Molecular basis of chromatin remodelling by DDM1 involved in plant DNA methylation.

Eukaryotic gene regulation occurs at the chromatin level, which requires changing the chromatin structure by a group of ATP-dependent DNA translocases-namely, the chromatin remodellers1. In plants, chromatin remodellers function in various biological processes and possess both conserved and plant-specific components2-5. DECREASE IN DNA METHYLATION 1 (DDM1) is a plant chromatin remodeller that plays a key role in the maintenance DNA methylation6-11. Here we determined the structures of Arabidopsis DDM1 in complex with nucleosome in ADP-BeFx-bound, ADP-bound and nucleotide-free conformations. We show that DDM1 specifically recognizes the H4 tail and nucleosomal DNA. The conformational differences between ADP-BeFx-bound, ADP-bound and nucleotide-free DDM1 suggest a chromatin remodelling cycle coupled to ATP binding, hydrolysis and ADP release. This, in turn, triggers conformational changes in the DDM1-bound nucleosomal DNA, which alters the nucleosome structure and promotes DNA sliding. Together, our data reveal the molecular basis of chromatin remodelling by DDM1.

Molecular mechanisms of the RNA polymerases in plant RNA-directed DNA methylation.

In plants, two atypical DNA-dependent RNA polymerases, RNA polymerase IV (Pol IV) and Pol V, and an RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) together produce noncoding RNAs (ncRNAs) to guide the plant-specific RNA-directed DNA methylation (RdDM). Although both Pol IV and Pol V have evolved from the canonical Pol II, they have adapted to different roles in RdDM. The mechanisms of their adaptation are key to understanding plant DNA methylation and the divergent evolution of polymerases. In this review, we summarize insights that have emerged from recent structural studies of Pol IV, Pol V, and RDR2 and discuss their structural features critical for efficient ncRNA production in RdDM.

Structure and mechanism of the plant RNA polymerase V.

In addition to the conserved RNA polymerases I to III (Pols I to III) in eukaryotes, two atypical polymerases, Pols IV and V, specifically produce noncoding RNA in the RNA-directed DNA methylation pathway in plants. Here, we report on the structures of cauliflower Pol V in the free and elongation conformations. A conserved tyrosine residue of NRPE2 stacks with a double-stranded DNA branch of the transcription bubble to potentially attenuate elongation by inducing transcription stalling. The nontemplate DNA strand is captured by NRPE2 to enhance backtracking, thereby increasing 3'-5' cleavage, which likely underpins Pol V's high fidelity. The structures also illuminate the mechanism of Pol V transcription stalling and enhanced backtracking, which may be important for Pol V's retention on chromatin to serve its function in tethering downstream factors for RNA-directed DNA methylation.

Molecular basis of the plant ROS1-mediated active DNA demethylation.

Active DNA demethylation plays a crucial role in eukaryotic gene imprinting and antagonizing DNA methylation. The plant-specific REPRESSOR OF SILENCING 1/DEMETER (ROS1/DME) family of enzymes directly excise 5-methyl-cytosine (5mC), representing an efficient DNA demethylation pathway distinct from that of animals. Here, we report the cryo-electron microscopy structures of an Arabidopsis ROS1 catalytic fragment in complex with substrate DNA, mismatch DNA and reaction intermediate, respectively. The substrate 5mC is flipped-out from the DNA duplex and subsequently recognized by the ROS1 base-binding pocket through hydrophobic and hydrogen-bonding interactions towards the 5-methyl group and Watson-Crick edge respectively, while the different protonation states of the bases determine the substrate preference for 5mC over T:G mismatch. Together with the structure of the reaction intermediate complex, our structural and biochemical studies revealed the molecular basis for substrate specificity, as well as the reaction mechanism underlying 5mC demethylation by the ROS1/DME family of plant-specific DNA demethylases.

Molecular basis of locus-specific H3K9 methylation catalyzed by SUVH6 in plants.

