Identification of plants' functional counterpart of the metazoan mediator of DNA Damage checkpoint 1.

Induction of DNA damage triggers rapid phosphorylation of the histone H2A.X (γH2A.X). In animals, mediator of DNA damage checkpoint 1 (MDC1) binds γH2A.X through a tandem BRCA1 carboxyl-terminal (tBRCT) domain and mediates recruitment of downstream effectors of DNA damage response (DDR). However, readers of this modification in plants have remained elusive. We show that from the Arabidopsis BRCT domain proteome, BCP1-4 proteins with tBRCT domains are involved in DDR. Through its tBRCT domain BCP4 binds γH2A.X in vitro and localizes to DNA damage-induced foci in an H2A.X-dependent manner. BCP4 also contains a domain that interacts directly with NBS1 and thus acts as a functional counterpart of MDC1. We also show that BCP1, that contains two tBRCT domains, co-localizes with γH2A.X but it does not bind γH2A.X suggesting functional similarity with human PAXIP1. A phylogenetic analysis supports that PAXIP1 and MDC1 in metazoa and their plant counterparts evolved independently from common ancestors with tBRCT domains. Collectively, our study reveals missing components and provides mechanistic and evolutionary insights into plant DDR.

Histone variants shape chromatin states in Arabidopsis.

How different intrinsic sequence variations and regulatory modifications of histones combine in nucleosomes remain unclear. To test the importance of histone variants in the organization of chromatin we investigated how histone variants and histone modifications assemble in the Arabidopsis thaliana genome. We showed that a limited number of chromatin states divide euchromatin and heterochromatin into several subdomains. We found that histone variants are as significant as histone modifications in determining the composition of chromatin states. Particularly strong associations were observed between H2A variants and specific combinations of histone modifications. To study the role of H2A variants in organizing chromatin states we determined the role of the chromatin remodeler DECREASED IN DNA METHYLATION (DDM1) in the organization of chromatin states. We showed that the loss of DDM1 prevented the exchange of the histone variant H2A.Z to H2A.W in constitutive heterochromatin, resulting in significant effects on the definition and distribution of chromatin states in and outside of constitutive heterochromatin. We thus propose that dynamic exchanges of histone variants control the organization of histone modifications into chromatin states, acting as molecular landmarks.

Crosstalk between H2A variant-specific modifications impacts vital cell functions.

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.

The histone variant H2A.W and linker histone H1 co-regulate heterochromatin accessibility and DNA methylation.

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.

The chromatin remodeler DDM1 prevents transposon mobility through deposition of histone variant H2A.W.

Mobile transposable elements (TEs) not only participate in genome evolution but also threaten genome integrity. In healthy cells, TEs that encode all of the components that are necessary for their mobility are specifically silenced, yet the precise mechanism remains unknown. Here, we characterize the mechanism used by a conserved class of chromatin remodelers that prevent TE mobility. In the Arabidopsis chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1), we identify two conserved binding domains for the histone variant H2A.W, which marks plant heterochromatin. DDM1 is necessary and sufficient for the deposition of H2A.W onto potentially mobile TEs, yet does not act on TE fragments or host protein-coding genes. DDM1-mediated H2A.W deposition changes the properties of chromatin, resulting in the silencing of TEs and, therefore, prevents their mobility. This distinct mechanism provides insights into the interplay between TEs and their host in the contexts of evolution and disease, and potentiates innovative strategies for targeted gene silencing.

The evolution and functional divergence of the histone H2B family in plants.

Chromatin regulation of eukaryotic genomes depends on the formation of nucleosome complexes between histone proteins and DNA. Histone variants, which are diversified by sequence or expression pattern, can profoundly alter chromatin properties. While variants in histone H2A and H3 families are well characterized, the extent of diversification of histone H2B proteins is less understood. Here, we report a systematic analysis of the histone H2B family in plants, which have undergone substantial divergence during the evolution of each major group in the plant kingdom. By characterising Arabidopsis H2Bs, we substantiate this diversification and reveal potential functional specialization that parallels the phylogenetic structure of emergent clades in eudicots. In addition, we identify a new class of highly divergent H2B variants, H2B.S, that specifically accumulate during chromatin compaction of dry seed embryos in multiple species of flowering plants. Our findings thus identify unsuspected diverse properties among histone H2B proteins in plants that has manifested into potentially novel groups of histone variants.

