KU70 and CAF-1 in Arabidopsis: Divergent roles in rDNA stability and telomere homeostasis.

Deficiency in chromatin assembly factor-1 (CAF-1) in plants through dysfunction of its components, FASCIATA1 and 2 (FAS1, FAS2), leads to the specific and progressive loss of rDNA and telomere repeats in plants. This loss is attributed to defective repair mechanisms for the increased DNA breaks encountered during replication, a consequence of impaired replication-dependent chromatin assembly. In this study, we explore the role of KU70 in these processes. Our findings reveal that, although the rDNA copy number is reduced in ku70 mutants when compared with wild-type plants, it is not markedly affected by diverse KU70 status in fas1 mutants. This is consistent with our previous characterisation of rDNA loss in fas mutants as a consequence part of the single-strand annealing pathway of homology-dependent repair. In stark contrast to rDNA, KU70 dysfunction fully suppresses the loss of telomeres in fas1 plants and converts telomeres to their elongated and heterogeneous state typical for ku70 plants. We conclude that the alternative telomere lengthening pathway, known to be activated in the absence of KU70, overrides progressive telomere loss due to CAF-1 dysfunction.

Chromatin during plant regeneration: Opening towards root identity?

Plants show exceptional developmental plasticity and the ability to reprogram cell identities during regeneration. Although regeneration has been used in plant propagation for decades, we only recently gained detailed cellular and molecular insights into this process. Evidently, not all cell types have the same regeneration potential, and only a subset of regeneration-competent cells reach pluripotency. Pluripotent cells exhibit transcriptional similarity to root stem cells. In different plant regeneration systems, transcriptional reprogramming involves transient release of chromatin repression during pluripotency establishment and its restoration during organ or embryo differentiation. Incomplete resetting of the epigenome leads to somaclonal variation in regenerated plants. As single-cell technologies advance, we expect novel, exciting insights into epigenome dynamics during the establishment of pluripotency.

Phylogenetic profiling resolves early emergence of PRC2 and illuminates its functional core.

Polycomb repressive complex 2 (PRC2) is involved in maintaining transcriptionally silent chromatin states through methylating lysine 27 of histone H3 by the catalytic subunit enhancer of zeste [E(z)]. Here, we report the diversity of PRC2 core subunit proteins in different eukaryotic supergroups with emphasis on the early-diverged lineages and explore the molecular evolution of PRC2 subunits by phylogenetics. For the first time, we identify the putative ortholog of E(z) in Discoba, a lineage hypothetically proximal to the eukaryotic root, strongly supporting emergence of PRC2 before the diversification of eukaryotes. Analyzing 283 species, we robustly detect a common presence of E(z) and ESC, indicating a conserved functional core. Full-length Su(z)12 orthologs were identified in some lineages and species only, indicating, nonexclusively, high divergence of VEFS-Box-containing Su(z)12-like proteins, functional convergence of sequence-unrelated proteins, or Su(z)12 dispensability. Our results trace E(z) evolution within the SET-domain protein family, proposing a substrate specificity shift during E(z) evolution based on SET-domain and H3 histone interaction prediction.

Polycomb Repressive Complex 2 in Eukaryotes-An Evolutionary Perspective.

Polycomb repressive complex 2 (PRC2) represents a group of evolutionarily conserved multi-subunit complexes that repress gene transcription by introducing trimethylation of lysine 27 on histone 3 (H3K27me3). PRC2 activity is of key importance for cell identity specification and developmental phase transitions in animals and plants. The composition, biochemistry, and developmental function of PRC2 in animal and flowering plant model species are relatively well described. Recent evidence demonstrates the presence of PRC2 complexes in various eukaryotic supergroups, suggesting conservation of the complex and its function. Here, we provide an overview of the current understanding of PRC2-mediated repression in different representatives of eukaryotic supergroups with a focus on the green lineage. By comparison of PRC2 in different eukaryotes, we highlight the possible common and diverged features suggesting evolutionary implications and outline emerging questions and directions for future research of polycomb repression and its evolution.

Plant chromatin, metabolism and development - an intricate crosstalk.

Chromatin structure influences DNA accessibility and underlying gene expression. Disturbances of chromatin structure often result in pleiotropic developmental phenotypes. Interactions between chromatin modifications and development have been the main focus of epigenetic studies. Recent years brought major advance in uncovering and understanding connections between chromatin organisation in the nucleus and metabolic processes that take place in the cytoplasm or other cellular compartments. Products of primary metabolism and cell redox states influence chromatin-modifying complexes, and chromatin modifiers in turn affect expression of metabolic genes. Current evidence indicates that complex interaction loops between these biological system layers exist. Applying interdisciplinary and holistic approaches will decipher causality and molecular mechanisms of the dynamic crosstalk between chromatin structure, metabolism and plant growth and development.

Tidying-up the plant nuclear space: domains, functions, and dynamics.

