Plant Cell Advance Publication. Published on February 13, 2017, doi:10.1105/tpc.16.00718
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RESEARCH ARTICLE
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INTRODUCTION
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Exogenous Transposable Elements Circumvent Identity-Based Silencing
Permitting the Dissection of Expression-Dependent Silencing
Dalen Fultz1,2 and R. Keith Slotkin1,3,4
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Department of Molecular Genetics, The Ohio State University
Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University
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Center for RNA Biology, The Ohio State University
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Corresponding Author: [email protected]
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Short Title: Silencing Exogenous Transposable Elements
One-sentence summary: Transformation of endogenous and exogenous transposable elements into
Arabidopsis distinguishes expression-dependent and identity-based mechanisms responsible for the
establishment, progression, and heritability of silencing.
The author responsible for distribution of materials integral to the findings presented in this article in
accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: R. Keith
Slotkin ([email protected]).
ABSTRACT
The propagation of epigenetic marks has received a great deal of attention, yet the initiation of epigenetic
silencing of a new transgene, virus, or transposable element (TE) remains enigmatic. The overlapping
and simultaneous function of multiple silencing mechanisms has obscured this area of investigation. Here,
we have revealed two broad mechanisms that can initiate silencing independently: identity-based and
expression-dependent silencing. We found that identity-based silencing is targeted by 21-22 or 24
nucleotide small interfering RNAs (siRNAs) generated from previously silenced regions of the genome.
By transforming exogenous TEs into Arabidopsis thaliana, we circumvented identity-based silencing
allowing us to isolate and investigate the molecular mechanism of expression-dependent silencing. We
found that several siRNA-generating mechanisms all trigger de novo expression-dependent RNAdirected DNA methylation (RdDM) through RNA Polymerase V. In addition, while full-length TEs quickly
progress beyond RdDM to heterochromatin formation and the final maintenance methylation state, TE
fragments stall at the RdDM phase. Lastly, we found that transformation into a mutant genotype followed
by introgression into wild type does not result in the same level of silencing as direct transformation into
wild type. This demonstrates that the plant genotype during a narrow window of time during TE insertion
(or transgene transformation) is key for establishing the transgenerational extent of epigenetic silencing.
Transgenes, viruses, and transposable elements (TEs) undergo three distinct phases of
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silencing in plants: initiation, establishment, and maintenance (Bond and Baulcombe, 2015).
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These pathways are likely linked in a linear progression leading to long-term epigenetic silencing
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(Panda and Slotkin, 2013); however, how each phase is activated is unclear. The maintenance
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phase responsible for epigenetic inheritance has been extensively investigated in plants and
©2017 American Society of Plant Biologists. All Rights Reserved
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shown to rely on the replication of symmetrical DNA methylation patterns across cell divisions
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(Kato et al., 2003; Feng et al., 2010). In addition, DNA methylation and histone 3 lysine 9
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dimethylation (H3K9me2) reinforce each other to generate a transcriptionally silent
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heterochromatic state (Du et al., 2012; Stroud et al., 2014). Although the mechanism(s) of
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silencing initiation and establishment have been investigated on regions of the genome such as
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endogenous TEs (Nuthikattu et al., 2013), pre-existing DNA methylation, and H3K9me2 directed
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by maintenance mechanisms confound this analysis. Therefore, the mechanism of de novo
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triggering (initiation) and placement (establishment) of chromatin modifications has remained
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enigmatic.
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Establishment of repressive chromatin modifications (including promoter DNA methylation
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and H3K9me2) is known to occur through several distinct small RNA-directed DNA methylation
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(RdDM) pathways in Arabidopsis (reviewed in Cuerda-Gil and Slotkin, 2016). A hallmark of
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RdDM is the equal targeting of all cytosine contexts (CG, CHG, CHH, where H=A, T, or C),
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whereas maintenance methylation occurs more efficiently at the symmetric CG context than
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asymmetric CHH sites (Lister et al., 2008; Zemach et al., 2013). RdDM can be subdivided into
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several distinct pathways. The canonical ‘Pol IV-RdDM’ pathway acts through the transcription
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of regions of the genome already associated with H3K9me2 by RNA Polymerase IV (Pol IV)
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(Law et al., 2013). Pol IV transcription, coupled with the activity of its binding partner, RDR2,
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produces double-stranded RNA substrates that are degraded by the Dicer protein DCL3 into 24
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nucleotide (nt) small interfering RNAs (siRNAs)(reviewed in Matzke and Mosher, 2014). These
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siRNAs are incorporated into either ARGONAUTE 4 (AGO4) or AGO6 and provide targeting
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specificity to downstream components of the RdDM pathway. Pol V generates nascent RNAs
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from methylated DNA regions to act as scaffolding transcripts, tethering the siRNA-bound AGO4
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or AGO6 to this region of chromatin by an RNA:RNA interaction (Wierzbicki et al., 2009), and
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triggering DNA methylation through DRM1/2 (Cao et al., 2003). Pol IV-RdDM occurs
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independently of Pol II expression and has been theorized to function as an identity sensor,
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using the 24 nt siRNAs produced from silenced TEs to target similar sequences across the
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genome for RdDM (Fultz et al., 2015).
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The field of RdDM has been recently transformed by the identification of non-canonical
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(non-Pol IV) mechanisms that are dependent on Pol II (reviewed in Cuerda-Gil and Slotkin,
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2016). For example, RDR6-RdDM functions on transcriptionally active TEs through the
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production of 21-22 nt siRNAs directly generated from the degradation of TE mRNAs (Nuthikattu
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et al., 2013; Panda et al., 2016). These 21-22 nt siRNAs guide RdDM through their incorporation
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into AGO6 (McCue et al., 2015). In addition to TEs, the non-protein coding single copy TAS loci
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are targets of this pathway in wild-type (wt) TE-silenced plants (Wu et al., 2012; Panda et al.,
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2016). Pol II mRNAs can also be directly degraded by DCL3 into 24 nt siRNAs, which target
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RdDM in a pathway termed DCL3-RdDM (Panda et al., 2016). Additionally, Dicer-independent
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small RNAs have been shown to function in RdDM (Ye et al., 2016; Yang et al., 2015). These
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three ‘non-canonical’ RdDM pathways have only limited roles in wt plants that lack TE activity,
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but play a considerable role in silencing transcriptionally reactivated TEs (Panda et al., 2016).
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Consequently, each of these non-canonical RdDM pathways has been hypothesized to function
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in the original establishment of TE silencing.
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To focus solely on the initiation and establishment phases of silencing, and separate
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these mechanisms from the maintenance of epigenetic marks, investigation has concentrated on
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transformation or infection of epigenetically naïve (chromatin modification-free) DNA into plants
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(Greenberg et al., 2011; Bond and Baulcombe, 2015; Jackel et al., 2016). Although transgenesis
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might be considered an artificial approach to investigate silencing, evidence suggests that
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transgenesis can occur naturally (Kyndt et al., 2015). In addition, TEs undergo widespread
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horizontal transfer into new genomes (Bartolomé et al., 2009; Walsh et al., 2013) akin to
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transgenesis. For example, the P element TE was horizontally transferred from Drosophila
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willistoni to D. melanogaster in the early 20th century after >40 million years of divergence
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between the two species (Daniels et al., 1990). Plant genomes display evidence of widespread
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and frequent horizontal transfer of TEs (El Baidouri et al., 2014). TE-transgenes have been
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previously generated as tools for gene tagging and mutant generation (Raizada et al., 2001; Li
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et al., 2013). TEs transgenes can be selected for activity, yet without selection for activity
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evidence suggests that they are recognized by host-defense mechanisms and silenced by
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RdDM (Hirochika et al., 2000; Pérez-Hormaeche et al., 2008). Like TEs, silencing of newly
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integrated transgenes can occur through homology to the existing genome (Matzke et al., 2000,
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1994), yet the detailed mechanism(s) of silencing establishment at the newly integrated TEs or
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transgenes is currently unknown.
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To dissect the mechanisms of establishment of TE silencing and model the horizontal
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transfer of a TE, we used floral dip transformation to stably integrate epigenetically naïve TEs
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into wt and mutant Arabidopsis genomes. We found that a naïve endogenous TE is subject to
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two distinct de novo silencing mechanisms: identity-based and expression-dependent silencing.
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We found that identity-based silencing, which can function independent of mRNA production, is
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targeted through 21-22 or 24 nt siRNAs produced from previously transcriptionally or post-
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transcriptionally silenced regions of the genome. We were able to circumvent identity-based
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silencing by introducing exogenous TEs into the Arabidopsis genome. Our system has therefore
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uniquely permitted the molecular characterization of the de novo establishment of TE chromatin
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modification by expression-dependent silencing without the obstruction of the identity-based
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pathway.
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RESULTS
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Endogenous TEs are the target of distinct identity-based and expression-dependent silencing
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mechanisms
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To investigate how epigenetically naïve TEs undergo de novo establishment of DNA
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methylation, we created a TE-transgene and inserted it into the Arabidopsis genome. The
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transgene contains a 395 base pair (bp) fragment of the Arabidopsis Athila6 long terminal repeat
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(LTR), which acts as the transcriptional control region for this TE family (Nuthikattu et al., 2013).
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Endogenous Arabidopsis Athila family TEs are transcriptionally silenced gypsy retrotransposons
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that produce abundant Pol IV-dependent 24 nt siRNAs (Nuthikattu et al., 2013). We placed an
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estradiol-inducible promoter upstream of the transgene LTR fragment (Figure 1A), providing the
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ability to control Pol II expression of the entire LTR (Figure 1B)(inducible-Athila LTR transgene =
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iAthila LTR). Upon induction of wt iAthila T1 plants, LTR biological replicate average mRNA
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levels increase 225-fold compared to the mock-induced control biological replicates (Figure 1B).
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Since the iAthila LTR includes the native Athila6 transcriptional start site (TSS)(Figure 1A), low
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but detectible levels of transgene mRNA accumulate from the native Athila TSS in wt Col without
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estradiol induction (Figure 1C), but not from upstream of the native TSS (Figure 1B). This
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demonstrates that the Athila LTR promoter within this transgene is both functional and weakly
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active. To assay silencing, we determined the DNA methylation level of this TE-transgene via
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bisulfite sequencing and found an intermediate level of DNA methylation on the iAthila LTR
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transgene in wt Col when it was not induced. This methylation increased in all sequence
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contexts when expression was induced in wt (Figure 1D). In the ago6 mutant, steady-state
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mRNA levels were higher from both the native Athila TSS and the induced promoter TSS
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(Figure 1B-C). However, the expression-dependent increase in DNA methylation found in wt Col
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did not occur in ago6 mutants (Figure 1D), recapitulating previous results demonstrating AGO6’s
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role in RdDM of expressed TEs (McCue et al., 2015). This analysis demonstrates that TE-
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transgenes are targeted by an expression-dependent RdDM mechanism in a similar fashion as
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endogenous TEs (Nuthikattu et al., 2013; McCue et al., 2015) and viruses (Herr et al., 2005).
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However, this transgene cannot be used to distinguish expression-dependent from expression-
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independent silencing mechanisms due to its identity to silenced endogenous TEs and the
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constant activity of its native Athila promoter (Figure 1C).
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To separate the expression-dependent and independent mechanisms functioning on the
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iAthila LTR transgene, we created an Athila LTR transgene without the inducible promoter
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(Figure 1A). This transgene was methylated (Figure 1E) with the sequence hallmarks of RdDM
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(CHH methylation greater than or equal to CG methylation, see Supplemental Figure 1), and
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matches abundant 24 nt siRNAs generated from silenced endogenous Athila TEs (Figure 1G).