Histone H2A variants confer specific properties to nucleosomes and impact on chromatin accessibility.

In eukaryotes, variants of core histone H2A are selectively incorporated in distinct functional domains of chromatin and are distinguished by conserved sequences of their C-terminal tail, the L1 loop and the docking domain, suggesting that each variant confers specific properties to the nucleosome. Chromatin of flowering plants contains four types of H2A variants, which biochemical properties have not been characterized. We report that in contrast with animals, in Arabidopsis thaliana H2A variants define only four major types of homotypic nucleosomes containing exclusively H2A, H2A.Z, H2A.X or H2A.W. In vitro assays show that the L1 loop and the docking domain confer distinct stability of the nucleosome. In vivo and in vitro assays suggest that the L1 loop and the docking domain cooperate with the C-terminal tail to regulate chromatin accessibility. Based on these findings we conclude that the type of H2A variant in the nucleosome impacts on its interaction with DNA and propose that H2A variants regulate the dynamics of chromatin accessibility. In plants, the predominance of homotypic nucleosomes with specific physical properties and their specific localization to distinct domains suggest that H2A variants play a dominant role in chromatin dynamics and function.

LHP1 Interacts with ATRX through Plant-Specific Domains at Specific Loci Targeted by PRC2.

Heterochromatin Protein 1 (HP1) is a major regulator of chromatin structure and function. In animals, the network of proteins interacting with HP1 is mainly associated with constitutive heterochromatin marked by H3K9me3. HP1 physically interacts with the putative ortholog of the SNF2 chromatin remodeler ATRX, which controls deposition of histone variant H3.3 in mammals. In this study, we show that the Arabidopsis thaliana ortholog of ATRX participates in H3.3 deposition and possesses specific conserved domains in plants. We found that plant Like HP1 (LHP1) protein interacts with ATRX through domains that evolved specifically in land plant ancestors. Loss of ATRX function in Arabidopsis affects the expression of a limited subset of genes controlled by PRC2 (POLYCOMB REPRESSIVE COMPLEX 2), including the flowering time regulator FLC. The function of ATRX in regulation of flowering time requires novel LHP1-interacting domain and ATPase activity of the ATRX SNF2 helicase domain. Taken together, these results suggest that distinct evolutionary pathways led to the interaction between ATRX and HP1 in mammals and its counterpart LHP1 in plants, resulting in distinct modes of transcriptional regulation.

Heterochromatin and DNA damage repair: Use different histone variants and relax.

Repair of damaged DNA requires the activation of kinases, which in turn phosphorylate diverse proteins including histone H2A.X, an event conserved from yeast to human. By combining genetics, biochemical, and cytological approaches, we recently reported that, in addition to H2A.X, phosphorylation of histone variant H2A.W.7 is required for DNA damage response in Arabidopsis. This work provides direct evidence for the functional diversification of plant-specific H2A.W histone variants, which are tightly associated with heterochromatin. We place our findings in perspective of other recent reports and discuss how DNA damage is being recognized and repaired in heterochromatin.

Compartmentalization of DNA Damage Response between Heterochromatin and Euchromatin Is Mediated by Distinct H2A Histone Variants.

DNA double-strand break (DSB) repair depends on the ataxia telangiectasia mutated (ATM) kinase that phosphorylates the conserved C-terminal SQ motif present in the histone variant H2A.X [1-7]. In constitutive heterochromatin of mammals, DSB repair is delayed and relies on phosphorylation of the proteins HP1 and KAP1 by ATM [2, 8-14]. However, KAP1 is not conserved in plants and the HP1-related protein Like-HP1 (LHP1) is not localized at constitutive heterochromatin [15], suggesting that in plants, alternative mechanisms could be responsible for repair of DSBs in heterochromatin. In Arabidopsis, constitutive heterochromatin is marked by H3K9 methylation and the plant-specific histone variants H2A.W, which are distinguished by their C-terminal motif KSPKK and required for heterochromatin compaction [16-18]. We report that the Arabidopsis histone variant H2A.W.7 is confined to heterochromatin and carries a SQ motif that is phosphorylated by ATM. In response to DNA damage, phosphorylation of H2A.W.7 takes place in heterochromatin, while H2A.X phosphorylation takes place primarily in euchromatin. We propose that H2A.W.7 evolved in addition to H2A.X to facilitate DNA damage response in highly condensed heterochromatin, thus playing a role similar to KAP1 and HP1 phosphorylation in mammals. These data support the idea of the functional diversification of histone variants and their role in spatial compartmentalization of chromatin-related functions in eukaryotes.

