Autophagy sustains cellular health by recycling damaged or excess components through autophagosomes. Autophagy is mediated by conserved ATG proteins, among which the ubiquitin-like ATG8 proteins play a central role by linking cargo to the growing autophagosomes. Unlike most ATG proteins, the ATG8 gene family is significantly expanded in vascular plants, but its functional specialization remains poorly understood. Using transcriptional and translational reporters in Arabidopsis thaliana, we revealed that ATG8 isoforms are differentially expressed across tissues and form distinct autophagosomes. To explore ATG8 specialization, we generated the nonuple Δatg8 mutant, lacking all nine ATG8 isoforms. The mutant displayed hypersensitivity to carbon and nitrogen starvation, coupled with defects in bulk and selective autophagy, as shown by biochemical and ultrastructural analyses. Complementation experiments demonstrated that ATG8A could rescue both carbon and nitrogen starvation phenotypes, whereas ATG8H could only complement carbon starvation. Proximity labeling proteomics further identified isoform-specific interactors under nitrogen starvation, underscoring their functional divergence. These findings provide genetic evidence for functional specialization of ATG8 isoforms in plants and lay the foundation for investigating their roles in diverse cell types and stress conditions.

SummaryTransposable elements (TEs) are marked by a complex array of chromatin modifications, but a central unifying mechanism for how they are silenced remains elusive. Histone H3 Lysine 9 methylation (H3K9me) is an important component of heterochromatin in most eukaryotes, including plants. In flowering plants, the specialized histone variant H2A.W occupies nucleosomes found at TE sequences. This variant is deposited by the chromatin remodeler DDM1 and confers specific biophysical properties to the nucleosomes.Here we use genetic and genomic strategies to evaluate the role of H2A.W in transposon silencing in Arabidopsis. Compared with mutants lacking either H2A.W or H3K9me, the combined loss of both H2A.W and H3K9me causes a dramatic increase in both the number of expressed TEs and their expression levels. Synergistic effects are also observed when H2A.W is lost in combination with histone H1 or CH methylation. Collectively, these TEs are also upregulated in mutants lacking DDM1, which are impaired in H2A.W deposition and lose heterochromatic marks.We conclude that H2A.W acts in combination with different elements of heterochromatin to maintain silencing across a large spectrum of TEs present primarily in pericentric heterochromatin in Arabidopsis. In mammals, the DDM1 ortholog LSH deposits macroH2A to heterochromatin and silences TEs. We thus propose that specialized H2A variants localized to heterochromatin interact with a complex array of histone modifications to silence TEs in eukaryotes.

In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create a h2a.w null mutant via CRISPR-Cas9, h2a.w-2 , to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.

Selection of C-terminal motifs participated in evolution of distinct histone H2A variants. Hybrid types of variants combining motifs from distinct H2A classes are extremely rare. This suggests that the proximity between the motif cases interferes with their function. We studied this question in flowering plants that evolved sporadically a hybrid H2A variant combining the SQ motif of H2A.X that participates in the DNA damage response with the KSPK motif of H2A.W that stabilizes heterochromatin. Our inventory of PTMs of H2A.W variants showed that in vivo the cell cycle-dependent kinase CDKA phosphorylates the KSPK motif of H2A.W but only in absence of an SQ motif. Phosphomimicry of KSPK prevented DNA damage response by the SQ motif of the hybrid H2A.W/X variant. In a synthetic yeast expressing the hybrid H2A.W/X variant, phosphorylation of KSPK prevented binding of the BRCT-domain protein Mdb1 to phosphorylated SQ and impaired response to DNA damage. Our findings illustrate that PTMs mediate interference between the function of H2A variant specific C-terminal motifs. Such interference could explain the mutual exclusion of motifs that led to evolution of H2A variants.

Background Chromatin organizes DNA and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent, while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. Results We isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome-wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, the release of heterochromatin silencing in POL2A-deficient mutants paradoxically occurs in a chromatin context of increased levels of these two repressive epigenetic marks. At the nuclear level, the POL2A defect is associated with fragmentation of heterochromatin. Conclusion These results indicate that POL2A is critical to heterochromatin structure and function, and that unhindered replisome progression is required for the faithful propagation of DNA methylation throughout the cell cycle.

In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, including DNA methylation and H3K9 methylation. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. To analyze the in vivo function of H2A.W, we created a h2a.w null mutant via CRISPR-Cas9, h2a.w-2. We found that H2A.W is not essential for plant development, and that loss of H2A.W did not perturb histone methylation patterns. In contrast, we found a reduction of non-CG DNA methylation in pericentromeric heterochromatin and an increase in DNA methylation at euchromatic sites targeted by the RNA-directed DNA methylation pathway. Loss of DNA methylation in h2a.w-2 correlated with both increased H1 occupancy and decreased DNA accessibility at heterochromatin. Our results indicate that H2A.W helps stabilize the accessibility of heterochromatin and facilitates efficient DNA methylation by fine tuning the genomic distribution of H1.