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As a control, we cloned the proximal promoter of the constitutively expressed gene AXS2 into
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the same transgene vector (Figure 1A). When transformed into wt, this region does not
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accumulate DNA methylation (Figure 1E), demonstrating that we are not investigating general
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transgene silencing, but rather the methylation of the Athila LTR transgene is specific to its
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identification as a TE. To assay the role of expression, we created an Athila LTR transgene with
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a 138 bp deletion of the native Athila TSS and upstream TATA box (Athila LTR Δpro, Figure 1A).
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This eliminated expression from the Athila LTR region of the transgene (Figure 1F), yet this
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deletion does not remove the targeting potential of 24 nt siRNAs produced from endogenous
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Athila TEs (Figure 1G). The level of DNA methylation was significantly reduced (t-test, p<0.05)
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but not absent, with 10.3% total cytosine methylation still targeted to the Athila LTR Δpro (Figure
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1E, Supplemental Figure 2A). Thus, for a newly introduced TE integration event that is identical
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to endogenous TEs, expression-dependent RdDM does not work alone, and there must an
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expression-independent identity-based pathway that targets DNA methylation even when the
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transgene lacks a core promoter and expression.
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Mechanism of identity-based silencing
When transformation into Arabidopsis, a naïve FWA transgene with a TE in its promoter
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is de novo silenced by the activity of the Pol IV-RdDM pathway (Chan et al., 2004; Greenberg et
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al., 2011; Kinoshita et al., 2007; Cao and Jacobsen, 2002). To determine and differentiate how
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the Pol IV-RdDM pathway functions in the silencing of TE transgenes, we transformed the Athila
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LTR and Athila LTR Δpro transgenes directly into the pol IV (nrpd1a) mutant background. Plants
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with mutated pol IV produce very few siRNAs that match the Athila LTR (Figure 1G), yet the
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integrated Athila LTR transgene is targeted for DNA methylation with the hallmarks of RdDM
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(CHH≥CG)(Figure 1E), confirming that Pol IV-RdDM is not solely responsible for the
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establishment of DNA methylation on an expressed TE (reviewed in Cuerda-Gil and Slotkin,
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2016). In contrast, the unexpressed Athila LTR Δpro transgene does not accumulate abundant
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DNA methylation (2.1% CHH)(Figure 1E), demonstrating that expression is required when Pol
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IV-RdDM fails. Without either expression (the Δpro transgene) or sequence-complementary
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siRNAs provided by the endogenous silenced TEs (in the pol IV mutant), the TE transgene is not
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targeted for abundant DNA methylation. Therefore, the Pol IV-RdDM pathway is sufficient to
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target an expressed or unexpressed naïve TE or transgene based on sequence identity via the
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abundant Pol IV-dependent 24 nt siRNAs that Athila and other endogenous silenced TEs
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produce. We refer to this expression-independent pathway as ‘identity-based’ silencing.
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We next aimed to determine whether 21-22 nt siRNAs could function in identity-based
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silencing in a similar fashion to Pol IV-dependent 24 nt siRNAs. The endogenous TRANS-
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ACTING SIRNA 3 A (TAS3A) locus is transcribed by Pol II into an mRNA that is targeted for
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post-transcriptional degradation triggered by microRNA cleavage and subsequent RDR6-
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dependent 21-22 nt tasiRNA production (Allen et al., 2005). Since TAS3 is an endogenous
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target of RDR6-RdDM (Wu et al., 2012; McCue et al., 2015)(Supplemental Figure 1C-D), we
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sought to determine whether the 21-22 nt siRNAs generated from this region could target RDR6-
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RdDM in trans to a separate naïve region of the genome and function in expression-independent
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identity-based silencing. We created a promoterless transgene of the TAS3A region that is
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internal to the two microRNA target sites which initiate tasiRNA production (Figure 1A)(Axtell et
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al., 2006). We found that our TAS3 transgene is not expressed (Figure 1H) but is targeted for
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DNA methylation (Figure 1E). This methylation pattern shows the sequence hallmarks of RdDM
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(CHH≥CG), demonstrating that the 21-22 nt tasiRNAs can function in trans on a newly
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introduced transgene that was not expressed. We conclude that identity-based RdDM can
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function in trans through 21-22 nt siRNAs in addition to 24 nt siRNAs. Therefore, as long as
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siRNAs are produced, transcriptionally active and silenced regions of the endogenous genome
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can both trigger identity-based silencing of a newly integrated TE/transgene.
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Exogenous TEs circumvent identity-based RdDM and are targeted solely by expression-
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dependent mechanisms
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To separate the activities of identity-based RdDM from expression-dependent RdDM, we
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used the exogenous Tto1 copia LTR retrotransposon from tobacco (Nicotiana tabacum –
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separated from Arabidopsis by ~118 million years). When a full-length copy of this TE was
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transferred into the Arabidopsis genome, the element was active and fully capable of
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transposition (Hirochika et al., 2000; Böhmdorfer et al., 2010). However, this transposition was
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only observed after selection in tissue culture (where TE silencing is compromised (Tanurdzic et
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al., 2008)), in a chromatin mutant, or when an inducible promoter is used to force TE expression,
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demonstrating that there is repression or silencing of this TE in wt Arabidopsis. To determine
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whether this silencing could be identity-based, we matched Tto1 to the Arabidopsis genome via
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BLAST and aligned Arabidopsis small RNA deep sequencing libraries to Tto1 via bowtie (see
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Methods and Supplemental Figure 3). We determined that no portion of the Tto1 sequence is
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identical to the Arabidopsis genome, and that there is only a slightly higher magnitude of
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similarity between Tto1 and the Arabidopsis genome compared to control alignments of
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scrambled DNA (Supplemental Figure 3). The most similar region between Tto and the
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Arabidopsis genome is 846 nt in length but only 64.7% similar, while the level of sRNA similarity
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between Tto1 and the Arabidopsis transcriptome is the same as matching to a scrambled
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sequence of the same length (Supplemental Figure 3). Therefore, our analysis demonstrates
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that Arabidopsis has only limited DNA sequence and sRNA identity to Tto1.
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We generated a Tto1 transgene representing the full 574 bp LTR, which includes the Tto1
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promoter and TSS (Hirochika, 1993; Takeda et al., 1999)(Figure 2A). When transformed into wt
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Col, this ‘solo Tto LTR’ transgene produces mRNA (Figure 2B) and is targeted for DNA
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methylation with the sequence context hallmarks of RdDM (CHH≥CG) over multiple generations
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(Figure 2C-D). To ensure that the solo Tto LTR transgene is targeted for RdDM because of its
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identification as a TE, and that we are not investigating general transgene silencing, we
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performed three controls. First, we demonstrated that transformation of an endogenous
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Arabidopsis genic promoter does not result in DNA methylation (Figure 1E). Second, to ensure
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that the solo Tto LTR is not targeted for RdDM due to its detection by RNA quality control
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mechanisms, we added an efficient terminator sequence (creating the transgene solo Tto
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LTR+term, Figure 2A). We found that the lack of a proper termination sequence in the solo Tto
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LTR transgene is not responsible for triggering the RdDM of this transgene (Figure 2C). Third,
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we investigated the NOS bacterial promoter driving the herbicide resistance gene on the same
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transgene as the solo Tto LTR and found no evidence of DNA methylation (Figure 2C),
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demonstrating that not all foreign transgene or non-Arabidopsis sequences are targeted for
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RdDM.
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To determine if RdDM of the solo Tto LTR transgene is expression-dependent, we
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created a transgene with a 142 bp deletion within the solo Tto LTR (solo Tto LTR Δpro)(Figure
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2A), which disables mRNA production (Figure 2B). In contrast to the solo Tto LTR, the solo Tto
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LTR Δpro transgene is not targeted for DNA methylation (Figure 2C, Supplemental Figure 2B).
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This result is in contrast to the Athila LTR Δpro transgene, which is targeted for DNA methylation
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via identity-based RdDM even when it is not expressed (Figure 1E, Supplemental Figure 2A).
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Thus, by using an exogenous TE without preexisting identical copies in the wt Arabidopsis
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genome, the solo Tto LTR transgene has circumvented identity-based RdDM, resulting in
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regulation solely by expression-dependent RdDM.
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To demonstrate that identity-based RdDM can target Tto, we tested whether a previously
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introduced and silenced Tto element in the Arabidopsis genome could trigger methylation of a
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new Tto integration event. We stably transformed wt Col with an inducible full-length form of Tto
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(Böhmdorfer et al., 2010), which is targeted for silencing (iTto, see below, Figures 3,4). We then
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re-transformed these iTto lines with the solo Tto LTR Δpro transgene from Figure 2A and found
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that this newly introduced TE is now targeted for identity-based (expression-independent) RdDM
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(Figure 2E). Therefore, the presence of a preexisting Tto copy in the Arabidopsis genome is
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sufficient to target this new TE integration event for RdDM, demonstrating that Tto can be
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subject to identity-based silencing. This correlates with the presence of abundant 24 nt siRNAs
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from the iTto background that match the Tto LTR, which are absent in the wt Col background
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without Tto1 (Figure 2F). The function of iTto 24 nt siRNAs in identity-based silencing strongly
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resembles the abundant 24 nt siRNAs that match the Athila LTR in wt Col (Figure 1G),
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demonstrating that the preexistence of matching siRNAs enables identity-based methylation of
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unexpressed TEs.
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Identity-based and expression-dependent RdDM can occur simultaneously on the same
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target, producing additive methylation levels. This is observed on both the iAthila LTR and Athila
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LTR transgenes that are targeted for identity-based silencing and display increased methylation
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when they are expressed (Figure 1D-E). When the solo Tto LTR was transformed into the iTto
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line (Figure 2E), increased CHG and CHH methylation occurs compared to either the solo Tto
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LTR Δpro transgene transformed into iTto (identity-based silencing only)(Figure 2E), or the solo
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Tto LTR transgene transformed into wt Col (expression-dependent silencing only)(Figure 2C).
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This is in line with the observation that transcriptionally active endogenous TEs are the strongest
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targets of RdDM (Panda et al., 2016), since both identity-based and expression-dependent
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mechanisms simultaneously target them.
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Multiple siRNA generating mechanisms redundantly target expression-dependent RdDM
To identify the mechanism(s) responsible for targeting expression-dependent RdDM
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without the confounding presence of identity-based RdDM, we directly transformed the solo Tto
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LTR transgene into a number of mutant Arabidopsis lines. When transformed into wt Col, the
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solo Tto LTR transgene produces a low level of mRNA as well as 21, 22, and 24 nt siRNAs
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(Figure 3A-B, Supplemental Figure 4). We found that multiple distinct mechanisms act to
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generate these siRNAs. We investigated the pol IV mutant to determine the role of Pol IV-RdDM.
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Pol IV-RdDM directs some, but not all, of the solo Tto LTR RdDM (Figure 3C). This incomplete
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dependence on Pol IV-RdDM correlated with the incomplete loss of 24 nt siRNAs in the pol IV
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mutant (Figure 3B). However, all 24 nt siRNAs were dependent on the activity of DCL3 (Figure
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3B). DCL3 is able to cleave Pol II transcripts into 24nt siRNAs, and this mechanism (termed
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DCL3-RdDM) has recently been linked to the RdDM of transcriptionally active TEs (Panda et al.,
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2016). We conclude that Tto LTR 24 nt siRNAs are generated from both Pol II and Pol IV
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transcripts processed by DCL3. Both of these mechanisms (Pol IV-RdDM and DCL3-RdDM)
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likely contribute to solo Tto LTR RdDM. Complicating this analysis, RdDM of the solo Tto LTR
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persists in dcl3 mutants (Figure 3C). The RdDM in dcl3 mutants is likely targeted by RDR6-
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RdDM, as only 21-22 nt siRNAs are present in the dcl3 mutant to perform RdDM (Figure 3B).