Dimethylated histone H3 Lys9 (H3K9me2) is a conserved heterochromatic mark catalyzed by SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG (SUVH) methyltransferases in plants. However, the mechanism underlying the locus specificity of SUVH enzymes has long been elusive. Here, we show that a conserved N-terminal motif is essential for SUVH6-mediated H3K9me2 deposition in planta. The SUVH6 N-terminal peptide can be recognized by the bromo-adjacent homology (BAH) domain of the RNA- and chromatin-binding protein ANTI-SILENCING 1 (ASI1), which has been shown to function in a complex to confer gene expression regulation. Structural data indicate that a classic aromatic cage of ASI1-BAH domain specifically recognizes an arginine residue of SUVH6 through extensive hydrogen bonding interactions. A classic aromatic cage of ASI1 specifically recognizes an arginine residue of SUVH6 through extensive cation-π interactions, playing a key role in recognition. The SUVH6-ASI1 module confers locus-specific H3K9me2 deposition at most SUVH6 target loci and gives rise to distinct regulation of gene expression depending on the target loci, either conferring transcriptional silencing or posttranscriptional processing of mRNA. More importantly, such mechanism is conserved in multiple plant species, indicating a coordinated evolutionary process between SUVH6 and ASI1. In summary, our findings uncover a conserved mechanism for the locus specificity of H3K9 methylation in planta. These findings provide mechanistic insights into the delicate regulation of H3K9 methylation homeostasis in plants.

Structure and Mechanism of Plant DNA Methyltransferases.

DNA methylation is an important epigenetic mark conserved in eukaryotes from fungi to animals and plants, where it plays a crucial role in regulating gene expression and transposon silencing. Once the methylation mark is established by de novo DNA methyltransferases, specific regulatory mechanisms are required to maintain the methylation state during chromatin replication, both during meiosis and mitosis. Plant DNA methylation is found in three contexts; CG, CHG, and CHH (H = A, T, C), which are established and maintained by a unique set of DNA methyltransferases and are regulated by plant-specific pathways. DNA methylation in plants is often associated with other epigenetic modifications, such as noncoding RNA and histone modifications. This chapter focuses on the structure, function, and regulatory mechanism of plant DNA methyltransferases and their crosstalk with other epigenetic pathways.

The H3K9me2-binding protein AGDP3 limits DNA methylation and transcriptional gene silencing in Arabidopsis.

DNA methylation, a conserved epigenetic mark, is critical for tuning temporal and spatial gene expression. The Arabidopsis thaliana DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1) initiates active DNA demethylation and is required to prevent DNA hypermethylation at thousands of genomic loci. However, how ROS1 is recruited to specific loci is not well understood. Here, we report the discovery of Arabidopsis AGENET Domain Containing Protein 3 (AGDP3) as a cellular factor that is required to prevent gene silencing and DNA hypermethylation. AGDP3 binds to H3K9me2 marks in its target DNA via its AGD12 cassette. Analysis of the crystal structure of the AGD12 cassette of AGDP3 in complex with an H3K9me2 peptide revealed that dimethylated H3K9 and unmodified H3K4 are specifically anchored into two different surface pockets. A histidine residue located in the methyllysine binding aromatic cage provides AGDP3 with pH-dependent H3K9me2 binding capacity. Our results uncover a molecular mechanism for the regulation of DNA demethylation by the gene silencing mark H3K9me2.

Dysfunction of histone demethylase IBM1 in Arabidopsis causes autoimmunity and reshapes the root microbiome.

Root microbiota is important for plant growth and fitness. Little is known about whether and how the assembly of root microbiota may be controlled by epigenetic regulation, which is crucial for gene transcription and genome stability. Here we show that dysfunction of the histone demethylase IBM1 (INCREASE IN BONSAI METHYLATION 1) in Arabidopsis thaliana substantially reshaped the root microbiota, with the majority of the significant amplicon sequence variants (ASVs) being decreased. Transcriptome analyses of plants grown in soil and in sterile growth medium jointly disclosed salicylic acid (SA)-mediated autoimmunity and production of the defense metabolite camalexin in the ibm1 mutants. Analyses of genome-wide histone modifications and DNA methylation highlighted epigenetic modifications permissive for transcription at several important defense regulators. Consistently, ibm1 mutants showed increased resistance to the pathogen Pseudomonas syringae DC3000 with stronger immune responses. In addition, ibm1 showed substantially impaired plant growth promotion in response to beneficial bacteria; the impairment was partially mimicked by exogenous application of SA to wild-type plants, and by a null mutation of AGP19 that is important for cell expansion and that is repressed with DNA hypermethylation in ibm1. IBM1-dependent epigenetic regulation imposes strong and broad impacts on plant-microbe interactions and thereby shapes the assembly of root microbiota.

A SYBR Gold-based Label-free in vitro Dicing Assay.

