronuclear destined sequences (MDSs) into functional genes. During Oxytricha conjugation, the parental macronucleus is broken down and degraded, while a new macronucleus, called the anlage, develops from one of the parental micronuclei. At this developmental stage, the anlage undergoes endoreplication, eliminates over 90% of its germline genome, breaks apart and fragments its chromosomes, and then ligates the thousands of remaining MDSs back together into functional genic reading frames (Adl and Berger 2000). A conjugation-specific class of 27 nt small RNAs called 27macRNAs has been identified and is highly upregulated during this process (peaking 24 h post-mixing of complementary mating types) (Fang et al. 2012; Zahler et al. 2012). These 27macRNAs are derived from the parental macronucleus, possess a strong 5′ U bias, and play a vital role during macronuclear development. The 27macRNAs associate with a PIWI protein called Otiwi1 and have been implicated in specifying which regions of the genome are protected from the DNA elimination occurring during this time (Fang et al. 2012). Microinjection of synthetic sRNAs corresponding to IES regions that are usually eliminated led to their retention in subsequent generations. Although little is known about the biogenesis of these RNAs or the exact mechanism by which DNA is protected, it has been suggested that this may occur through methylation of cytosine residues within IES regions (Bracht et al. 2012). In addition to the 27macRNAs necessary for DNA retention throughout macronuclear development, long maternal guide RNA templates transcribed from macronuclear nanochromosomes have also been shown to mediate genomic rearrangements (Nowacki et al. 2008). Long RNA transcripts (both sense and antisense), corresponding to entire macronuclear DNA molecules, can be detected for a brief period during conjugation and it is hypothesized that these act as templates for the correct unscrambling of MDSs. Microinjection of synthetic double-stranded nanochromosomes (DNA or RNA versions) with alternatively arranged MDSs led to defects in the proper reordering of MDSs in subsequent generations, indicating epigenetic inheritance through these RNAs (Nowacki et al. 2008). In a recent study, RNA-cached copies of over half of Oxytricha nanochromosomes have been identified during macronuclear development, supporting the model in which maternal guide RNA templates are transmitted to the progeny (Lindblad et al. 2017). Although it has been suggested that the long guide RNAs may act as precursors for the biogenesis of 27macRNAs, the relationship between these two classes of RNAs remains unknown. Interestingly, a striking number of the genes upregulated during Oxytricha macronuclear development encode well-conserved proteins with links to germline function and development in higher-level eukaryotes (Neeb et al. 2017).
To be considered truly transgenerational, these DNA methylation landscapes must be heritable and maintained in subsequent generations after their initial establishment. Maintenance of DNA methylation patterns through DNA replication requires the cooperation of several protein factors (Law and Jacobsen 2010). In Arabidopsis, DNA methylation is well maintained across TEs and genes and relies primarily on the de novo DNA methyltransferase (DMTase) MET1 for maintenance of CG methylation (Vongs et al. 1993). For maintenance of CHG methylation, an additional chromomethylase called CMT3 is necessary and specifically binds histone H3 lysine 9 dimethylation, while asymmetric CHH methylation maintenance relies on DRM2 and CMT2, only requiring CMT3 at specific loci (Cao et al. 2003; Du et al. 2015; Johnson et al. 2007; Lindroth et al. 2001; Lindroth et al. 2004; Stroud et al. 2014; Stroud et al. 2013). Evidence suggests that methylation patterns across TEs and repetitive sequence elements are transmitted from parent to offspring upon fertilization, although CHH methylation must be reestablished as the embryo develops (Hsieh et al. 2009; Jullien et al. 2012). CHH methylation is guided by maternally inherited 24 nt siRNAs that are present upon fertilization (Calarco et al. 2012; Lu et al. 2012; Mosher et al. 2009). Plants tend to undergo significantly less germline reprogramming of DNA methylation patterns than mammals, displaying an excellent example of transgenerational epigenetic inheritance (Heard and Martienssen 2014).