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When these 21-22 nt siRNAs are removed in the dcl2/3/4 triple mutant (Figure 3B), RdDM of the
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solo Tto LTR does not occur (Figure 3C). We only detected RDR6-RdDM in the dcl3 mutant and
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not in wt Col (Figure 3C), suggesting that for this transgene, RDR6-RdDM acts as a secondary
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pathway which functions only when other pathways are disabled. RDR6 does function post-
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transcriptionally in wt Col to degrade the solo Tto LTR transcripts into 21-22 nt siRNAs, as in the
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rdr6 mutant the mRNA level is increased (Figure 3A) and 21-22 nt siRNAs are reduced (Figure
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3B). Lastly, in contrast to recent reports that suggested Dicer-independent siRNAs are
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responsible for the initiation of TE silencing (Ye et al., 2016; Yang et al., 2015), our data
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demonstrate that (multiple) Dicer-dependent mechanisms are necessary for TE RdDM, as
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transformation into the dcl2/3/4 triple mutant resulted in a loss of all siRNAs (Figure 3B) and
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RdDM (Figure 3C). In summary, Pol IV-RdDM, DCL3-RdDM, and RDR6-RdDM (but not Dicer9
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independent RdDM) all function in an overlapping manner to direct the methylation of the newly
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inserted solo Tto LTR transgene in T1 plants.
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In contrast to the redundant mechanisms responsible for siRNA production, we found that
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Pol V is responsible for all expression-dependent RdDM (Figure 3C). Using pol V (nrpe1) T1
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seedlings, we confirmed that Pol V is necessary for DNA methylation from as early as we can
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assay transgene germinal integration (Figure 3D). The Pol V transcript is likely targeted by both
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AGO6 and AGO4, as ago6 mutants only display a mild (not statistically significant) reduction in
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Tto LTR methylation (Figure 3C). Similar to the pol V mutant, the drm1/2 double mutant does not
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target RdDM to the solo Tto LTR (Figure 3D). Therefore, we conclude that multiple siRNA
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generating mechanisms redundantly target one downstream RdDM chromatin-bound complex
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requiring Pol V and DRM1/2. Pol V has been shown to be recruited to methylated DNA and
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RdDM loci by the SUVH2 and SUVH9 proteins (Johnson et al., 2014). Surprisingly, in a suvh2/9
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double mutant, solo Tto LTR RdDM occurs at levels similar to wt Col (Figure 3E). This suggests
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the existence of a yet-to-be-defined SUVH2/9-independent mechanism responsible for recruiting
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Pol V.
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We also tested the role of primary (independent of RNA-dependent RNA polymerase
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activity) small RNAs in targeting solo Tto LTR RdDM, since these small RNAs are thought to
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initiate the first stages of TE transcript recognition, RDR6 activity and RdDM (Creasey et al.,
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2014). We detected no loss of solo Tto LTR RdDM in ago1, ago2, or dcl1 mutants (Figure 3E),
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although the ago1 and dcl1 mutations do not result in the complete loss of protein function due
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to their requirement in embryonic development (Nodine and Bartel, 2010). In addition, we tested
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AtRRP6L1, a trimmer protein necessary for Dicer-independent small RNA processing (Ye et al.,
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2016). Similar to ago1, ago2 and dcl1, RdDM persists at near wt levels in the atrrp6l1 mutant,
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suggesting that either primary small RNAs are not driving the recognition of the solo Tto LTR for
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initial silencing or that redundant mechanisms exist.
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Full Tto elements transition to maintenance methylation and heterochromatin formation
When a TE invades a new genome by horizontal transfer, evidence suggests that a full-
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length element is transferred, not simply an LTR-only fragment (solo LTR)(El Baidouri et al.,
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2014; Pace et al., 2008). Although the solo Tto LTR provides a tool to investigate expression-
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dependent RdDM, it does not recapitulate the silencing dynamics of a full element. In addition,
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recent data have determined that TE structure plays a key role in targeting specific silencing
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mechanisms (Panda et al., 2016). We therefore aimed to determine how a full-length exogenous
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TE is silenced using two versions of a full (or nearly full) length Tto1 element. The ‘full Tto’
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transgene includes both complete LTRs and the entire Tto1 internal coding sequence (CDS)
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(Figure 4A), as in the native N. tabacum. Two short introns were added in the CDS as tools to
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measure RNA processing and retrotransposon-induced cDNA formation (Böhmdorfer et al.,
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2010). Similar to the solo Tto LTR transgene, which shares the same LTR promoter, the full Tto
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transgene is expressed when transformed into wt Col (Figure 4B). In contrast to the solo Tto
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LTR, which produces both 21-22 and 24nt siRNAs (Figure 3B and 4C), the full Tto element
345
produces low levels of only 24 nt siRNAs in the inflorescence tissue of T1 plants (Figure 4C,
346
Supplemental Figure 5). A second transgene, iTto, was previously constructed using a full Tto1
347
element with added CDS introns and a truncated 5’ LTR (Böhmdorfer et al., 2010). This element
348
is not able to produce its own coding transcripts, but rather is under the control of an inducible
349
promoter and has a transcriptional terminator (Figure 4A). We confirmed that this transgene
350
does not accumulate abundant mRNA transcripts without induction (Figure 4B). Like the full Tto
351
transgene, the iTto transgene only generates 24 nt LTR siRNAs in T1 inflorescence tissue,
352
however the levels are higher regardless of induction of expression (Figure 4C, Supplemental
353
Figure 5).
354
To examine the DNA methylation targeted to these transgenes we performed bisulfite
355
sequencing. As a control, we used an endogenous Arabidopsis gene, RAS-RELATED
356
NUCLEAR PROTEIN1 (RAN1), cloned into the same inducible vector backbone as iTto (iRAN1
357
(Nuthikattu et al., 2013)). The low level of CHH DNA methylation of iRAN1 (4.7%, Figure 4D)
358
demonstrates that the Tto transgenes are being targeted for silencing due to their TE structure /
359
sequence and not due to the process of transformation. Compared to the DNA methylation of
360
the solo Tto LTR transgene, there is higher methylation of the LTR sequences of both the full
361
Tto and iTto transgenes (Figure 4D). In contrast to the RdDM pattern of solo Tto LTR
362
methylation, the CHH sequence context methylation is low compared to CG for three out of the
363
four full-element LTRs, indicating a maintenance methylation state (Figure 4E)(Supplemental
364
Figure 1). Methylation of the Tto internal CDS region is consistently lower than methylation at
365
the LTRs (Figure 4D), suggesting that the CDS does not cause element recognition and that the
366
LTRs drive silencing. This correlates with published data demonstrating that the ends of
367
endogenous long TEs are preferentially targeted by RdDM mechanisms (Zemach et al., 2013;
368
Böhmdorfer et al., 2016).
11
369
Since we detect maintenance methylation patterns in the T1 generation transformed with
370
full-length elements, we aimed to verify that full-length Tto transgene silencing was not
371
dependent on pre-existing identity to the Arabidopsis genome. First, we tested and confirmed
372
that there is only slight sequence similarity between the Arabidopsis genome or small RNAs and
373
the full Tto1 sequence beyond what is expected by chance (Supplemental Figure 3B,D). Second,
374
we created the iTf1 transgene using a truncated fission yeast (Schizosaccharomyces pombe –
375
separated from Arabidopsis by ~1624 million years) Tf1 LTR retrotransposon placed into the
376
same inducible vector backbone as iTto. We found that the Tf1 TE has no substantial sequence
377
identity to the Arabidopsis genome compared to what is expected by chance (Supplemental
378
Figure 3D), and the top BLAST hit is 87 bp with 71% similarity. The iTf1 transgene displays the
379
same silencing characteristics as full-length Tto elements: higher methylation levels of the LTRs
380
compared to the internal CDS, and a CG≥CHH methylation pattern of the LTRs (Figure 4F).
381
Therefore, the maintenance methylation pattern of full-length Tto elements is not due to identity-
382
based silencing depending on existing similarity to the Arabidopsis genome, but rather a general
383
feature of the silencing of full-length exogenous TEs.
384
To test if heterochromatin formation is occurring on full-length elements, we measured
385
the heterochromatic mark H3K9me2 of the solo Tto LTR and iTto transgenes. The solo Tto LTR
386
element accumulates very little H3K9me2, while iTto accumulates substantial enrichment
387
throughout the element (Figure 4G), suggesting that the structure of a full-length TE is
388
necessary for abundant heterochromatin formation. To test whether the presence of the internal
389
CDS is a critical structural feature triggering maintenance methylation, we created a Tto
390
transgene that has the full 5’ LTR and full CDS, but lacks the 3’ LTR (Figure 4H). This transgene
391
is targeted by RdDM, but fails to activate maintenance methylation (CHH≥CG, Figure 4E,H),
392
similar to the solo Tto LTR transgene. Therefore, the CDS is not critical in the silencing of the
393
full-length elements, and we conclude that either the full TE structure or the presence of two
394
LTRs is critical for the cell to target maintenance methylation and heterochromatin formation.
395
Intrigued by the possibility of the unique regulation of full-length retrotransposons
396
compared to their fragmented derivatives, we searched the Arabidopsis genome for endogenous
397
TE subfamilies with both full-length elements and solo LTRs. Of the 121 TE subfamilies
398
identified with full-length elements, 45 contained at least one solo LTR copy. Of these 45, we
399
found 8 that show the same pattern as the exogenous Tto: the solo LTRs are regulated by
400
RdDM (reduced methylation in pol V mutants compared to wt) and full-length elements are
12
401
regulated by maintenance methylation and not RdDM. Overall, solo LTRs were significantly
402
more likely to be regulated by RdDM compared to full-length elements (t-test, p<0.001)(Figure
403
4I).
404
405
TE detection by RdDM is required for subsequent maintenance methylation and
406
heterochromatin formation
407
Like an endogenous full-length TE, iTto is targeted by maintenance methylation and
408
heterochromatin formation (Figure 4D, E, G). It is therefore an excellent model for identity-
409
independent silencing that progresses to the full maintenance methylation and heterochromatin
410
formation stage. To investigate this mechanism, we transformed iTto directly into pol IV and pol
411
V mutants. Upon induction of expression and accumulation of iTto mRNAs (Figure 5A), iTto
412
produces transposition intermediates (Figure 5B). We used the engineered introns (which are
413
spliced from retrotransposon-induced cDNA) to determine the level of transposition
414
intermediates and found significantly higher levels in pol V mutants (Figure 5B). This result was
415
independently verified by identification of circularized cDNA retrotransposition intermediates in
416
the pol V mutant (Supplemental Figure 6)(Hirochika and Otsuki, 1995), demonstrating that iTto
417
progresses further into the retrotransposition mechanism in pol V mutants.