Understanding how the packaging of chromatin in the nucleus is regulated and organized to guide complex cellular and developmental programmes, as well as responses to environmental cues is a major question in biology. Technological advances have allowed remarkable progress within this field over the last years. However, we still know very little about how the 3D genome organization within the cell nucleus contributes to the regulation of gene expression. The nuclear space is compartmentalized in several domains such as the nucleolus, chromocentres, telomeres, protein bodies, and the nuclear periphery without the presence of a membrane around these domains. The role of these domains and their possible impact on nuclear activities is currently under intense investigation. In this review, we discuss new data from research in plants that clarify functional links between the organization of different nuclear domains and plant genome function with an emphasis on the potential of this organization for gene regulation.

Convergent evolution of complex genomic rearrangements in two fungal meiotic drive elements.

Meiotic drive is widespread in nature. The conflict it generates is expected to be an important motor for evolutionary change and innovation. In this study, we investigated the genomic consequences of two large multi-gene meiotic drive elements, Sk-2 and Sk-3, found in the filamentous ascomycete Neurospora intermedia. Using long-read sequencing, we generated the first complete and well-annotated genome assemblies of large, highly diverged, non-recombining regions associated with meiotic drive elements. Phylogenetic analysis shows that, even though Sk-2 and Sk-3 are located in the same chromosomal region, they do not form sister clades, suggesting independent origins or at least a long evolutionary separation. We conclude that they have in a convergent manner accumulated similar patterns of tandem inversions and dense repeat clusters, presumably in response to similar needs to create linkage between genes causing drive and resistance.

Transgenerational phenotype aggravation in CAF-1 mutants reveals parent-of-origin specific epigenetic inheritance.

Chromatin is assembled by histone chaperones such as chromatin assembly factor CAF-1. We had noticed that vigor of Arabidopsis thaliana CAF-1 mutants decreased over several generations. Because changes in mutant phenotype severity over generations are unusual, we asked how repeated selfing of Arabidopsis CAF-1 mutants affects phenotype severity. CAF-1 mutant plants of various generations were grown, and developmental phenotypes, transcriptomes and DNA cytosine-methylation profiles were compared quantitatively. Shoot- and root-related growth phenotypes were progressively more affected in successive generations of CAF-1 mutants. Early and late generations of the fasciata (fas)2-4 CAF-1 mutant displayed only limited changes in gene expression, of which increasing upregulation of plant defense-related genes reflects the transgenerational phenotype aggravation. Likewise, global DNA methylation in the sequence context CHG but not CG or CHH (where H = A, T or C) changed over generations in fas2-4. Crossing early and late generation fas2-4 plants established that the maternal contribution to the phenotype severity exceeds the paternal contribution. Together, epigenetic rather than genetic mechanisms underlie the progressive developmental phenotype aggravation in the Arabidopsis CAF-1 mutants and preferred maternal transmission reveals a more efficient reprogramming of epigenetic information in the male than the female germline.

Arabidopsis Chromatin Assembly Factor 1 is required for occupancy and position of a subset of nucleosomes.

Chromatin Assembly Factor 1 (CAF-1) is a major nucleosome assembly complex which functions particularly during DNA replication and repair. Here we studied how the nucleosome landscape changes in a CAF-1 mutant in the model plant Arabidopsis thaliana. Globally, most nucleosomes were not affected by loss of CAF-1, indicating the presence of efficient alternative nucleosome assemblers. Nucleosomes that we found depleted in the CAF-1 mutant were enriched in non-transcribed regions, consistent with the notion that CAF-1-independent nucleosome assembly can compensate for loss of CAF-1 mainly in transcribed regions. Depleted nucleosomes were particularly enriched in proximal promoters, suggesting that CAF-1-independent nucleosome assembly mechanisms are often not efficient upstream of transcription start sites. Genes related to plant defense were particularly prone to lose nucleosomes in their promoters upon CAF-1 depletion. Reduced nucleosome occupancy at promoters of many defense-related genes is associated with a primed gene expression state that may considerably increase plant fitness by facilitating plant defense. Together, our results establish that the nucleosome landscape in Arabidopsis is surprisingly robust even in the absence of the dedicated nucleosome assembly machinery CAF-1 and that CAF-1-independent nucleosome assembly mechanisms are less efficient in particular genome regions.

PRC2 Represses Hormone-Induced Somatic Embryogenesis in Vegetative Tissue of Arabidopsis thaliana.

Many plant cells can be reprogrammed into a pluripotent state that allows ectopic organ development. Inducing totipotent states to stimulate somatic embryo (SE) development is, however, challenging due to insufficient understanding of molecular barriers that prevent somatic cell dedifferentiation. Here we show that Polycomb repressive complex 2 (PRC2)-activity imposes a barrier to hormone-mediated transcriptional reprogramming towards somatic embryogenesis in vegetative tissue of Arabidopsis thaliana. We identify factors that enable SE development in PRC2-depleted shoot and root tissue and demonstrate that the establishment of embryogenic potential is marked by ectopic co-activation of crucial developmental regulators that specify shoot, root and embryo identity. Using inducible activation of PRC2 in PRC2-depleted cells, we demonstrate that transient reduction of PRC2 activity is sufficient for SE formation. We suggest that modulation of PRC2 activity in plant vegetative tissue combined with targeted activation of developmental pathways will open possibilities for novel approaches to cell reprogramming.