Both plants and ciliates exhibit a case of RNA-mediated epigenetic inheritance, utilizing classes of small RNAs, but the mechanisms by which they perform such a feat are quite different. While RNA polymerase IV and RNA polymerase V are involved in the transcription of sRNA precursors and the nascent transcript targets in plants, this process is much less clear in ciliates. Plants use an RNA-dependent RNA polymerase (RDRP) to transcribe the second strand of RNA before Dicer-like cleavage, but it is hypothesized that in ciliates, transcription occurs bidirectionally to form dsRNA substrates, although the polymerase responsible has yet to be identified. Interestingly, however, additional RNA polymerase II subunits have also been identified in ciliates that may play similar roles to plant Pols IV and V. For example, Oxytricha has additional largest and second largest Pol II subunit paralogs (RPB1b and RPB2b) that are highly upregulated during macronuclear development and likely play roles in the transcription of either small RNA precursors or the guide RNAs described in the process of gene unscrambling (Khurana et al. 2014; Neeb et al. 2017). These systems also use two distinct types of Argonaute family RNA-binding proteins to target regions of the genome for silencing or excision, using the AGOs or Piwis, respectively. AGOs are completely absent from ciliate genomes and Piwis have taken on the roles of these proteins. It remains unclear how Piwi-bound small RNAs “target” particular regions of the genome, but one can imagine the involvement of nascent transcripts within the developing macronucleus. Interestingly, plant AGO4 which binds siRNAs is most similar to Ptiwi10, known to bind iesRNAs in Paramecium during the second IES removal step of the “scanning model,” suggesting a possible similar mechanism (Furrer et al. 2017). Potentially, the most striking difference between these two systems is the fact that while both plants and ciliates silence particular regions of the genome using repressive histone modifications, plants merely form heterochromatin, while ciliates like Tetrahymena use repressive histone marks to excise and degrade large segments of the genome entirely, taking this process to the extreme. It remains to be elucidated whether ciliates and plants share additional commonalities in how they accomplish epigenetic inheritance and additional studies are needed to fill in the remaining gaps in our understanding of these models.
Ciliates and plants represent unique and fascinating systems to study RNA-mediated transgenerational epigenetic inheritance. Both genomes must protect against the invasion of transposable elements and other foreign DNA, and this comes in the form of silencing expression, and in some cases excision, of entire DNA sequences to carefully defend the germline and subsequent generations. With the power of next-generation sequencing (NGS) of entire genomes and epigenomes, along with reverse genetic approaches, it will be possible to investigate the roles of epigenetic inheritance in other biological processes and contexts. Although transgenerational inheritance is clearly demonstrated and well described in ciliates and plants, further work is needed to investigate the implications in mammalian systems and how widespread this process is among other eukaryotes.
Compliance with ethical standards
This research does not include human participants or animals.
Conflict of interest
The authors declare that they have no conflict of interest.
- Adl SM, Berger JD (2000) Timing of life cycle morphogenesis in synchronous samples of Sterkiella histriomuscorum. II. The sexual pathway. J Eukaryot Microbiol 47(5):443–449. https://doi.org/10.1111/j.1550-7408.2000.tb00073.x CrossRefPubMedGoogle Scholar
- Arnaiz O, Mathy N, Baudry C, Malinsky S, Aury JM, Denby Wilkes C, Garnier O, Labadie K, Lauderdale BE, Le Mouel A et al (2012) The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences. PLoS Genet 8(10):e1002984. https://doi.org/10.1371/journal.pgen.1002984 CrossRefPubMedPubMedCentralGoogle Scholar
- Aronica L, Bednenko J, Noto T, DeSouza LV, Siu KW, Loidl J, Pearlman RE, Gorovsky MA, Mochizuki K (2008) Study of an RNA helicase implicates small RNA-noncoding RNA interactions in programmed DNA elimination in Tetrahymena. Genes Dev 22(16):2228–2241. https://doi.org/10.1101/gad.481908 CrossRefPubMedPubMedCentralGoogle Scholar