418
When induced in wt Col, iTto undergoes a slight (1.33-fold) increase in 21-22 nt siRNAs
419
(365 to 486 RPM, Figure 5C,D) and expression-dependent methylation which is observed as
420
increased levels of CHH methylation (Figure 5E-F). This increase in 21-22 nt siRNAs is only
421
detectable by deep sequencing, while not detectible by RNA gel blot (Figure 5C, Supplemental
422
Figure 7). The increase in transposition intermediates in pol V mutants correlates with an
423
increase in 21-22 nt siRNAs produced throughout the iTto element (Figure 5C-D, Supplemental
424
Figure 7) and low methylation levels comparable to the baseline level of the control iRAN1
425
transgene (Figure 5E-F). The low level of methylation in pol V mutants is recapitulated with the
426
iTf1 full-length TE (Figure 5G). This low methylation suggests that full-length exogenous TEs are
427
not recognized as TEs for RdDM without a functional Pol V chromatin-modifying complex. Like
428
pol V mutants, drm1/2 double mutants do not accumulate iTto methylation (Figure 5E-F),
429
confirming that RdDM is required to initiate the silencing of iTto. Therefore, the iTto and iTf1
430
maintenance methylation pattern (CG>CHH) observed in the wt Col T1 inflorescence (Figure
431
5E-G) required the activity of RdDM, even though the RdDM pattern (CHH≥CG) is no longer
432
observed in this T1 inflorescence tissue.
13
433
To determine if iTto methylation can be established in pol V mutants after multiple
434
generations, we self-fertilized the T1 iTto plants and investigated the T2 generation. We
435
propagated both expression-induced and mock-induced plants, however, the induced plants
436
were not re-induced in the T2 generation and did not accumulate iTto mRNAs in the T2
437
generation (Figure 5H). The iTto transgene remains unrecognized as a TE in the T2 of pol V
438
mutants, in contrast to the continued methylation observed in wt Col lines (Figure 5I). To test the
439
formation of heterochromatin (H3K9me2) in the T2 generation, we performed ChIP and found
440
that iTto accumulates H3K9me2 over the entire element in wt Col, although induced expression
441
of iTto in the previous generation results in reduced levels (Figure 5J). In contrast, pol V mutants
442
fail to accumulate H3K9me2 (Figure 5J). Since both iTto DNA methylation levels and H3K9me2
443
are at background levels in pol V (Figure 5I-J), we conclude that pol V is fully responsible for the
444
triggering of silencing on exogenous TE chromatin. In addition, H3K9me2 does not accumulate
445
in drm1/2 double mutants (Figure 5J), proving that the establishment of TE DNA methylation via
446
RdDM is necessary before the establishment of H3K9me2. Since iTto and iTf1 have a
447
maintenance methylation pattern (CG>CHH) in T1 wt Col inflorescence tissue (Figure 5E-G), our
448
results suggest that full-length elements are dependent on RdDM to reach the ultimate silencing
449
state of maintenance methylation and heterochromatin formation. Even though we do not
450
observe RdDM methylation patterns for iTto or iTf1 after they have been transformed into wt Col,
451
these transgenes were likely expressed, identified as a TE, and subjected to RdDM at a
452
developmental time point prior to our analysis.
453
Pol IV is not required to establish silencing or chromatin modification at exogenous full-
454
length TEs, but only to transition to a maintenance methylation phase of silencing. We find that
455
pol IV mutants are able to perform RdDM on the iTto and iTf1 transgenes, demonstrated by the
456
intermediate levels of DNA methylation with the signature of RdDM (CHH≥CG) in this mutant
457
(Figure 5E-G). While the TEs are recognized in pol IV mutants, maintenance methylation fails to
458
activate and establish high CG levels compared to CHH (as in wt Col). Even in the T2
459
generation, iTto remains only partially CG methylated in pol IV mutants compared to wt Col
460
(Figure 5I). In pol IV mutants, H3K9me2 is only detected if iTto was expressed in the previous
461
generation (Figure 5J), demonstrating that Pol IV is not absolutely required to establish
462
H3K9me2, but there are two distinct routes for H3K9me2 deposition: 1) a Pol IV mechanism that
463
is expression-independent and 2) a Pol IV-independent mechanism that requires expression.
464
14
465
466
The genotype of TE insertion/transformation is critical for de novo silencing
Since the iTto transgene fails to correctly proceed to the maintenance methylation phase
467
of silencing when integrated into a pol IV genome, we aimed to determine if reintroduction of a
468
functional Pol IV copy would result in this progression of silencing. We crossed mock-induced
469
iTto T1 pol IV plants to wt Col, and then self-fertilized the T2 progeny to produce T3 individuals,
470
which were genotyped and selected for Pol IV/Pol IV wt plants (blue labels, Figure 6A). We
471
found that T3 wt plants in which iTto had been integrated into pol IV mutants had equal levels of
472
DNA methylation in all sequence contexts (CHH≥CG), and thus were never able to transition
473
from RdDM to maintenance methylation (Figure 6B-C). We biologically replicated the T1 cross
474
with a distinct iTto insertion event (Figure 6B, replicate B) and in addition performed exact
475
reciprocal crosses with a third unique insertion site (Figure 6B, replicate C). We consistently
476
found a lack of maintenance methylation across replicates. Therefore, when a functional copy of
477
Pol IV is crossed to the pol IV iTto line, Pol IV does not act on iTto. This is possibly due to the
478
lack of H3K9me2 (the heterochromatic signal that recruits Pol IV (Law et al., 2013)) in pol IV
479
mock-induced plants (Figure 5J). As a control, we crossed wt Col mock-induced iTto T1 plants
480
to pol IV mutants and then self-fertilized the progeny (orange labels, Figure 6A). This results in
481
T3 plants that have the same genotype, but in which the transgene was inserted into a wt rather
482
than mutant genome. These plants displayed a maintenance methylation pattern
483
(CG>CHH)(Figure 6B-C), similar to plants constantly in the wt genotype (Figure 5E, F, I). We
484
also selected pol IV / pol IV homozygous mutants from the T3 generation (purple labels, Figure
485
6A) and surprisingly these plants also demonstrated a maintenance methylation pattern
486
(CG>CHH) similar to wt plants (Figure 6B-C). This indicates that after maintenance methylation
487
occurs in the wt T1 plants (Figure 5E-F), Pol IV is no longer required for full maintenance-level
488
methylation. Pol IV’s function, therefore, is to transition the silencing from the RdDM phase to
489
the maintenance phase in the T1 generation. We conclude that a plant’s current wt or pol IV
490
genotype is not as important as the genotype that was initially transformed.
491
To determine if the lack of H3K9me2 deposition is restored upon addition of a functional
492
Pol IV copy into pol IV mutant lines, we performed ChIP of the T3 wt/wt plants from the pol IV
493
mutant cross (blue label, replicate A, Figure 6A-B). As a control, we compared these plants to
494
the self-fertilized T2 and T3 generation of iTto directly integrated into wt Col or pol IV from Figure
495
5. We found that only low levels of H3K9me2 accumulate if iTto was inserted into a pol IV
15
496
mutant genotype, irrespective of the crossing scheme or current genotype of the T3 plants
497
(Figure 6D).
498
We repeated our experimental design with iTto integrated into pol V mutants (Figure 6A),
499
which do not show iTto methylation in the T1 or T2 generation (Figure 5E,F,I), and found that
500
methylation appears in wt Pol V/Pol V T3 plants (blue labels, Figure 6B). This indicates that,
501
when a functional copy of Pol V is crossed into the T2 generation, Pol V acts on iTto without any
502
previous DNA or H3K9me2 marks. However, the methylation of iTto in these plants remains in
503
the RdDM phase (CHH≥CG) and is unable to progress to maintenance methylation (Figure 6B-
504
C), similar to the results of the pol IV cross. As a control, we performed the control cross with
505
transgene insertion directly into the wt genotype (orange labels, Figure 6A). Again, we found that
506
if transformed into a wt genome, the silencing progresses to the maintenance methylation phase
507
(CG>CHH) regardless of crossing scheme (Figure 6B-C). Similar to our findings with Pol IV, our
508
Pol V data demonstrate that the genotype of genome insertion / T1 is critical for transitioning to
509
maintenance methylation, and when transformed into a pol IV or pol V mutant, the maintenance
510
methylation phase is never obtained. These results confirm that the genotype of element
511
insertion (or at least soon after this event in the T1 generation), and not the crossing scheme or
512
current genotype, is the critical factor responsible for dictating the level of maintenance
513
methylation and heterochromatin formation in the T3 generation.
514
515
DISCUSSION
516
Separation of Identity-Based Silencing from Expression-Dependent Silencing
517
Multiple silencing mechanisms target active endogenous TEs to generate high levels of
518
DNA methylation (Panda et al., 2016). Through the analysis of exogenous TEs integrated into
519
the Arabidopsis genome, we have functionally distinguished two broad mechanisms of silencing
520
initiation. Plant transformation is an imperfect system, as multiple extra-chromosomal copies of
521
all transgenes are likely present within the cell at the time of genome insertion. However, this
522
process will be the same for all the transgenes used in this study, and the comparison of TE
523
transgenes to genic control transgenes has allowed us to dissect not the process of
524
transformation itself, but rather specific TE recognition mechanisms. Identity-based silencing
525
requires small RNAs produced from one transcriptionally or post-transcriptionally silenced TE (or
526
other region of the genome) to match a second incoming TE or transgene. Identity-based
527
silencing may form a cellular ‘immune system’ that is able to store a history of the TEs that the
16
528
plant has previously silenced and use this repository to silence a transposing or horizontally
529
transferred TE with sequence similarity. This mechanism may be the reason why Pol IV
530
transcription and 24 nt siRNA production continues from silenced TEs in most cells (Lu et al.,
531
2005), even for TEs that do not require RdDM reinforcement of their silenced state (Zemach et
532
al., 2013; Li et al., 2015; McCue et al., 2015). Identity-based silencing is able to establish DNA
533
methylation that is subject to heterochromatin formation and maintenance methylation. This
534
mechanism is likely more efficient at targeting regions for methylation compared to expression-
535
dependent RdDM, particularly in the CG sequence context (compare Figure 2C to 2E).
536
The mechanism of identity-based silencing broadly resembles the homology-dependent
537
phenomena known to affect non-TE transgenes when they become silenced (Matzke et al.,
538
2002, 2000). Identity-based silencing has been utilized as a tool to target silencing of an
539
endogenous gene using a transgene or viral DNA trigger (Jakowitsch et al., 1999; Ruiz et al.,
540
1998). However, it is unclear what level of homology (sequence similarity) is required to target
541
this mechanism. The amount of sequence similarity required for identity-based silencing likely
542
follows similar dynamics to the targeting of endogenous mRNAs by siRNAs or microRNAs (Liu
543
et al., 2014). Because we are certain that identical sequences target this mechanism, but we do
544
not know the level of sequence similarity required, we favor describing this pathway as ‘identity-
545
based’ rather than ‘homology-based’ silencing. In contrast to the trans-acting nature of identity-
546
based silencing, expression-dependent silencing occurs in cis – the same region that produces
547
the siRNAs is the target of RdDM, so the siRNA sequence is likely identical to its target
548
transcript. Our investigation determined that both 24 nt and 21-22 nt siRNAs can separately
549
function in identity-based silencing, providing a robust small RNA defense against new copies of
550
DNA that are similar to previously transcriptionally or post-transcriptionally silenced regions of
551
the genome.
