In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on Mediator of Paramutation 1 (Mop1), a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via histone H3 lysine 9 dimethylation (H3K9me2), and histone H3 lysine27 dimethylation (H3K27me2), even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by histone H3 lysine 27 trimethylation (H3K27me3), a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild-type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.
Although transposable elements (TEs) comprise a major fraction of many higher eukaryotic genomes, most TEs are silenced by host defense mechanisms. The means by which otherwise active TEs are recognized and silenced remains poorly understood. Here we analyzed two independent cases of spontaneous silencing of the active maize Ac/Ds transposon system. This silencing is initiated by alternative transposition, a type of aberrant transposition event that engages the termini of two nearby separate TEs. Alternative transposition during DNA replication can generate Composite Insertions that contain inverted duplications of the transposon sequences. We show that the inverted duplications of two Composite Insertions are transcribed to produce double-stranded RNAs that trigger the production of two distinct classes of small interfering RNAs: a 24-nt class complementary to the TE terminal inverted repeats and noncoding subterminal regions, and a 21- to 22-nt class corresponding to the TE transcribed regions. Plants containing these small interfering RNA-generating Composite Insertions exhibit decreased levels of Ac transcript and heritable repression of Ac/Ds transposition. Further, we demonstrate that Composite Insertions can heritably silence otherwise active elements in trans. This study documents the first case of transposon silencing induced by alternative transposition and may represent a general initiating mechanism for silencing of DNA transposons.
Eukaryotic Macrotransposons (MTns) can be formed by two nearby elements flanking a segment of host DNA. The maize Ac transposon can form Ac::MTns, but little is known about Ac::MTn transposition activities. Here we studied three Ac::MTns at the maize p1 locus, each of which is composed of a segment of maize p1 genomic DNA (up to 15 kb) bounded by a fractured Ac element (fAc, 2039 bp), and a full-length Ac element in direct orientation. The resulting Ac::MTns are of 16, 16.5 and 22 kb total length. From these three Ac::MTns, we identified 10 independent cases of macrotransposition, and observed similar features of transposition between Ac::MTn and standard Ac/Ds, including characteristic excision footprints and insertion target site duplications (TSD). Nine out of the 10 Ac::MTn reinsertion targets were genetically linked to the donor sites, another similarity with Ac/Ds standard transposition. We also identified a MTn-like structure in the maize B73 reference genome and 5 NAM founder lines. The MTn in diverse lines is flanked by target site duplications (TSD), confirming the historic occurrence of MTn transposition during genome evolution. Our results show that Ac::MTns are capable of mobilizing segments of DNA long enough to include a typical full-length plant gene and in theory could erode gene co-linearity in syntenic regions during plant genome evolution.
In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on Modifer of Paramutation 1 (Mop1), a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via H3K9 and H3K27 dimethylation, even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by H3K27 trimethylation, a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.Author SummaryMost plant genomes are mostly transposable elements (TEs), most of which are held in check by modifications of both DNA and histones. The bulk of silenced TEs are associated with methylated DNA and histone H3 lysine 9 demethylation (H3K9me2). In contrast, epigenetically silenced genes are often associated with histone lysine 27 trimethylation (H3K27me3). Although stress can affect each of these modifications, plants are generally competent to rapidly reset them following that stress. Here we demonstrate that although DNA methylation is not required to maintain silencing of the MuDR element, it is essential for preventing heat-induced, stable and heritable changes in both H3K9me2 and H3K27me3 at this element, and for concomitant changes in transcriptional activity. These finding suggest that RdDM acts to buffer the effects of heat on silenced transposable elements, and that a loss of DNA methylation under conditions of stress can have profound and long lasting effects on epigenetic silencing in maize.
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