- Baudry C, Malinsky S, Restituito M, Kapusta A, Rosa S, Meyer E, Betermier M (2009) PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia. Genes Dev 23(21):2478–2483. https://doi.org/10.1101/gad.547309 CrossRefPubMedPubMedCentralGoogle Scholar
- Bouhouche K, Gout JF, Kapusta A, Betermier M, Meyer E (2011) Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling. Nucleic Acids Res 39(10):4249–4264. https://doi.org/10.1093/nar/gkq1283 CrossRefPubMedPubMedCentralGoogle Scholar
- Calarco JP, Borges F, Donoghue MT, Van Ex F, Jullien PE, Lopes T, Gardner R, Berger F, Feijo JA, Becker JD et al (2012) Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA. Cell 151(1):194–205. https://doi.org/10.1016/j.cell.2012.09.001 CrossRefPubMedPubMedCentralGoogle Scholar
- Chen X, Bracht JR, Goldman AD, Dolzhenko E, Clay DM, Swart EC, Perlman DH, Doak TG, Stuart A, Amemiya CT, Sebra RP, Landweber LF (2014) The architecture of a scrambled genome reveals massive levels of genomic rearrangement during development. Cell 158(5):1187–1198. https://doi.org/10.1016/j.cell.2014.07.034 CrossRefPubMedPubMedCentralGoogle Scholar
- Cheng CY, Vogt A, Mochizuki K, Yao MC (2010) A domesticated piggyBac transposase plays key roles in heterochromatin dynamics and DNA cleavage during programmed DNA deletion in Tetrahymena thermophila. Mol Biol Cell 21(10):1753–1762. https://doi.org/10.1091/mbc.E09-12-1079 CrossRefPubMedPubMedCentralGoogle Scholar
- Duret L, Cohen J, Jubin C, Dessen P, Gout JF, Mousset S, Aury JM, Jaillon O, Noel B, Arnaiz O, Betermier M, Wincker P, Meyer E, Sperling L (2008) Analysis of sequence variability in the macronuclear DNA of Paramecium tetraurelia: a somatic view of the germline. Genome Res 18(4):585–596. https://doi.org/10.1101/gr.074534.107 CrossRefPubMedPubMedCentralGoogle Scholar
- Eisen JA, Coyne RS, Wu M, Wu D, Thiagarajan M, Wortman JR, Badger JH, Ren Q, Amedeo P, Jones KM, Tallon LJ, Delcher AL, Salzberg SL, Silva JC, Haas BJ, Majoros WH, Farzad M, Carlton JM, Smith RK, Garg J, Pearlman RE, Karrer KM, Sun L, Manning G, Elde NC, Turkewitz AP, Asai DJ, Wilkes DE, Wang Y, Cai H, Collins K, Stewart BA, Lee SR, Wilamowska K, Weinberg Z, Ruzzo WL, Wloga D, Gaertig J, Frankel J, Tsao CC, Gorovsky MA, Keeling PJ, Waller RF, Patron NJ, Cherry JM, Stover NA, Krieger CJ, del Toro C, Ryder HF, Williamson SC, Barbeau RA, Hamilton EP, Orias E (2006) Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. PLoS Biol 4(9):e286. https://doi.org/10.1371/journal.pbio.0040286 CrossRefPubMedPubMedCentralGoogle Scholar
- Fass JN, Joshi NA, Couvillion MT, Bowen J, Gorovsky MA, Hamilton EP, Orias E, Hong K, Coyne RS, Eisen JA, Chalker DL, Lin D, Collins K (2011) Genome-scale analysis of programmed DNA elimination sites in Tetrahymena thermophila. G3 (Bethesda) 1(6):515–522. https://doi.org/10.1534/g3.111.000927 CrossRefPubMedCentralGoogle Scholar
- Guerin F, Arnaiz O, Boggetto N, Denby Wilkes C, Meyer E, Sperling L, Duharcourt S (2017) Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements. BMC Genomics 18(1):327. https://doi.org/10.1186/s12864-017-3713-7 CrossRefPubMedPubMedCentralGoogle Scholar
- Hamilton, E.P., Kapusta, A., Huvos, P.E., Bidwell, S.L., Zafar, N., Tang, H., Hadjithomas, M., Krishnakumar, V., Badger, J.H., Caler, E.V., et al. (2016). Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome. Elife 5 Google Scholar
- Havecker ER, Wallbridge LM, Hardcastle TJ, Bush MS, Kelly KA, Dunn RM, Schwach F, Doonan JH, Baulcombe DC (2010) The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22(2):321–334. https://doi.org/10.1105/tpc.109.072199 CrossRefPubMedPubMedCentralGoogle Scholar
- Kanno T, Bucher E, Daxinger L, Huettel B, Kreil DP, Breinig F, Lind M, Schmitt MJ, Simon SA, Gurazada SG et al (2010) RNA-directed DNA methylation and plant development require an IWR1-type transcription factor. EMBO Rep 11(1):65–71. https://doi.org/10.1038/embor.2009.246 CrossRefPubMedGoogle Scholar
- Kapusta A, Matsuda A, Marmignon A, Ku M, Silve A, Meyer E, Forney JD, Malinsky S, Betermier M (2011) Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining. PLoS Genet 7(4):e1002049. https://doi.org/10.1371/journal.pgen.1002049 CrossRefPubMedPubMedCentralGoogle Scholar