552
553
554
Mechanism of Expression-Dependent Silencing
Both identity-based and expression-dependent mechanisms function on endogenous TEs,
555
confounding the interpretation of their silencing dynamics. The solo Tto LTR transgene is useful
556
because 1) it is only targeted by expression-dependent RdDM, and 2) it never progresses
557
beyond the RdDM phase, remaining targeted by expression-dependent RdDM over multiple
558
generations. We used the solo Tto LTR transgene to probe the mechanisms expression-
559
dependent silencing and found that AGO2, RDR6-generated 24 nt siRNAs, and high TE copy
17
560
numbers were not required to initiate TE silencing, refuting previous reports (Hirochika et al.,
561
2000; Pontier et al., 2012; Marí-Ordóñez et al., 2013). Instead, we identified multiple upstream
562
small RNA-generating mechanisms (including Pol IV-RdDM, DCL3-RdDM, and RDR6-RdDM)
563
that all redundantly function to produce the siRNAs necessary for RdDM, while one downstream
564
silencing complex dependent on Pol V is necessary for all RdDM. The dependency on a single
565
chromatin-bound Pol V complex, yet multiple small RNA biogenesis pathways, is recapitulated in
566
the initiation of viral silencing (Bond and Baulcombe, 2015), which also likely functions through
567
expression-dependent RdDM. Interestingly, expression-dependent RdDM of the solo Tto LTR
568
transgene did not require the SUVH2/9 proteins, which are known to mediate the recruitment of
569
Pol V (Johnson et al., 2014). This suggests that an independent mechanism exists to recruit Pol
570
V to newly inserted or expressed TEs or transgenes. In line with the theory of an alternate route
571
of Pol V recruitment, the addition of a functional Pol V copy into pol V mutant line restores iTto
572
RdDM, suggesting that Pol V is recruited to an unmethylated TE. The only other known
573
recruitment mechanism of Pol V is through double-strand DNA breaks (but Pol V was shown not
574
to function in RdDM at these sites (Wei et al., 2012)). The double-strand break recruitment of
575
Pol V remains an intriguing possibility, particularly for TEs and transgenes that insert into the
576
genome via DNA breaks.
577
578
Progression of Silencing
579
We found that RdDM is necessary for the initiation of silencing on TE fragments and full-
580
length TEs, while only full-length elements (and not solo LTRs or LTR+CDS elements with only
581
one LTR) progress beyond RdDM to heterochromatin formation and a maintenance methylation
582
state within the T1 generation. Our data suggest that full-length TEs are regulated differently
583
than their derived fragments, and that solo LTRs are more likely to be regulated by RdDM
584
genome-wide. Full-length TEs may progress via expression-dependent silencing more efficiently
585
due to the presence of two LTRs (Figure 4), and the ability of siRNAs produced from either LTR
586
(including the 3’LTR that is partially transcribed in the retrotransposon coding transcript) to target
587
the 5’LTR promoter.
588
Transformation of the full-length iTto into mutants demonstrated that both Pol IV and Pol
589
V are required in the T0 or T1 generation to progress beyond RdDM to the maintenance
590
methylation and heterochromatin formation phase. In pol V mutants, RdDM does not occur and
591
the TE cannot accumulate DNA or H3K9 methylation. We find that the role of Pol IV on iTto is to
18
592
transition from the establishment phase via RdDM to the end-point maintenance methylation
593
phase. This suggests that maintenance methylation originally triggered by expression-
594
dependent RdDM occurs through the intermediate step of Pol IV-RdDM, forming a linear
595
‘initiation-establishment-reinforcement-maintenance’ progression of TE silencing (Figure 7).
596
597
Genotype of Transformation
598
Surprisingly, upon outcrossing a mutant T1 generation and thereby complementing the
599
T2 generation with a functional Pol IV or Pol V, the silencing of the integrated TE was not fully
600
restored to wt Col levels. When inserted into pol IV mutants and outcrossed, the TE DNA
601
methylation remained at intermediate levels established by RdDM, and only minimal levels of
602
H3K9me2 were induced. This demonstrates that the initial signal that recruits Pol IV to a new TE
603
insertion must be transient and limited to a narrow window of time during insertion or within the
604
T1 generation. This signal is likely H3K9me2 itself, as without the formation of H3K9me2 at the
605
TE in the T1 generation of pol IV mutants, functional Pol IV is not recruited to the TE in later
606
generations. In contrast, when iTto was inserted into pol V mutants, no methylation was
607
detected, however an intermediate level returns when a functional Pol V copy is introduced. This
608
intermediate level of methylation has the sequence context hallmarks of RdDM (CHH≥CG),
609
suggesting that Pol V is successfully recruited back to the inserted TE (unlike Pol IV) and
610
functions there to target RdDM. As in the pol IV cross, the silencing cannot progress to the
611
maintenance methylation phase. This is likely due to the lack of H3K9me2 in pol V mutants that
612
again prohibits Pol IV recruitment during a critical window, stalling the activation of maintenance
613
methylation and heterochromatin formation. In addition to the analysis performed in Figure 6 on
614
iTto un-induced for expression, we have not yet determined how inducing expression in the T1
615
generation (or later) affects these trans-generational silencing dynamics.
616
Our genotype-of-transformation finding has implications for crop improvement. In many
617
crops, a specialized genotype is used for transformation, followed by introgression of the
618
transgene into an elite performance line. Our finding suggests that the genotype of first insertion
619
is critical for the establishment of trans-generational silencing. Transformation directly into a
620
silencing mutant (such as pol IV or pol V) followed by segregation away of these mutant alleles
621
during introgression into an elite performance line may be a route to avoid the complete
622
silencing of transgenes.
623
19
624
METHODS
625
Plant Materials
626
Plants were grown in long‐day (18 h cool white (4100K) fluorescent light at 120 μmol m-2 s-1
627
provided by Sylvania Octron Eco bulbs) conditions at 23°C. See Supplemental Table 1 for a list
628
of mutant alleles used and their sources. Biological replicates were grown side-by-side. Plants
629
were stably (germinal) transformed via Agrobacterium floral dip and T1 events selected on
630
appropriate antibiotics. Selection on antibiotics was continued each generation. Unless
631
otherwise noted, all experiments use inflorescence tissue (stage 1 -12 unopened flower buds).
632
633
634
Transgene Generation and Expression
The iTto and iRAN transgenes have been previously described (Böhmdorfer et al., 2010;
635
Nuthikattu et al., 2013). For other transgenes, TE sequences were amplified from the
636
Arabidopsis genome (iAthila LTR, Athila LTR, At1g08200 pro, TAS3A internal), the iTto plasmid
637
(solo Tto LTR), or the pHL414 (Levin et al., 1993) plasmid (iTf1) using Phusion High-Fidelity
638
DNA Polymerase (NEB) and primers from Supplemental Table 2. The iAthila LTR and iTf1 TE
639
sequences were cloned into pMDC7 (Curtis and Grossniklaus, 2003) at the restriction sites AscI
640
and PacI using In-Fusion HD cloning (Clontech), removing the ccdB region. The remaining
641
amplicons were cloned into pBGW (Karimi et al., 2002) at the restriction sites XbaI and SpeI
642
using In-Fusion HD cloning (Clontech), removing the ccdB region. The promoter deletion
643
constructs (Athila LTR Δpro, Tto LTR Δpro) were generated by performing inverse PCR on their
644
respective parent constructs with primers upstream of the TATA box and downstream of the
645
TSS (primers in Supplemental Table 2). These amplicons were then blunted and self-ligated
646
using Quick Blunting Kit (NEB) and T4 DNA ligase (NEB). This method was also used to delete
647
the neomycin resistance from the Tf1 sequence derived from pHL414 before cloning the iTf1
648
construct. To generate the Tto LTR+term construct, the 35S terminator from pB2GW7 was
649
amplified using Phusion polymerase and cloned into the PstI site of the solo Tto LTR construct
650
using In-Fusion cloning (Clontech). To generate the full Tto construct, iTto was digested with
651
Bpu10I (NEB) and the resulting TE portion was gel extracted and purified. The solo Tto LTR
652
construct was linearized with Bpu10I and treated with Shrimp Alkaline Phosphatase (NEB).
653
These two products were then ligated together using T4 DNA ligase, and colonies were
654
screened for the appropriate direction of ligation. See Supplemental Table 2 for all primers used
20
655
in cloning. Induction of expression-inducible transgenes was performed as in (Nuthikattu et al.,
656
2013).
657
658
Reverse Transcription-quantitative PCR (RT-qPCR)
659
Tissue from multiple sibling individuals was pooled for RNA isolations. At least three distinct
660
biological replicate pools were performed for each genotype. Each replicate consisted of a non-
661
overlapping pool from at least 6 individuals. RT-qPCR was performed and analyzed as
662
described in (Nuthikattu et al., 2013). RT-qPCR primers are listed in Supplemental Table 2.
663
664
Transposition Assay
665
An exon-exon spanning primer (see Supplemental Table 2) was used to amplify DNA directly
666
extracted from the indicated plants in Figure 5B, as spliced DNA is only generated from
667
retrotransposon-induced cDNA formation.
668
669
Bisulfite Sequencing
670
Tissue from at least six T1 individuals per each transgene construct / genotype / treatment
671
combination were pooled for each experiment. DNA extraction was performed on the pooled
672
tissue and bisulfite sequencing was performed and analyzed as in (McCue et al., 2015), with the
673
exception that PCR amplification of the bisulfite converted DNA was performed using MyTaq
674
DNA polymerase (Bioline). See Supplemental Table 2 for all primers used in bisulfite PCR.
675
Bisulfite primers are anchored to non-TE sequences of the chromosomal transgene, and
676
therefore do not detect the extra-chromosomal retrotransposon cDNA generated in Figure 5B or
677
potential transposed copies. PCR products were cloned using the TOPO-TA pCR4 vector
678
(Invitrogen). At least 6 clones were sequenced per sample. 95% Wilson confidence intervals and
679
t-tests were calculated as in (McCue et al., 2015).
680
681
Chromatin Immunoprecipitation
682
Chromatin was isolated from pooled sibling plants of the same genotype. Chromatin
683
Immunoprecipitation was performed as in (McCue et al., 2015). Each ChIP and mock-ChIP was
684
performed on three distinct and non-overlapping biological replicate samples per
685
genotype/treatment. ChIP used the H3K9me2 antibody (Abcam #ab1220). qPCR was performed
686
with primers shown in Supplemental Table 2. Three technical replicate qPCR amplifications
21
687
were performed for each of the biological replicates. The data reported represents the average
688
and standard error between distinct biological replicate pools.
689
690
Small RNA Analysis
691
RNA was extracted from pooled sibling plants of the same genotype. For RNA gel blots, RNA
692
preparation, gel electrophoresis, blotting, and probe production were performed as in (Nuthikattu
693
et al., 2013), with the exception that nine micrograms of small RNA-enriched RNA were loaded
694
in each lane. See Supplemental Table 2 for all primers used for probe production.
695
For the small RNA-seq in Figure 5, small RNA preparation and sequencing were
696
performed as in (McCue et al., 2015). The number of reads used for reads per million
697
normalization was determined by filtering each library for size (18–28 nt), exact matches to the
698
Arabidopsis genome (TAIR10) plus any transgene present in the line, and the removal of tRNA /
699
rRNA reads. Sequencing tracks were generated for iTto allowing up to 3 mismatches using
700
Bowtie (Langmead et al., 2009) and small RNA reads that where only filtered for size (18-28 nt)
701
and not filtered for genome-matching or tRNA/rRNA. For small RNA sequencing in Figure 2F,
702
the wt Col (no transgene) library from GSM2102963 and the wt Col iTto mock-induced library
703
(GSM2308720, described above) were aligned to the respective LTRs using Bowtie and
704
allowing for 3 mismatches.
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Genomic Informatics Analysis
707
To compare full-length TEs versus solo LTRs for dependence on RdDM, we selected TEs with
708
two LTRs and an intact coding sequence (full-length TEs), or containing only a single LTR with
709
no nearby TE coding sequence (solo LTRs). We averaged the total percent cytosine methylation
710
of these TEs by subfamily in both wt Col and pol V (Panda et al., 2016). We took the difference
711
of these averages (wt Col – pol V percent methylation) and performed a two-tailed Student’s t-
712
test between all full-length TEs and all solo LTRs.
