We previously reported that methylation reprogramming in the Arabidopsis male germline regulates gene expression and meiosis (Walker et al. 2018 Nature Genetics). However, how transgenerational epigenetic inheritance is governed in plants is still unclear due to the lack of understanding of the reprogramming mechanism. In this project, we developed techniques to isolate early reproductive cells, and combined single-cell-type genomics, genome editing, genetic mosaics and live imaging to comprehensively investigate DNA methylation reprogramming in the male Arabidopsis germline. We found that male meiocytes (the origin of the male germline) are quiescent in sRNA biogenesis and gene methylation in meiocytes is induced by 24-nt sRNAs transported from tapetal nurse cells. We further demonstrated that gene-targeting sRNAs are produced by TEs with imperfectly matching sequences, and meiocytes can use imperfectly matching sRNAs to target DNA methylation. Furthermore, we found that a chromatin remodeler specific to tapetal cells, CLSY3, drives the biogenesis of gene-targeting sRNAs. We demonstrated that tapetal sRNAs can specify the entire de novo methylome of the male germline, including the sperm. Finally, besides their gene regulatory functions, we showed that tapetal sRNAs silence transposable elements (TEs) in the germline and protect genome integrity. These results (now published: Long et al. 2021 Science) demonstrated that paternal epigenetic inheritance is determined by tapetal nurse cells, which drive reprogramming at a scale unprecedented in plants. The unique ability of 24-nt sRNAs to induce methylation at mismatched targets in meiocytes indicates that meiosis is a key stage for genome surveillance. This discovery also provides a potential solution to a mystery in crop genetics – the large accumulation of tapetal sRNAs, the 24-nt phasiRNAs, in monocots. PhasiRNAs are essential for male fertility, but the biological mechanism is unknown as perfectly matching genomic targets are lacking. The ability of tapetal 24-nt sRNAs to silence TEs and control gene expression despite mismatches presents a likely mechanism for phasiRNA activity.
It was previously showed that meiotic crossovers (COs) are mis-localized in the absence of AXR1, an enzyme involved in the neddylation/rubylation protein modification pathway in Arabidopsis. Our collaboration with two French labs showed that in the axr1 mutant, COs are redistributed towards subtelomeric chromosomal ends where they frequently form clusters, in contrast to large central regions depleted in recombination. The CO suppressed regions correlate with DNA hypermethylation of TEs in the CHH context in axr1 male meiocytes. Additionally, we found axr1 affects DNA methylation in somatic tissues, causing hypermethylation in all sequence contexts (CG, CHG and CHH) at TEs. Impairment of DNA methylation pathways is epistatic over the axr1 mutation for DNA methylation in somatic cells but does not restore regular chromosome segregation during meiosis. Collectively, our findings revealed that the neddylation pathway not only regulates CO distribution but is also, directly or indirectly, a major limiting pathway of TE DNA methylation in somatic cells (Christophorou et al. 2020 PLos Genetics).
Together with Ian Henderson’s lab in Cambridge, we discovered a novel mode of meiotic recombination control via a meiocyte-enriched subunit of the general RNA polymerase II transcription factor TFIID, TBP-ASSOCIATED FACTOR 4b (TAF4b). Using genetics, genomics and immunocytology, we demonstrated a genome-wide decrease in crossovers in the taf4b mutant, with strongest reduction in the sub-telomeric regions. Using RNA-seq from purified meiocytes, we showed that TAF4b expression is meiocyte-enriched, whereas its paralog TAF4 is broadly expressed. Consistent with the role of TFIID in promoting gene expression, RNA-seq of wild type and taf4b meiocytes identified widespread transcriptional changes, including in genes that regulate the meiotic cell cycle and recombination. Therefore, we showed that TAF4b duplication is associated with acquisition of meiocyte-specific expression and promotion of germline transcription, which acts directly or indirectly to elevate crossovers (Lawrence et al. 2019 Current Biology).
TEs, the movement of which can damage the genome, are epigenetically silenced in eukaryotes. Intriguingly, TEs are activated in the sperm companion cell – vegetative cell (VC) – of the flowering plant Arabidopsis thaliana. However, the extent and mechanism of this activation were unknown. We showed that about 100 heterochromatic TEs are activated in VCs, mostly by DEMETER-catalyzed DNA demethylation. We further demonstrated that DEMETER access to some of these TEs is permitted by the natural depletion of linker histone H1 in VCs. Ectopically expressed H1 suppresses TEs in VCs by reducing DNA demethylation and via a methylation-independent mechanism. We demonstrated that H1 is required for heterochromatin condensation in plant cells and showed that H1 overexpression creates heterochromatic foci in the VC progenitor cell. Taken together, our results demonstrated that the natural depletion of H1 during male gametogenesis facilitates DEMETER-directed DNA demethylation, heterochromatin relaxation, and TE activation (He et al. 2019 eLife).