Diversification of histone H2A variants during plant evolution.

Among eukaryotes, the four core histones show an extremely high conservation of their structure and form nucleosomes that compact, protect, and regulate access to genetic information. Nevertheless, in multicellular eukaryotes the two families, histone H2A and histone H3, have diversified significantly in key residues. We present a phylogenetic analysis across the green plant lineage that reveals an early diversification of the H2A family in unicellular green algae and remarkable expansions of H2A variants in flowering plants. We define motifs and domains that differentiate plant H2A proteins into distinct variant classes. In non-flowering land plants, we identify a new class of H2A variants and propose their possible role in the emergence of the H2A.W variant class in flowering plants.

The ability to form homodimers is essential for RDM1 to function in RNA-directed DNA methylation.

RDM1 (RNA-DIRECTED DNA METHYLATION1) is a small plant-specific protein required for RNA-directed DNA methylation (RdDM). RDM1 interacts with RNA polymerase II (Pol II), ARGONAUTE4 (AGO4), and the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) and binds to methylated single stranded DNA. As the only protein identified so far that interacts directly with DRM2, RDM1 plays a pivotal role in the RdDM mechanism by linking the de novo DNA methyltransferase activity to AGO4, which binds short interfering RNAs (siRNAs) that presumably base-pair with Pol II or Pol V scaffold transcripts synthesized at target loci. RDM1 also acts together with the chromatin remodeler DEFECTIVE IN RNA-DIRECTED DNA METHYLATION1 (DRD1) and the structural-maintenance-of-chromosomes solo hinge protein DEFECTIVE IN MERISTEM SILENCING3 (DMS3) to form the DDR complex, which facilitates synthesis of Pol V scaffold transcripts. The manner in which RDM1 acts in both the DDR complex and as a factor bridging DRM2 and AGO4 remains unclear. RDM1 contains no known protein domains but a prior structural analysis suggested distinct regions that create a hydrophobic pocket and promote homodimer formation, respectively. We have tested several mutated forms of RDM1 altered in the predicted pocket and dimerization regions for their ability to complement defects in RdDM and transcriptional gene silencing, support synthesis of Pol V transcripts, form homodimers, and interact with DMS3. Our results indicate that the ability to form homodimers is essential for RDM1 to function fully in the RdDM pathway and may be particularly important during the de novo methylation step.

Interplay among RNA polymerases II, IV and V in RNA-directed DNA methylation at a low copy transgene locus in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is an epigenetic process whereby small interfering RNAs (siRNAs) guide cytosine methylation of homologous DNA sequences. RdDM requires two specialized RNA polymerases: Pol IV transcribes the siRNA precursor whereas Pol V generates scaffold RNAs that interact with siRNAs and attract the methylation machinery. Recent evidence also suggests the involvement of RNA polymerase II (Pol II) in recruiting Pol IV and Pol V to low copy, intergenic loci. We demonstrated previously that Pol V-mediated methylation at a transgene locus in Arabidopsis spreads downstream of the originally targeted region by means of Pol IV/RNA-DEPENDENT RNA POLYMERASE2 (RDR2)-dependent 24-nt secondary siRNAs. Here we show that these secondary siRNAs can not only induce methylation in cis but also in trans at an unlinked target site, provided this sequence is transcribed by Pol II to produce a non-coding RNA. The Pol II transcript appears to be important for amplification of siRNAs at the unlinked target site because its presence correlates not only with methylation but also with elevated levels of 24-nt siRNAs. Potential target sites that lack an overlapping Pol II transcript and remain unmethylated in the presence of trans-acting 24-nt siRNAs can nevertheless acquire methylation in the presence of 21-24-nt hairpin-derived siRNAs, suggesting that RdDM of non-transcribed target sequences requires multiple size classes of siRNA. Our findings demonstrate that Pol II transcripts are not always needed for RdDM at low copy loci but they may intensify RdDM by facilitating amplification of Pol IV-dependent siRNAs at the DNA target site.