713
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Accession Numbers
715
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717
718
719
720
RAN1 – AT5G20010; AXS2 – AT1G08200; TAS3A – AT3G17185; AGO6 – AT2G32940; POL IV
(NRPD1) – AT1G63020; DCL3 – AT3G43920; POL V (NRPE1) – AT2G40030; RDR6 –
AT3G49500; DCL2 – AT3G03300; DCL4 – AT5G20320; DRM1 – AT5G15380; DRM2 –
AT5G14620; SUVH2 – AT2G33290; SUVH9 – AT4G13460; AGO1 – AT1G48410; DCL1 –
AT1G01040; AGO2 – AT1G31280; RRP6L1 – AT1G54440
22
721
Small RNA-sequencing raw data can be accessed at NCBI GEO as GSE86787 using the
722
following GSM numbers:
723
wt Col inflorescence (non-transgenic) – GSM2308719
724
wt Col inflorescence with T1 iTto, mock induced – GSM2308720
725
wt Col inflorescence with T1 iTto, expression induced – GSM2308721
726
pol IV inflorescence with T1 iTto, expression induced – GSM2308722
727
pol V inflorescence with T1 iTto, expression induced – GSM2308723
728
729
Supplemental Data
730
Supplemental Figure 1 - CHH/CG methylation vs. total methylation scatter plots accurately
731
define the methylation type observed from bisulfite sequencing data (supports Figures 1-2).
732
Supplemental Figure 2 – Bisulfite cytosine methylation results linearly displayed on the Athila
733
LTR and solo Tto LTR sequences, with and without the Δpro deletion (supports Figures 1-2).
734
Supplemental Figure 3 - The Tto1 / Tf1 sequences are not identical to the Arabidopsis genome
735
(Supports Figures 2-4).
736
Supplemental Figure 4 - Full scanned images of the RNA gel blots supporting Figure 3B.
737
Supplemental Figure 5 - Full scanned images of the RNA gel blots supporting Figure 4C.
738
Supplemental Figure 6 - iTto retrotransposon-induced cDNA forms in pol V mutants (supports
739
Figure 5).
740
Supplemental Figure 7 - Full scanned images of the RNA gel blots supporting Figure 5C.
741
Supplemental Table 1 - Mutant alleles used this report
742
Supplemental Table 2 – Primer and probe sequences used this report
743
Supplemental Methods
744
745
ACKNOWLEDGEMENTS
746
The authors thank Andrea McCue, Eric Roose, Josquin Daron, Kaushik Panda, and Shobhana
747
Rajasenan for their data contributions. We thank Mikel Zaratiegui for the Tf1 plasmid, Andreas
748
Bachmair for the iTto plasmid, and Qikun Liu & Steve Jacobsen for the suvh2/9 mutant line. D.F.
749
is supported by the Presidential and University Fellowships and the Cellular, Molecular and
750
Biochemical Sciences T32 Training Program (NIH-GM086252) at The Ohio State University.
751
This work is supported by National Science Foundation grants MCB-1252370 and MCB-
752
1608392 to R.K.S.
23
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AUTHOR CONTRIBUTIONS
755
D.F and R.K.S. designed the research. D.F. performed research. D.F and R.K.S. analyzed data.
756
D.F and R.K.S. wrote the paper.
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Figure Legends
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Figure 1. Transgenes of endogenous TEs are targeted by identity-based and expressiondependent silencing.
A. Transgenes generated and transformed into Arabidopsis. Black
arrows indicate transcriptional start sites (TSS), green are PCR primers used in RT-qPCR, and
orange are PCR primers used for bisulfite sequencing of DNA methylation levels. B. RT-qPCR
detecting mRNA accumulation from the iAthila LTR transgene 5’ of the native Athila TSS in
either mock (not) induced or transcription-induced plants (primer set “B” in part A). For all RTqPCR, bar height represents the average calculated relative expression (compared to the
constitutively expressed UDP-D-APIOSE/UDP-D-XYLOSE SYNTHASE 2 (AXS2)) from
biological replicate samples of non-overlapping pools of sibling plants with the same genotype /
treatment. Error bars represent the standard error of the mean between biological replicate
samples. C. RT-qPCR of the iAthila LTR transgene 3’ of the native Athila TSS (primer set “C” in
part A). For all RT-qPCR, primers only amplify the transgene copy and not the endogenous
Athila elements, which are not activated by estradiol-induction (Nuthikattu et al., 2013). D.
Bisulfite sequencing of DNA methylation levels of the mock or transcription-induced iAthila LTR
transgene in T1 plants. H=A,T or C. For all bisulfite sequencing, bar height represents the
average methylation level, and bars represent 95% Wilson score confidence intervals, which
display the sequencing depth.
For all bisulfite PCR, primers only amplify the transgene copy
and not the endogenous Athila elements. E. Bisulfite sequencing of DNA methylation levels of
the transgenes depicted in part A in the T1 generation. The TAS3 internal CHG methylation has
a large confidence interval due to the low number (1) of CHG context cytosines in this PCR
product, while the number of CHH sites is high (38) and is used for all data comparisons. F. RTqPCR of the Athila LTR and Athila LTR Δpro T1 transgenes. G. Endogenous small RNAs from
inflorescence tissue without the transgene aligned to the indicated transgene. H. RT-qPCR of
the endogenous TAS3a locus and the TAS3 internal region transgene.
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787
Figure 2. The exogenous Tto TE circumvents identity-based silencing
A. Transgenes
generated and transformed into Arabidopsis. Arrows are the same as described in Figure 1. B.
RT-qPCR detecting steady-state mRNA accumulation in T1 inflorescence tissue of plants with
the indicated transgene. C. Bisulfite sequencing of T1 DNA methylation levels of the indicated
24
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793
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795
transgenes. The NOS promoter (pro) region is from the same transgene and integration events
as the solo Tto LTR T1. D. Categorization of methylation type (no methylation, no CHH, RdDM,
or maintenance methylation) based on the CHH/CG methylation context ratio (see Supplemental
Figure 1). Results from panels C and E are plotted. E. Bisulfite sequencing of T1 DNA
methylation levels of the indicated transgenes transformed into a wt Col line carrying the
silenced iTto TE transgene. F. Small RNA deep sequencing reads that match the indicated LTR
sequence. The small RNA library used is indicated above each bar, and the LTR used for
alignment is labeled below.
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Figure 3. Mechanism of expression-dependent RdDM. A. RT-qPCR of mutant T1 plants
transformed with the solo Tto LTR transgene. B. Small RNA gel blot of the individuals depicted
in part A. For all RNA gel blot assays, lanes are generated from pooled sibling plants. 21 vs 22
nt small RNAs are not distinguishable in this assay. Control Athila LTR siRNAs are absent in
dcl3 and pol IV mutants, yet only reduced in the dcl2/3/4 triple mutant due to the activation of the
Dicer-independent pathway (Ye et al., 2016). C. Bisulfite sequencing of T1 DNA methylation
levels of plants depicted in part A in inflorescence, or in T1 seedlings or juvenile leaves (D). E.
Bisulfite sequencing of T1 DNA methylation levels in inflorescence tissue of the solo Tto LTR
transgene transformed into various single and double mutant combinations.
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813
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817
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Figure 4. Full-length transposable elements are targeted for maintenance methylation and
heterochromatin formation.
A. Transgenes generated and transformed into
Arabidopsis.
Arrows are the same as described in Figure 1. B. RT-qPCR of the Tto LTR region
in T1 plants transformed with the indicated transgene. C. Small RNA gel blot of plants depicted
in part B.
The iRAN1 transgene serves as a negative control for plants that do not contain Tto1
sequences. D. Bisulfite sequencing of T1 DNA methylation levels of plants depicted in part B.
For the full Tto and iTto transgenes, multiple regions are assayed, including both LTRs. The
‘LTRs’ lane is the combination of both 5’ and 3’ LTRs for equal comparison to the solo Tto LTR
transgene. E. Categorization of methylation type based on the CHH/CG methylation context
ratio. LTR results from panel D and H are plotted. F. Bisulfite sequencing of T1 DNA methylation
levels of plants carrying the mock-induced iTf1 fission yeast TE-transgene. G. H3K9me2 ChIP of
plants carrying either the solo Tto LTR or iTto transgene. For all ChIP experiments, three
biological replicate samples were isolated from non-overlapping pools of sibling plants with the
same genotype. ChIP qPCR fold-enrichment is normalized to the constitutively expressed gene
AXS2. Bar height represents the average between biological replicate samples, and the error
bars represent the standard error of the mean between biological replicates. N/A indicates that
the indicated region does not exist in that transgene.
H. Bisulfite sequencing of T1 DNA
methylation levels of plants carrying the Tto LTR:CDS transgene.
I. Comparison of total DNA
methylation levels of endogenous LTR retrotransposons in the Arabidopsis genome in wt Col
25
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825
826
827
and pol V mutants. Each dot represents the average methylation of a single TE family for either
full-length copies (open circles) or solo LTR copies (red dots). Text labels indicate solo LTRs
and full-length elements that are regulated by RdDM (decrease of at least 4% methylation in pol
V compared to wt).
828
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832
833
834
Figure 5. Silencing of exogenous TEs requires RdDM to progress to maintenance
methylation. A. RT-qPCR of iTto steady-state mRNA levels in T1 plants of the indicated
genotype. Plants were either induced for iTto expression or mock-induced. B. qPCR of spliced
retrotransposon-induced cDNA formation in the same plants as part A. Relative copy number is
calculated based on the amount of retrotransposon-induced cDNA compared to the single copy
chromosomal gene AXS2. Bar height represents the average calculated from three biological
replicate samples of non-overlapping pools of sibling plants with the same genotype / treatment.
835
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Error bars represent the standard error of the mean between biological replicate samples. C.
Small RNA gel blot of the plants from part A. D. Results of small RNA deep sequencing aligned
to the iTto sequence. The Y-axes are all to the same scale, with the maximum abundance
adjusted to the highest peak by genotype, as indicated. Small RNAs have been library-size
normalized by calculating the reads per million of genome-matched (including the transgene
sequence) 18-28 nt reads. E. Bisulfite sequencing of T1 DNA methylation levels of plants
depicted in part A. F. Categorization of methylation type based on the CHH/CG methylation
context ratio. LTR results from panel E (orange) and H (blue) are plotted. G. Bisulfite sequencing
of T1 DNA methylation levels of plants with the iTf1 TE. H. RT-qPCR of iTto mRNA
accumulation from T2 plants, some of which were produced from expression-induced T1 plants.
The X-axis labels refer to the induction of the T1 parent of the assayed T2 progeny. I. Bisulfite
sequencing of T2 of plants depicted in part H. In parts H-I, PCR primers only detect the
transgene copy of iTto and not any potential extra-chromosomal or transposed copies. J.
H3K9me2 ChIP of T2 of plants depicted in part H. 5’ and 3’ LTR PCR primers are anchored to
the transgene, while the CDS primers could potentially amplify extra-chromosomal or
transposed iTto copies.
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Figure 6. The genotype of direct transformation dictates the final level of transgene
silencing.
A. Experimental crossing scheme. The right side indicates control crosses. Colors
denote data labels in parts B-C. B. Bisulfite sequencing of DNA methylation levels of genotyped
T3 plant lines depicted in part A. These T3 plants were generated by performing the T1 cross
indicated on the X-axis followed by selfing of the T2 generation. Replicates (rep A-C) denote
different transgene insertion events used in the crossing scheme. Brackets under plant lines
represent reciprocal crosses performed with the same T1 individual. C. Categorization of
methylation type based on the CHH/CG methylation context ratio using the results of panel B. D.