Background Chromatin organizes the DNA molecule and regulates its transcriptional activity through epigenetic modifications. Heterochromatic regions of the genome are generally transcriptionally silent while euchromatin is more prone to transcription. During DNA replication, both genetic information and chromatin modifications must be faithfully passed on to daughter strands. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. Results We isolate a strong hypomorphic Arabidopsis thaliana mutant of the POL2A catalytic subunit of DNA polymerase epsilon and show that POL2A is required to stabilize heterochromatin silencing genome wide, likely by preventing replicative stress. We reveal that POL2A inhibits DNA methylation and histone H3 lysine 9 methylation. Hence, release of heterochromatin silencing in POL2A deficient mutants paradoxically occurs in a chromatin context of increased level of these two repressive epigenetic marks. At the nuclear level, POL2A defect is associated with fragmentation of heterochromatin. Conclusion These results indicate that POL2A is critical to secure both heterochromatin structure and function. We also reveal that unhindered replisome progression is required for the faithful propagation of DNA methylation through the cell cycle. ### Competing Interest Statement The authors have declared no competing interest.

Constitutive heterochromatin is associated with repressive epigenetic modifications of histones and DNA which silence transcription. Yet, particular mutations or environmental changes can destabilize heterochromatin-associated silencing without noticeable changes in repressive epigenetic marks. Factors allowing transcription in this nonpermissive chromatin context remain poorly known. Here, we show that the transcription factor IIH component UVH6 and the mediator subunit MED14 are both required for heat stress-induced transcriptional changes and release of heterochromatin transcriptional silencing in Arabidopsis thaliana. We find that MED14, but not UVH6, is required for transcription when heterochromatin silencing is destabilized in the absence of stress through mutating the MOM1 silencing factor. In this case, our results raise the possibility that transcription dependency over MED14 might require intact patterns of repressive epigenetic marks. We also uncover that MED14 regulates DNA methylation in non-CG contexts at a subset of RNA-directed DNA methylation target loci. These findings provide insight into the control of heterochromatin transcription upon silencing destabilization and identify MED14 as a regulator of DNA methylation.

Transposable elements (TEs) are prevalent in most eukaryotes, and host genomes have devised silencing strategies to rein in TE activity. One of these, transcriptional silencing, is generally associated with DNA methylation and short interfering RNAs. Here we show that the Arabidopsis genes MAIL1 and MAIN define an alternative silencing pathway independent of DNA methylation and short interfering RNAs. Mutants for MAIL1 or MAIN exhibit release of silencing and appear to show impaired condensation of pericentromeric heterochromatin. Phylogenetic analysis suggests not only that MAIL1 and MAIN encode a retrotransposon-related plant mobile domain, but also that host plant mobile domains were captured by DNA transposons during plant evolution. Our results reveal a role for Arabidopsis proteins with a transposon-related domain in gene silencing.

Le silencing transcriptionnel limite la transcription des genes et des elements transposables dont l’expression pourrait etre deletere a la cellule. Il depend d’une diversite de modifications de la chromatine comme la methylation ADN ou les marques repressives des histones. De facon a mieux comprendre les mecanismes moleculaires a l’origine du silencing transcriptionnel, nous avons mene une approche de genetique directe a l’aide d’un transgene soumis au silencing dans la plante modele Arabidopsis thaliana. Cette strategie nous a permis d'isoler a la fois des mutants deficients pour le maintien du silencing transcriptionnel et des mutations qui empechent la reactivation transcriptionnelle des elements transposables en reponse a un stress thermique. Nous avons caracterise les defauts provoques par ces mutations en combinant des approches de biologie moleculaire, de cytologie et de genomique.Nous montrons ainsi que MED14, la sous-unite centrale du complexe Mediator, et UVH6, composant du complexe TFIIH, sont requis pour la transcription de l'heterochromatine en stress thermique. MED14 stimule aussi la transcription de l'heterochromatine en l'absence de stress, mais ne semble fonctionner qu'en presence de la methylation ADN. En plus de cette fonction originale, nous identifions un nouveau role de MED14 dans le maintien de la methylation ADN, possiblement via la voie de methylation ADN dirigee par les petits ARN.Par ailleurs, nos resultats nous ont permis d’identifier le role des proteines MAIN et MAIL1, qui definissent une voie de silencing transcriptionnelle independante des voies connues jusqu'alors. De facon interessante, MAIN et MAIL1 possedent un domaine proteique partage avec les elements transposables, qui aurait successivement ete capture par les elements transposables et leur hote au cours de l’histoire evolutive des plantes a fleurs.Enfin, en isolant une nouvelle mutation du gene POL2A, nous confirmons le role de l’ADN polymerase epsilon dans le silencing transcriptionnel et caracterisons les proprietes chromatiniennes qui dependent de POL2A. Nous montrons que les defauts de silencing des mutants pol2a correlent avec une desorganisation importante de l’heterochromatine sans diminution drastique des marques qui y sont associees. Au contraire, nous detectons une hypermethylation ADN prononcee dans le mutant, et explorons differentes hypotheses pour expliquer ce phenotype particulier. Nos donnees suggerent que plusieurs mecanismes moleculaires sont a l’origine des defauts des mutants pol2a. Elles confirment le role preponderant de la chromomethylase CMT3 dans la regulation de la methylation ADN, et suggerent qu’un stress replicatif pourrait causer une hypermethylation de l’ADN.Dans l’ensemble, ces travaux de these proposent des pistes de travail dont l’exploration pourrait permettre d’expliquer les effets des deficiences replicatives dans le maintien du silencing transcriptionnel et de l’homeostasie de la methylation ADN. Ils suggerent en outre que MED14 a une fonction dediee a la transcription de l’heterochromatine qui pourrait stimuler le maintien de la methylation ADN.