MORC proteins and epigenetic regulation.

Two recent studies in Arabidopsis implicated MORC proteins, which contain a GHKL ATPase domain, in transcriptional gene silencing. Here, these studies are compared and contrasted to discuss the roles of MORC proteins in epigenetic regulation. Although MORC proteins are likely to catalyze changes in chromatin structure in response to epigenetic signals, their precise mode of action and target site-specificity still remain unclear.

Involvement of a GHKL ATPase in RNA-directed DNA methylation in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery comprising two RNA polymerase II-related RNA polymerases, called Pol IV and Pol V, as well as chromatin remodelers, transcription factors, and other novel proteins whose roles in the RdDM mechanism remain poorly understood. We have identified a new component of the RdDM machinery, DMS11 (defective in meristem silencing 11), which has a GHKL (gyrase, Hsp90, histidine kinase, MutL) ATPase domain. siRNAs accumulate in the dms11 mutant, and repressive epigenetic modifications undergo only modest reductions at target sequences. DMS11 interacts physically with another RdDM component, DMS3, an unusual structural maintenance of chromosomes (SMC) hinge domain-containing protein that lacks the ATPase motifs of authentic SMC proteins. The hinge region of DMS3 resembles that of the mammalian epigenetic factor SMCHD1, which also has a GHKL-type ATPase. In vitro, DMS11 has ATPase activity that is stimulated by DMS3. We suggest that DMS11 provides the missing ATPase function for DMS3 and that these proteins cooperate in the RdDM pathway to promote transcriptional repression. GHKL ATPases are thus emerging as new players in epigenetic regulation in plants and mammals.

AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

Genetic evidence that DNA methyltransferase DRM2 has a direct catalytic role in RNA-directed DNA methylation in Arabidopsis thaliana.

RNA-directed DNA methylation (RdDM) is a small RNA-mediated epigenetic modification in plants. We report here the identification of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) in a forward screen for mutants defective in RdDM in Arabidopsis thaliana. The finding of a mutation in the presumptive active site argues in favor of direct catalytic activity for DRM2.

An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation.

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

RNA-directed DNA methylation and plant development require an IWR1-type transcription factor.

RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase (Pol) II-related RNA polymerases, namely Pol IV and Pol V. A genetic screen designed to reveal factors that are important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype. The DMS4 protein is similar to yeast IWR1 (interacts with RNA polymerase II), a conserved putative transcription factor that interacts with Pol II subunits. The DMS4 complementary DNA partly complements the K1 killer toxin hypersensitivity of a yeast iwr1 mutant, suggesting some functional conservation. In the transgenic system studied, mutations in DMS4 directly or indirectly affect Pol IV-dependent secondary short interfering RNAs, Pol V-mediated RdDM, Pol V-dependent synthesis of intergenic non-coding RNA and expression of many Pol II-driven genes. These data suggest that DMS4 might be a regulatory factor for several RNA polymerases, thus explaining its diverse roles in the plant.

Role of plant RNA-binding proteins in development, stress response and genome organization.

RNA-binding proteins (RBPs) in eukaryotes have crucial roles in all aspects of post-transcriptional gene regulation. They are important governors of diverse developmental processes by modulating expression of specific transcripts. The Arabidopsis (Arabidopsis thaliana) genome encodes for more than 200 different RBPs, most of which are plant specific and are therefore likely to perform plant-specific functions. Indeed, recent identification and analysis of plant RBPs clearly showed that, in addition to the important role in diverse developmental processes, they are also involved in adaptation of plants to various environmental conditions. Clearly, they act by regulating pre-mRNA splicing, polyadenylation, RNA stability and RNA export, as well as by influencing chromatin modification.