26
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H3K9me2 ChIP of plants with the iTto transgene directly transformed into the current genotype
(wt Col T2, wt Col T3, pol IV T2) compared to plants in which the transgene was transformed
into pol IV and introgressed wt Col (replicate A from part B).
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Figure 7. The Initiation-Establishment-Reinforcement-Maintenance model of TE silencing.
Incoming unique TEs must be expressed, go through post-transcriptional silencing via RNAi
(initiation), and establishment of DNA methylation though either the RDR6-RdDM or DCL3RdDM mechanism of expression-dependent RdDM (Cuerda-Gil and Slotkin, 2016). Incoming
TEs that share identity to the pool of siRNAs in the existing genome short-circuit this pathway
and enter directly into Pol IV-RdDM. A functional Pol IV-RdDM pathway is necessary in the
transformed T0 cells or subsequent T1 generation to reinforce DNA methylation and transition
the TE from RdDM to the fully silenced maintenance of DNA methylation.
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30
3x10-6
2x10-6
1x10-6
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25
24
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40
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60
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80
Constructs
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Constructs
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E100
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400
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0.6
Relative Expression
Athila LTR
Ttransgene sRNA matches (RPM)
At At
hi hil
la a
LT LT
R R
At Δp
hi ro
la
LT
R
0.5
800 wt Col pol IV
sRNA sRNA
En
TAS3a
G
600
o6
tC
ol
ag
o6
TAS3 internal region
1.0
0.8
w
At1g08200 promoter
1.5
3’ of Athila TSS
ag
term
1.0
Relative Expression
Athila6 LTR
Relative Expression
C
C
2.0 5’ of Athila TSS
Relative Expression
At1g08200 pro
B
Native Athila
TSS
Inducible
TSS
iAthila LTR
B
Inducible Promoter
R
Figure 1
A
Figure 1. Transgenes of endogenous TEs are targeted by identity-based and expression-dependent silencing.
A. Transgenes generated and transformed into Arabidopsis. Black arrows indicate transcriptional start sites (TSS), green are PCR primers used in RT-qPCR, and orange are
PCR primers used for bisulfite sequencing of DNA methylation levels. B. RT-qPCR detecting mRNA
accumulation from the iAthila LTR transgene 5’ of the native Athila TSS in either mock (not) induced
or transcription-induced plants (primer set “B” in part A). For all RT-qPCR, bar height represents the
average calculated relative expression (compared to the constitutively expressed UDP-D-APIOSE/
UDP-D-XYLOSE SYNTHASE 2 (AXS2)) from biological replicate samples of non-overlapping pools
of sibling plants with the same genotype / treatment. Error bars represent the standard error of the
mean between biological replicate samples. C. RT-qPCR of the iAthila LTR transgene 3’ of the native
Athila TSS (primer set “C” in part A). For all RT-qPCR, primers only amplify the transgene copy and
not the endogenous Athila elements, which are not activated by estradiol-induction (Nuthikattu et al.,
2013). D. Bisulfite sequencing of DNA methylation levels of the mock or transcription-induced iAthila
LTR transgene in T1 plants. H=A,T or C. For all bisulfite sequencing, bar height represents the average methylation level, and bars represent 95% Wilson score confidence intervals, which display the
sequencing depth.
For all bisulfite PCR, primers only amplify the transgene copy and not the endogenous Athila elements. E. Bisulfite sequencing of DNA methylation levels of the transgenes depicted
in part A in the T1 generation. The TAS3 internal CHG methylation has a large confidence interval due
to the low number (1) of CHG context cytosines in this PCR product, while the number of CHH sites
is high (38) and is used for all data comparisons. F. RT-qPCR of the Athila LTR and Athila LTR Δpro
T1 transgenes. G. Endogenous small RNAs from inflorescence tissue without the transgene aligned
to the indicated transgene. H. RT-qPCR of the endogenous TAS3a locus and the TAS3 internal region
transgene.
B
solo Tto LTR T2
0.6
solo Tto LTR ∆pro
0.4
solo Tto LTR in iTto
no CHH
20
30
40
total methylation (%)
% Methylation
40
20
50
0
∆p
LT
R
o
Tt
Tt
o
lo
so
lo
so
o
pr
O
S
N
∆p
ro
m
LT
R
+t
er
Tt
o
LT
R
so
1000
i
sRTto
N in
A w
tC
ol
F
lo
Tt
o
lo
so
so
lo
Tt
o
LT
R
T2
T1
60
LT
10
Constructs
transformed
into iTto
100
80
solo Tto LTR ∆pro in iTto
0
E
w
sR t C
N ol
A
solo Tto
LTR+term
maintenance
0.2
0.0
solo Tto LTR
Transgene sRNA matches (RPM)
0.8
no methylation
RdDM
0
0.00
800
600
400
sRNA
size
(nt)
25
24
23
22
21
20
200
0
so so
lo lo
Tt Tto
o
L LT
so TR R
lo ∆p
Tt ro
o
LT
R
1.0
20
R
CHH/CG methylation
D
term
40
LT
R
Tto1 LTR
0.05
CG
CHG
CHH
60
Tt
o
Tto1 LTR
promoter
deletion
solo Tto LTR+term
Constructs
transformed
into wt Col
80
lo
soloTto LTR ∆pro
83-225
0.10
so
Relative Expression
Tto1 LTR
C 100
Tto LTR
ro
TSS
% Methylation
solo Tto LTR
so so
lo lo
Tt Tt
o o
LT LT
R R
∆p
ro
Figure 2
A
Figure 2. The exogenous Tto TE circumvents identity-based silencing
A. Transgenes generated
and transformed into Arabidopsis. Arrows are the same as described in Figure 1. B. RT-qPCR detecting steady-state mRNA accumulation in T1 inflorescence tissue of plants with the indicated transgene.
C. Bisulfite sequencing of T1 DNA methylation levels of the indicated transgenes. The NOS promoter
(pro) region is from the same transgene and integration events as the solo Tto LTR T1. D. Categorization of methylation type (no methylation, no CHH, RdDM, or maintenance methylation) based on
the CHH/CG methylation context ratio (see Supplemental Figure 1). Results from panels C and E are
plotted. E. Bisulfite sequencing of T1 DNA methylation levels of the indicated transgenes transformed
into a wt Col line carrying the silenced iTto TE transgene. F. Small RNA deep sequencing reads that
match the indicated LTR sequence. The small RNA library used is indicated above each bar, and the
LTR used for alignment is labeled below.
E
/3
/4
1
6l
o2
ol
tC
w
1/
m
dr
tC
w
po
tC
w
/3
l2
dc
rd
lI
po
po
ag
dc
ol
tC
rrp
0
ag
0
l1
0
2
20
ol
20
lV
20
ol
40
/4
40
r6
40
V
60
lV
60
o6
60
l3
80
% Methylation
solo Tto LTR in Inflorescence
100
80
w
dc
l2
w
tC
ol
po
lI
V
rd
r6
lV
o6
ag
solo Tto LTR
Leaf
Seedling
dc
100
o1
80
miR161
D
solo Tto LTR in Inflorescence
CG
CHG
CHH
100
endogenous
Athila LTR
9
w
C
Tto LTR
antisense
ag
o
dc l
l
ag 3
po o6
w lV
tC
po ol
lI
V
dc rdr
l2 6
/3
/4
0.0
24nt
21nt
24nt
21nt
21nt
2/
0.2
Tto LTR
sense
vh
0.4
24nt
21nt
su
0.6
po
solo Tto LTR in:
w
tC
ol
dc
l3
0.8
B
solo Tto LTR
tC
Relative Expression
Figure 3
A 1.0
Figure 3. Mechanism of expression-dependent RdDM. A. RT-qPCR of mutant T1 plants transformed with the solo Tto LTR transgene. B. Small RNA gel blot of the individuals depicted in part A.
For all RNA gel blot assays, lanes are generated from pooled sibling plants. 21 vs 22 nt small RNAs
are not distinguishable in this assay. Control Athila LTR siRNAs are absent in dcl3 and pol IV mutants, yet only reduced in the dcl2/3/4 triple mutant due to the activation of the Dicer-independent
pathway (Ye et al., 2016). C. Bisulfite sequencing of T1 DNA methylation levels of plants depicted in
part A in inflorescence, or in T1 seedlings or juvenile leaves (D). E. Bisulfite sequencing of T1 DNA
methylation levels in inflorescence tissue of the solo Tto LTR transgene transformed into various single and double mutant combinations.
engineered introns
iTto
LTR
Tto1 LTR
Tto1 CDS
Term
iTf1
LTR
Tf1 LTR
Term
0.00
oc
k
In Ind
u
d
iR
AN uce ced
d
1
M
to
,
iT
to
,
LT
R
to
Tt
o
lT
o
0.05
24nt
21nt
24nt
21nt
Tto LTR
sense
21nt
miR161
Tto LTR
antisense
so
l
o
Tf1 CDS
0.10
Tt
o
LT
fu R
iT ll T
t
Ino, M to
du o
ce ck
d
inducible promoter
in wt Col:
iT
Tto1 LTR
Tto1 CDS
C
0.15
fu
l
full Tto Tto1 LTR
LTR Relative Expression
B
solo Tto LTR Tto1 LTR
0.20
so
l
Figure 4
A
100
80
% Methylation
E
CG
CHG
CHH
CHH/CG Methylation
D
60
40
20
20
40
20
40
total methylation (%)
no CHH
60
Tto1 CDS
50
solo LTRs
full-length
40
30
ATCOPIA26
ATCOPIA49
ENDOVIR1
ATLANTYS3
ATGP1
ATCOPIA15
META1
ATCOPIA39
ATRE1
ATCOPIA82
20
10
ATCOPIA31
D
:C
LT
LT
R
Tt
o
o
Tt
lo
D
Tt
so
3'
C
LT
R
o
Tt
Tt
o
5’
LT
R
2
tin
la
hi
Ac
0
S
0
R
N/A
LT
R
0
N/A
10
S
20
00
maintenance
pol V % Methylation
30
60
full Tto, 3'LTR
iTto, 5'LTR
0.2
LTRs
3'LTR
% Methylation
40
At
iTf1
50
82
CDS
5'LTR
20
iTto, 3'LTR
0.4
I
solo Tto LTR 100
iTto,
Mock Induced 80
60
g0
40
CDS
5'LTR
H
400
At
1
60
solo Tto LTR
0.6
iTto, Mock Induced Tto LTR:CDS Tto1 LTR
100
80
% Methylation
LTRs
full Tto
o
G
100
H3K9me2 Relative Fold Enrichment
F
3’LTR
CDS
5’LTR
LTR
TSS
solo
Tto LTR
Transgene: iRAN
full Tto, 5'LTR
Tto LTR:CDS
0.8
0.0
0
Target
region:
0
no methylation
1.0
RdDM
0
0
10
20
30
40
wt Col % Methylation
META1
50
Figure 4. Full-length transposable elements are targeted for maintenance methylation and heterochromatin formation.
A. Transgenes generated and transformed into Arabidopsis.
Arrows are the
same as described in Figure 1. B. RT-qPCR of the Tto LTR region in T1 plants transformed with the
indicated transgene. C. Small RNA gel blot of plants depicted in part B.