In Arabidopsis, DICER-LIKE PROTEIN 3 (DCL3) cuts the substrate pre-siRNA into a product siRNA duplex, encompassing one 23-nt strand and one 24-nt strand. To monitor the separation of the siRNA duplex with only 1-nt difference, we developed this protocol to evaluate the in vitro dicing activity of DCL3. The method can be applied for measuring the lengths of single-stranded RNA separated through denaturing urea polyacrylamide gel electrophoresis (urea PAGE), which are visualized by a label-free fluorescence SYBR Gold, and quantified in a multi-function imager. This label-free method is easy to conduct, has low cost, and lacks the hazard of the traditional radio-labeled method. This method can also be adapted to the other Dicers and small RNAs.

Progressive chromatin silencing of ABA biosynthesis genes permits seed germination in Arabidopsis.

Seed germination represents a major developmental switch in plants that is vital to agriculture, but how this process is controlled at the chromatin level remains obscure. Here we demonstrate that successful germination in Arabidopsis thaliana requires a chromatin mechanism that progressively silences 9-CIS-EPOXYCAROTENOID DIOXYGENASE 6 (NCED6), which encodes a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, through the cooperative action of the RNA-binding protein RZ-1 and the polycomb repressive complex 2 (PRC2). Simultaneous inactivation of RZ-1 and PRC2 blocked germination and synergistically derepressed NCEDs and hundreds of genes. At NCED6, in part by promoting H3 deacetylation and suppressing H3K4me3, RZ-1 facilitates transcriptional silencing and also an H3K27me3 accumulation process that occurs during seed germination and early seedling growth. Genome-wide analysis revealed that RZ-1 is preferentially required for transcriptional silencing of many PRC2 targets early during seed germination, when H3K27me3 is not yet established. We propose RZ-1 confers a novel silencing mechanism to compensate for and synergize with PRC2. Our work highlights the progressive chromatin silencing of ABA biosynthesis genes via the RNA-binding protein RZ-1 and PRC2 acting in synergy, a process that is vital for seed germination.

Structure and mechanism of histone methylation dynamics in Arabidopsis.

Histone methylation plays a central role in regulating chromatin state and gene expression in Arabidopsis and is involved in a variety of physiological and developmental processes. Dynamic regulation of histone methylation relies on both histone methyltransferase "writer" and histone demethylases "eraser" proteins. In this review, we focus on the four major histone methylation modifications in Arabidopsis H3, H3K4, H3K9, H3K27, and H3K36, and summarize current knowledge of the dynamic regulation of these modifications, with an emphasis on the biochemical and structural perspectives of histone methyltransferases and demethylases.

Structure of plant RNA-DEPENDENT RNA POLYMERASE 2, an enzyme involved in small interfering RNA production.

In plants, the biogenesis of small interfering RNA (siRNA) requires a family of RNA-dependent RNA polymerases that convert single-stranded RNA (ssRNA) into double-stranded RNA (dsRNA), which is subsequently cleaved into defined lengths by Dicer endonucleases. Here, we determined the structure of maize (Zea mays) RNA-DEPENDENT RNA POLYMERASE 2 (ZmRDR2) in the closed and open conformations. The core catalytic region of ZmRDR2 possesses the canonical DNA-dependent RNA polymerase (DdRP) catalytic sites, pointing to a shared RNA production mechanism between DdRPs and plant RDR-family proteins. Apo-ZmRDR2 adopts a highly compact structure, representing an inactive closed conformation. By contrast, adding RNA induced a significant conformational change in the ZmRDR2 Head domain that opened the RNA binding tunnel, suggesting this is an active elongation conformation of ZmRDR2. Overall, our structural studies trapped both the active and inactive conformations of ZmRDR2, providing insights into the molecular mechanism of dsRNA synthesis during plant siRNA production.

Molecular basis of CONSTANS oligomerization in FLOWERING LOCUS T activation.

The transcription factor CONSTANS (CO) integrates day-length information to induce the expression of florigen FLOWERING LOCUS T (FT) in Arabidopsis. We recently reported that the C-terminal CCT domain of CO forms a complex with NUCLEAR FACTOR-YB/YC to recognize multiple cis-elements in the FT promoter, and the N-terminal tandem B-box domains form a homomultimeric assembly. However, the mechanism and biological function of CO multimerization remained unclear. Here, we report that CO takes on a head-to-tail oligomeric configuration via its B-boxes to mediate FT activation in long days. The crystal structure of B-boxesCO reveals a closely connected tandem B-box fold forming a continuous head-to-tail assembly through unique CDHH zinc fingers. Mutating the key residues involved in CO oligomerization resulted in a non-functional CO, as evidenced by the inability to rescue co mutants. By contrast, a transgene encoding a human p53-derived tetrameric peptide in place of the B-boxesCO rescued co mutant, emphasizing the essential role of B-boxesCO -mediated oligomerization. Furthermore, we found that the four TGTG-bearing cis-elements in FT proximal promoter are required for FT activation in long days. Our results suggest that CO forms a multimer to bind to the four TGTG motifs in the FT promoter to mediate FT activation.