- Khurana JS, Wang X, Chen X, Perlman DH, Landweber LF (2014) Transcription-independent functions of an RNA polymerase II subunit, Rpb2, during genome rearrangement in the ciliate, Oxytricha trifallax. Genetics 197(3):839–849. https://doi.org/10.1534/genetics.114.163279 CrossRefPubMedPubMedCentralGoogle Scholar
- Lhuillier-Akakpo M, Frapporti A, Denby Wilkes C, Matelot M, Vervoort M, Sperling L, Duharcourt S (2014) Local effect of enhancer of zeste-like reveals cooperation of epigenetic and cis-acting determinants for zygotic genome rearrangements. PLoS Genet 10(9):e1004665. https://doi.org/10.1371/journal.pgen.1004665 CrossRefPubMedPubMedCentralGoogle Scholar
- Li CF, Pontes O, El-Shami M, Henderson IR, Bernatavichute YV, Chan SW, Lagrange T, Pikaard CS, Jacobsen SE (2006) An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126(1):93–106. https://doi.org/10.1016/j.cell.2006.05.032 CrossRefPubMedGoogle Scholar
- Lindblad, K.A., Bracht, J.R., Williams, A.E., and Landweber, L. (2017). Thousands of RNA-cached copies of whole chromosomes are present in the ciliate Oxytricha during development. RNAGoogle Scholar
- Lindroth AM, Shultis D, Jasencakova Z, Fuchs J, Johnson L, Schubert D, Patnaik D, Pradhan S, Goodrich J, Schubert I, Jenuwein T, Khorasanizadeh S, Jacobsen SE (2004) Dual histone H3 methylation marks at lysines 9 and 27 required for interaction with CHROMOMETHYLASE3. EMBO J 23(21):4286–4296. https://doi.org/10.1038/sj.emboj.7600430 CrossRefPubMedGoogle Scholar
- Malone CD, Anderson AM, Motl JA, Rexer CH, Chalker DL (2005) Germ line transcripts are processed by a Dicer-like protein that is essential for developmentally programmed genome rearrangements of Tetrahymena thermophila. Mol Cell Biol 25(20):9151–9164. https://doi.org/10.1128/MCB.25.20.9151-9164.2005 CrossRefPubMedPubMedCentralGoogle Scholar
- Mochizuki K, Gorovsky MA (2004b) RNA polymerase II localizes in Tetrahymena thermophila meiotic micronuclei when micronuclear transcription associated with genome rearrangement occurs. Eukaryot Cell 3(5):1233–1240. https://doi.org/10.1128/EC.3.5.1233-1240.2004 CrossRefPubMedPubMedCentralGoogle Scholar
- Neeb ZT, Hogan DJ, Katzman S, Zahler AM (2017) Preferential expression of scores of functionally and evolutionarily diverse DNA and RNA-binding proteins during Oxytricha trifallax macronuclear development. PLoS One 12(2):e0170870. https://doi.org/10.1371/journal.pone.0170870 CrossRefPubMedPubMedCentralGoogle Scholar
- Pontier D, Yahubyan G, Vega D, Bulski A, Saez-Vasquez J, Hakimi MA, Lerbs-Mache S, Colot V, Lagrange T (2005) Reinforcement of silencing at transposons and highly repeated sequences requires the concerted action of two distinct RNA polymerases IV in Arabidopsis. Genes Dev 19(17):2030–2040. https://doi.org/10.1101/gad.348405 CrossRefPubMedPubMedCentralGoogle Scholar
- Quadrana L, Colot V (2016) Plant transgenerational epigenetics. Annu Rev Genet 50(1):467–491. https://doi.org/10.1146/annurev-genet-120215-035254 CrossRefPubMedGoogle Scholar
- Swart EC, Bracht JR, Magrini V, Minx P, Chen X, Zhou Y, Khurana JS, Goldman AD, Nowacki M, Schotanus K, Jung S, Fulton RS, Ly A, McGrath S, Haub K, Wiggins JL, Storton D, Matese JC, Parsons L, Chang WJ, Bowen MS, Stover NA, Jones TA, Eddy SR, Herrick GA, Doak TG, Wilson RK, Mardis ER, Landweber LF (2013) The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes. PLoS Biol 11(1):e1001473. https://doi.org/10.1371/journal.pbio.1001473 CrossRefPubMedPubMedCentralGoogle Scholar
- Tucker SL, Reece J, Ream TS, Pikaard CS (2010) Evolutionary history of plant multisubunit RNA polymerases IV and V: subunit origins via genome-wide and segmental gene duplications, retrotransposition, and lineage-specific subfunctionalization. Cold Spring Harb Symp Quant Biol 75(0):285–297. https://doi.org/10.1101/sqb.2010.75.037 CrossRefPubMedGoogle Scholar
- Yao MC, Chao JL (2005) RNA-guided DNA deletion in Tetrahymena: an RNAi-based mechanism for programmed genome rearrangements. Annu Rev Genet 39(1):537–559. https://doi.org/10.1146/annurev.genet.39.073003.095906 CrossRefPubMedGoogle Scholar
- Zhong X, Du J, Hale CJ, Gallego-Bartolome J, Feng S, Vashisht AA, Chory J, Wohlschlegel JA, Patel DJ, Jacobsen SE (2014) Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell 157(5):1050–1060. https://doi.org/10.1016/j.cell.2014.03.056 CrossRefPubMedPubMedCentralGoogle Scholar
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.