The iRAN1 transgene serves
as a negative control for plants that do not contain Tto1 sequences. D. Bisulfite sequencing of T1 DNA
methylation levels of plants depicted in part B. For the full Tto and iTto transgenes, multiple regions
are assayed, including both LTRs. The ‘LTRs’ lane is the combination of both 5’ and 3’ LTRs for equal
comparison to the solo Tto LTR transgene. E. Categorization of methylation type based on the CHH/CG
methylation context ratio. LTR results from panel D and H are plotted. F. Bisulfite sequencing of T1 DNA
methylation levels of plants carrying the mock-induced iTf1 fission yeast TE-transgene. G. H3K9me2
ChIP of plants carrying either the solo Tto LTR or iTto transgene. For all ChIP experiments, three biological replicate samples were isolated from non-overlapping pools of sibling plants with the same genotype. ChIP qPCR fold-enrichment is normalized to the constitutively expressed gene AXS2. Bar height
represents the average between biological replicate samples, and the error bars represent the standard
error of the mean between biological replicates. N/A indicates that the indicated region does not exist in
that transgene.
H. Bisulfite sequencing of T1 DNA methylation levels of plants carrying the Tto LTR:CDS
transgene.
I. Comparison of total DNA methylation levels of endogenous LTR retrotransposons in the
Arabidopsis genome in wt Col and pol V mutants. Each dot represents the average methylation of a
single TE family for either full-length copies (open circles) or solo LTR copies (red dots). Text labels
indicate solo LTRs and full-length elements that are regulated by RdDM (decrease of at least 4%
methylation in pol V compared to wt).
M
ed
V
lI
ORF
TSS
LTR Term
iTto 5’LTR
80
60
40
20
0
1400
300
500
150
300
LTR
ORF
J
1000
600
200
60
40
0
At1g08200
LTR Term
T2
Actin2
Mock Induced
0.6
0.0
0.4
pol V Ind
0.2
pol V Ind
pol V Mock
drm1/2 Ind
drm1/2 Mock
G 100
0
20
80
iTf1 5’LTR
4
60
3
40
2
20
1
0
0
endogenous
Athila LTR
w
t
w Col
tC I
iTto Relative Expression
o nd
w l I uc
po t C ndu ed
l I ol ce
V M d
p In o
po ol I duc ck
l V V ed
M
po Ind ock
l V uc
M ed
oc
k
1.0
oc
k
F
M
300
50
50
300
oc
k
21-22 nt
24 nt
M
1000
100
V
LTR
E
V
0
% Methylation
5
po
l
iR
iR AN
AN 1 M
1
oc
I
w ndu k
t
w Co ced
tC l
ol Mo
In ck
po du
c
l
po IV ed
lI M
V
o
In ck
po duc
po l V ed
l V Mo
c
dr Ind k
m u
dr 1/ ce
m 2 d
1/ M
2
o
In ck
du
ce
d
50
50
50
50
50
50
300
to
to
in
in
w
tC
o
w l
iT
to t C
o
in
p l le
iT
to ol I af
V
in
iR p o l
AN
V
1
in
iT
w
to
tC
in
o
w
iT
to t C l
ol
in
w
iT
to t C
ol
in
p o le a
iT
f
to
l
in IV
iR p o
AN l V
1
in
w
tC
ol
iT
iT
C
oc
k
150
p < 0.05
po
lI
0
0.8
CHH/CG Methylation
iTto Relative Expression
10
w wt C
tC o
iTto Relative Mobile Copy #
ol l M
In o
p
o
c
po l du k
l I IV ce
V M d
In o
po pol du ck
l V V ce
In Mo d
du ck
ce
d
w wt C
tC o
ol l M
I
po pol ndu ock
l I IV ce
V M d
In o
po pol du ck
l V V ce
In Mo d
du ck
ce
d
3
% Methylation
D
wt Col
no transgene
6
M
300
500
iTto pol V Induced
iTto wt Col Mock
9
tC
ol
iTto wt Col Induced
12
15
w
iTto pol IV Induced
B
H3K9eme2 Relative Fold Enrichment
T2
oc
k
In
du
c
po
e
lV d
M
po
oc
lV
k
In
du
ce
d
po
V
k
oc
du
c
In
ol
M
po
lI
ol
tC
oc
k
M
% Methylation
100
w
tC
1
AN
I
w
iR
Figure 5
A 15
24nt
21nt
iTto LTR
sense
24nt
21nt
iTto CDS
sense
21nt
miR161
Induced
TSS
iTto 5’LTR
80
no methylation
pol V Mock
wt Col Mock
40
60
Total Methylation (%)
iTto 5'LTR
H
5
iTto CDS
CG
CHG
CHH
60
40
20
0
orange = T1
blue = T2
pol IV Ind
RdDM
pol IV Mock wt Col Ind
pol IV Mock
wt Col Ind
pol IV Ind
wt Col Mock
maintenance
no CHH
80
T1
T2
wt Col Mock
wt Col Induced
pol IV Mock
pol IV Induced
pol V Mock
pol V Induced
drm1/2 Mock
drm1/2 Induced
20
iTto 3'LTR
Figure 5. Silencing of exogenous TEs requires RdDM to progress to maintenance methylation.
A. RT-qPCR of iTto steady-state mRNA levels in T1 plants of the indicated genotype. Plants were either induced for iTto expression or mock-induced. B. qPCR of spliced retrotransposon-induced cDNA
formation in the same plants as part A. Relative copy number is calculated based on the amount of
retrotransposon-induced cDNA compared to the single copy chromosomal gene AXS2. Bar height
represents the average calculated from three biological replicate samples of non-overlapping pools of
sibling plants with the same genotype / treatment. Error bars represent the standard error of the mean
between biological replicate samples. C. Small RNA gel blot of the plants from part A. D. Results of
small RNA deep sequencing aligned to the iTto sequence. The Y-axes are all to the same scale, with
the maximum abundance adjusted to the highest peak by genotype, as indicated. Small RNAs have
been library-size normalized by calculating the reads per million of genome-matched (including the
transgene sequence) 18-28 nt reads. E. Bisulfite sequencing of T1 DNA methylation levels of plants
depicted in part A. F. Categorization of methylation type based on the CHH/CG methylation context
ratio. LTR results from panel E (orange) and H (blue) are plotted. G. Bisulfite sequencing of T1 DNA
methylation levels of plants with the iTf1 TE. H. RT-qPCR of iTto mRNA accumulation from T2 plants,
some of which were produced from expression-induced T1 plants. The X-axis labels refer to the
induction of the T1 parent of the assayed T2 progeny. I. Bisulfite sequencing of T2 of plants depicted in part H. In parts H-I, PCR primers only detect the transgene copy of iTto and not any potential
extra-chromosomal or transposed copies. J. H3K9me2 ChIP of T2 of plants depicted in part H. 5’ and
3’ LTR PCR primers are anchored to the transgene, while the CDS primers could potentially amplify
extra-chromosomal or transposed iTto copies.
100
CG
CHG
CHH
LT
R
3'
60
to
20
40
Total Methylation (%)
0
iT
0
no CHH
S
maintenance
100
LT
R
wt Col x pol IV
wt Col x pol IV
pol IV x wt Col
wt Col x pol V
pol IV x wt Col
pol V x wt Col
200
5'
pol IV x wt Col, rep A
pol V x wt Col, rep B
pol IV x wt Col, rep B
pol IV x wt Col, rep C
to
pol V x wt Col, rep A
iT
0.5
wt Col x pol IV, rep C
wt Col, selfed T2
wt Col, selfed T3
pol IV, selfed T2
T3: pol IV x wt Col, rep A
wt/wt
0
1.0
300
2
RdDM
D
tin
wt Col x pol V, rep B
wt Col x pol V, rep A
Ac
no methylation
20
iTto from wild-type
grandparent
0
08
CHH/CG Methylation
1.5
screen mutant
for
mutant
20
1g
wt
wt
iTto from mutant
grandparent
C
wt
(T2)
mutant
(T3)
(T3)
40
At
screen wt
for
wt
mutant
wt
mutant
(T2)
wt
60
mutant
mutant
H3K9me2 Relative
Fold Enrichment
wt
mutant
mutant
mutant
lI
V
po x
l I wt
V
po x Col
l I wt , re
V
w x Col p A
tC w ,
ol t C rep
x ol, B
po re
lI p
po
V, C
lV
re
p
w xw
C
tC t
C
ol
po x ol,
l V po re
p
w x w l V, A
tC t
re
C
ol
p
x ol, A
po re
lV pB
,r
ep
w
B
tC
o
po l x
l I po
V
l
w x w IV
tC
t
C
o
po l x ol
l V po
x lV
w
tC
w
ol
tC
ol
po x
l I po
V
l
x IV
w
tC
ol
wt
wt
(T1)
po
wt
wt
wt
wt
mockinduced
% Methylation
80
mock- mutant
induced mutant (T1)
D
iTto
T3
pol IV
pol IV
T3 wt/wt
C
iTto
B
to
mutant
transform
wild-type
iT
Figure 6
A transform
Figure 6. The genotype of direct transformation dictates the final level of transgene silencing.
A. Experimental crossing scheme. The right side indicates control crosses. Colors denote data
labels in parts B-C. B. Bisulfite sequencing of DNA methylation levels of genotyped T3 plant lines
depicted in part A. These T3 plants were generated by performing the T1 cross indicated on the
X-axis followed by selfing of the T2 generation. Replicates (rep A-C) denote different transgene insertion events used in the crossing scheme. Brackets under plant lines represent reciprocal crosses
performed with the same T1 individual. C. Categorization of methylation type based on the CHH/
CG methylation context ratio using the results of panel B. D. H3K9me2 ChIP of plants with the iTto
transgene directly transformed into the current genotype (wt Col T2, wt Col T3, pol IV T2) compared
to plants in which the transgene was transformed into pol IV and introgressed wt Col (replicate A from
part B).
Figure 7
Establishment by
Expressiondependent RdDM
TE
Pol II
AAAAA
RDR6
Reinforcement by
Pol IV-RdDM
Pol V
RD
R6
-
Rd
DM
Initiation
DCL3-RdDM
AGO
TE
Pol IV
DCL3
AGO1
AAAAA
DRM2
TE
RDR2
Maintenance
DRM2
TE
Pol V
AGO4/6
Pol IV-RdDM
24 nt
siRNAs
MET1, CMT2,
CMT3, and DDM1
Epigenetic inheritance
of the transcriptionallysilenced TE
21 & 22 nt
siRNAs
plant cell
DCL2, DCL4
Expressiondependent
silencing
Identitybased
silencing
TE
Unique
Incoming TEs
TE
Homologous
Figure 7. The Initiation-Establishment-Reinforcement-Maintenance model of TE silencing. Incoming unique TEs must be expressed, go through post-transcriptional silencing via RNAi (initiation),
and establishment of DNA methylation though either the RDR6-RdDM or DCL3-RdDM mechanism of
expression-dependent RdDM (Cuerda-Gil and Slotkin, 2016). Incoming TEs that share identity to the
pool of siRNAs in the existing genome short-circuit this pathway and enter directly into Pol IV-RdDM.
A functional Pol IV-RdDM pathway is necessary in the transformed T0 cells or subsequent T1 generation to reinforce DNA methylation and transition the TE from RdDM to the fully silenced maintenance
of DNA methylation.
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Exogenous Transposable Elements Circumvent Identity-Based Silencing Permitting the Dissection of
Expression Dependent Silencing
Dalen Fultz and R. Keith Slotkin
Plant Cell; originally published online February 13, 2017;
DOI 10.1105/tpc.16.00718
This information is current as of June 18, 2017
Supplemental Data
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