Variation of 45S rDNA intergenic spacers in Arabidopsis thaliana.

Approximately seven hundred 45S rRNA genes (rDNA) in the Arabidopsis thaliana genome are organised in two 4 Mbp-long arrays of tandem repeats arranged in head-to-tail fashion separated by an intergenic spacer (IGS). These arrays make up 5 % of the A. thaliana genome. IGS are rapidly evolving sequences and frequent rearrangements inside the rDNA loci have generated considerable interspecific and even intra-individual variability which allows to distinguish among otherwise highly conserved rRNA genes. The IGS has not been comprehensively described despite its potential importance in regulation of rDNA transcription and replication. Here we describe the detailed sequence variation in the complete IGS of A. thaliana WT plants and provide the reference/consensus IGS sequence, as well as genomic DNA analysis. We further investigate mutants dysfunctional in chromatin assembly factor-1 (CAF-1) (fas1 and fas2 mutants), which are known to have a reduced number of rDNA copies, and plant lines with restored CAF-1 function (segregated from a fas1xfas2 genetic background) showing major rDNA rearrangements. The systematic rDNA loss in CAF-1 mutants leads to the decreased variability of the IGS and to the occurrence of distinct IGS variants. We present for the first time a comprehensive and representative set of complete IGS sequences, obtained by conventional cloning and by Pacific Biosciences sequencing. Our data expands the knowledge of the A. thaliana IGS sequence arrangement and variability, which has not been available in full and in detail until now. This is also the first study combining IGS sequencing data with RFLP analysis of genomic DNA.

H2A deubiquitinases UBP12/13 are part of the Arabidopsis polycomb group protein system.

Polycomb group (PcG) proteins form an epigenetic memory system in plants and animals, but interacting proteins are poorly known in plants. Here, we have identified Arabidopsis UBIQUITIN SPECIFIC PROTEASES (USP; UBP in plant and yeasts) 12 and 13 as partners of the plant-specific PcG protein LIKE HETEROCHROMATIN PROTEIN 1 (LHP1). UBP12 binds to chromatin of PcG target genes and is required for histone H3 lysine 27 trimethylation and repression of a subset of PcG target genes. Plants lacking UBP12 and UBP13 developed autonomous endosperm in the absence of fertilization. We have identified UBP12 and UBP13 as new proteins in the plant PcG regulatory network. UBP12 and UBP13 belong to an ancient gene family and represent plant homologues of metazoan USP7. We have found that Drosophila USP7 shares a function in heterochromatic gene repression with UBP12/13 and their homologue UBP26. In summary, we demonstrate that USP7-like proteins are essential for gene silencing in diverse genomic contexts.

Phenotypic reversion in fas mutants of Arabidopsis thaliana by reintroduction of FAS genes: variable recovery of telomeres with major spatial rearrangements and transcriptional reprogramming of 45S rDNA genes.

Arabidopsis thaliana mutants dysfunctional in the evolutionarily conserved protein complex chromatin assembly factor-1 (CAF-1), which deposits the canonical histone H3 variant H3.1 during DNA synthesis-dependent chromatin assembly, display complex phenotypic changes including meristem and growth alterations, sensitivity to DNA-damaging agents, and reduced fertility. We reported previously that mutants in the FAS1 subunit of CAF-1 progressively lose telomere and 45S rDNA repeats. Here we show that multiple aspects of the fas phenotype are recovered immediately on expression of a reintroduced FAS1 allele, and are clearly independent of the recovery of rDNA copy-numbers and telomeres. In reverted lines, 45S rDNA genes are recovered to diverse levels with a strikingly different representation of their variants, and the typical association of nucleolar organizing region 4 with the nucleolus is perturbed. One of 45S rDNA variants (VAR1), which is silenced in wild-type (WT) plants without mutation history (Col-0 WT), dominates the expression pattern, whereas VAR2 is dominant in Col-0 WT plants. We propose an explanation for the variability of telomere and 45S rDNA repeats associated with CAF-1 function, suggesting that the differences in nuclear partitioning and expression of the rDNA variants in fas mutants and their revertants provide a useful experimental system to study genetic and epigenetic factors in gene dosage compensation.

DNA-sequence-specific erasers of epigenetic memory.

How epigenetic regulators find their specific targets remains a challenging question. Two parallel studies show that REF6, a plant H3K27me3 demethylase, binds a specific DNA motif via its zinc-finger domains and recruits the SWI/SNF-type ATPase BRAHMA, demonstrating a sequence-specific recruitment mechanism for a chromatin-modifying complex.