Acetylproteomics analyses reveal critical features of lysine-ε-acetylation in Arabidopsis and a role of 14-3-3 protein acetylation in alkaline response.

Lysine-ε-acetylation (Kac) is a post-translational modification (PTM) that is critical for metabolic regulation and cell signaling in mammals. However, its prevalence and importance in plants remain to be determined. Employing high-resolution tandem mass spectrometry, we analyzed protein lysine acetylation in five representative Arabidopsis organs with 2 ~ 3 biological replicates per organ. A total of 2887 Kac proteins and 5929 Kac sites were identified. This comprehensive catalog allows us to analyze proteome-wide features of lysine acetylation. We found that Kac proteins tend to be more uniformly expressed in different organs, and the acetylation status exhibits little correlation with the gene expression level, indicating that acetylation is unlikely caused by stochastic processes. Kac preferentially targets evolutionarily conserved proteins and lysine residues, but only a small percentage of Kac proteins are orthologous between rat and Arabidopsis. A large portion of Kac proteins overlap with proteins modified by other PTMs including ubiquitination, SUMOylation and phosphorylation. Although acetylation, ubiquitination and SUMOylation all modify lysine residues, our analyses show that they rarely target the same sites. In addition, we found that "reader" proteins for acetylation and phosphorylation, i.e., bromodomain-containing proteins and GRF (General Regulatory Factor)/14-3-3 proteins, are intensively modified by the two PTMs, suggesting that they are main crosstalk nodes between acetylation and phosphorylation signaling. Analyses of GRF6/14-3-3λ reveal that the Kac level of GRF6 is decreased under alkaline stress, suggesting that acetylation represses plant alkaline response. Indeed, K56ac of GRF6 inhibits its binding to and subsequent activation of the plasma membrane H+-ATPase AHA2, leading to hypersensitivity to alkaline stress. These results provide valuable resources for protein acetylation studies in plants and reveal that protein acetylation suppresses phosphorylation output by acetylating GRF/14-3-3 proteins.

Mechanism of siRNA production by a plant Dicer-RNA complex in dicing-competent conformation.

In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are a central part of sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nucleotide (nt) small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation pathway. Here, we determined the structure of a DCL3­pre-siRNA complex in an active dicing-competent state. The 5'-phosphorylated A1 of the guide strand and the 1-nt 3' overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5'-phosphorylated end of the guide RNA and dual cleavage sites formed by the paired ribonuclease III domains. These structural studies, complemented by functional data, provide insight into the dicing principle for Dicers in general.

TEM1 combinatorially binds to FLOWERING LOCUS T and recruits a Polycomb factor to repress the floral transition in Arabidopsis.

Arabidopsis TEMPRANILLO 1 (TEM1) is a transcriptional repressor that participates in multiple flowering pathways and negatively regulates the juvenile-to-adult transition and the flowering transition. To understand the molecular basis for the site-specific regulation of FLOWERING LOCUS T (FT) by TEM1, we determined the structures of the two plant-specific DNA-binding domains in TEM1, AP2 and B3, in complex with their target DNA sequences from the FT gene 5'-untranslated region (5'-UTR), revealing the molecular basis for TEM1 specificity for its DNA targets. In vitro binding assays revealed that the combination of the AP2 and B3 binding sites greatly enhanced the overall binding of TEM1 to the FT 5'-UTR, indicating TEM1 combinatorically recognizes the FT gene 5'-UTR. We further showed that TEM1 recruits the Polycomb repressive complex 2 (PRC2) to the FT 5'-UTR. The simultaneous binding of the TEM1 AP2 and B3 domains to FT is necessary for deposition of H3K27me3 at the FT 5'-UTR and for the flowering repressor function of TEM1. Overall, our data suggest that the combinatorial recognition of FT 5'-UTR by TEM1 ensures H3K27me3 deposition to precisely regulate the floral transition.