No abstract

In Arabidopsis, LTR retrotransposons are activated by mutations in the chromatin gene DECREASE in DNA METHYLATION 1 (DDM1), giving rise to 21-to 22-nt epigenetically activated siRNA (easiRNA) that depend on RNA DEPENDENT RNA POLYMERASE 6 (RDR6). We purified virus-like particles (VLPs) from ddm1 and ddm1rdr6 mutants in which genomic RNA is reverse transcribed into complementary DNA. High-throughput short-read and long-read sequencing of VLP DNA (VLP DNA-seq) revealed a comprehensive catalog of active LTR retrotransposons without the need for mapping transposition, as well as independent of genomic copy number. Linear replication intermediates of the functionally intact COPIA element EVADE revealed multiple central polypurine tracts (cPPTs), a feature shared with HIV in which cPPTs promote nuclear localization. For one member of the ATCOPIA52 subfamily (SISYPHUS), cPPT intermediates were not observed, but abundant circular DNA indicated transposon "suicide" by auto-integration within the VLP. easiRNA targeted EVADE genomic RNA, polysome association of GYPSY (ATHILA) subgenomic RNA, and transcription via histone H3 lysine-9 dimethylation. VLP DNA-seq provides a comprehensive landscape of LTR retrotransposons and their control at transcriptional, post-transcriptional, and reverse transcriptional levels.

15In Arabidopsis, LTR-retrotransposons are activated by mutations in the chromatin remodeler 16 DECREASE in DNA METHYLATION 1 (DDM1), giving rise to 21-22nt epigenetically 17 79 retrotransposons by stress, or by loss of histone methylation, also requires loss of 24nt small RNA 80 and RDR2/RNA polymerase IV (Ito et al., 2011; Mirouze et al., 2009). By contrast, in ddm1 81 mutants and wild-type pollen, most transposons are transcriptionally activated and 24nt siRNA 82 are partly replaced by 21-22nt easiRNA (Slotkin et al., 2009). In ddm1 mutants, easiRNA are 83 generated by RDR6 (Creasey et al., 2014; Nuthikattu et al., 2013) from the non-functional 84 ATHILA2 and ATHILA6 Ty3/gypsy retrotransposons but also from the functional, TY1/copia 85 element EVADE, and are triggered by diverse miRNA. In wild-type, retroelements generate 86 easiRNA only in pollen, where they are targeted at the PBS by miR845, and biogenesis occurs via 87 a non-canonical pathway (Borges et al., 2018).88 89 In order to develop a comprehensive catalog of functional retrotransposons in Arabidopsis, we 90 performed VLP DNA sequencing from ddm1 mutants, as well as genome-wide polysomal RNA 91 (translatome) and chromatin immunoprecipitation (ChIP) sequencing. VLP sequencing recovered 92 all known active retrotransposons in Arabidopsis, without the need for genome sequencing of 93 5 transposition events. Replication intermediates revealed profound differences between elements 94with multiple cPPT and high integration rates, and elements with no cPPT which preferentially 95 integrated into themselves ("suicidal" auto-integration within the VLP). We examined the roles of 96 easiRNA in retrotransposon control by investigating ddm1rdr6 double mutants (Creasey et al., 97 2014; Lippman et al., 2004; Vongs et al., 1993). We found that some retrotransposons are 98 regulated post-transcriptionally by RNA interference, while others are regulated at the 99 transcriptional level by histone H3 lysine-9 methylation guided by small RNA. We conclude that 100 easiRNA inhibits retrotransposition at multiple levels in the replication cycle and identify features 101 of active retrotransposons that promote activity and escape from silencing.

Epigenetic inheritance refers to the faithful replication of DNA methylation and histone modification independent of DNA sequence. Nucleosomes block access to DNA methyltransferases, unless they are remodeled by DECREASE IN DNA METHYLATION1 (DDM1[Lsh/HELLS]), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 activity results in replacement of the transcriptional histone variant H3.3 for the replicative variant H3.1 during the cell cycle. In ddm1 mutants, DNA methylation can be restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals direct engagement at SHL2 with histone H3.3 at or near variant residues required for assembly, as well as with the deacetylated H4 tail. An N-terminal autoinhibitory domain binds H2A variants to allow remodeling, while a disulfide bond in the helicase domain is essential for activity in vivo and in vitro. We show that differential remodeling of H3 and H2A variants in vitro reflects preferential deposition in vivo. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1[Dnmt1]. DDM1 localization to the chromosome is blocked by H4K16 acetylation, which accumulates at DDM1 targets in ddm1 mutants, as does the sperm cell specific H3.3 variant MGH3 in pollen, which acts as a placeholder nucleosome in the germline and contributes to epigenetic inheritance.