H1 restricts euchromatin-associated methylation pathways from heterochromatic encroachment.

Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT DNA methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an h1 mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase, Pol V. Loss of H1 resulted in NRPE1 enrichment predominantly in heterochromatic TEs. Increased NRPE1 binding was associated with increased chromatin accessibility in h1, suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an h1/nrpe1 double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in h1 corresponded to the loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 similarly restricts the occupancy of the methylation reader, SUVH1, and polycomb-mediated H3K27me3. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.

H1 restricts euchromatin-associated methylation pathways from heterochromatic encroachment.

Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT DNA methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localized to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localization in an h1 mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase, Pol V. Loss of H1 resulted in NRPE1 enrichment predominantly in heterochromatic TEs. Increased NRPE1 binding was associated with increased chromatin accessibility in h1 , suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an h1/nrpe1 double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in h1 corresponded to loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposition correlated with increased methylation specifically in the CHH context. Additionally, we found that H1 similarly restricts the occupancy of the methylation reader, SUVH1, and polycomb-mediated H3K27me3. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.

Epigenetic processes in plant stress priming: Open questions and new approaches.

Priming reflects the capacity of plants to memorise environmental stress experience and improve their response to recurring stress. Epigenetic modifications in DNA and associated histone proteins may carry short-term and long-term memory in the same plant or mediate transgenerational effects, but the evidence is still largely circumstantial. New experimental tools now enable scientists to perform targeted manipulations that either prevent or generate a particular epigenetic modification in a particular location of the genome. Such 'reverse epigenetics' approaches allow for the interrogation of causality between individual priming-induced modifications and their role for altering gene expression and plant performance under recurring stress. Furthermore, combining site-directed epigenetic manipulation with conditional and cell-type specific promoters creates novel opportunities to test and engineer spatiotemporal patterns of priming.

DDT-RELATED PROTEIN4-IMITATION SWITCH alters nucleosome distribution to relieve transcriptional silencing in Arabidopsis.

DNA methylation is a conserved epigenetic modification that is typically associated with silencing of transposable elements and promoter methylated genes. However, some DNA-methylated loci are protected from silencing, allowing transcriptional flexibility in response to environmental and developmental cues. Through a genetic screen in Arabidopsis (Arabidopsis thaliana), we uncovered an antagonistic relationship between the MICRORCHIDIA (MORC) protein and the IMITATION SWITCH (ISWI) complex in regulating the DNA-methylated SUPPRESSOR OF DRM1 DRM2 CMT3 (SDC) reporter. We demonstrate that components of the plant-specific ISWI complex, including CHROMATIN REMODELING PROTEIN11 (CHR11), CHR17, DDT-RELATED PROTEIN4 (DDR4), and DDR5, function to partially derepress silenced genes and transposable elements (TEs), through their function in regulating nucleosome distribution. This action also requires the known transcriptional activator DNAJ proteins, providing a mechanistic link between nucleosome remodeling and transcriptional activation. Genome-wide studies revealed that DDR4 causes changes in nucleosome distribution at numerous loci, a subset of which is associated with changes in DNA methylation and/or transcription. Our work reveals a mechanism for balancing transcriptional flexibility and faithful silencing of DNA-methylated loci. As both ISWI and MORC family genes are widely distributed across plant and animal species, our findings may represent a conserved eukaryotic mechanism for fine-tuning gene expression under epigenetic regulation.

MORC proteins regulate transcription factor binding by mediating chromatin compaction in active chromatin regions.

BACKGROUND: The microrchidia (MORC) proteins are a family of evolutionarily conserved GHKL-type ATPases involved in chromatin compaction and gene silencing. Arabidopsis MORC proteins act in the RNA-directed DNA methylation (RdDM) pathway, where they act as molecular tethers to ensure the efficient establishment of RdDM and de novo gene silencing. However, MORC proteins also have RdDM-independent functions although their underlying mechanisms are unknown. RESULTS: In this study, we examine MORC binding regions where RdDM does not occur in order to shed light on the RdDM-independent functions of MORC proteins. We find that MORC proteins compact chromatin and reduce DNA accessibility to transcription factors, thereby repressing gene expression. We also find that MORC-mediated repression of gene expression is particularly important under conditions of stress. MORC-regulated transcription factors can in some cases regulate their own transcription, resulting in feedback loops. CONCLUSIONS: Our findings provide insights into the molecular mechanisms of MORC-mediated chromatin compaction and transcription regulation.

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.

Arabidopsis MORC proteins function in the efficient establishment of RNA directed DNA methylation.

The Microrchidia (MORC) family of ATPases are required for transposable element (TE) silencing and heterochromatin condensation in plants and animals, and C. elegans MORC-1 has been shown to topologically entrap and condense DNA. In Arabidopsis thaliana, mutation of MORCs has been shown to reactivate silent methylated genes and transposons and to decondense heterochromatic chromocenters, despite only minor changes in the maintenance of DNA methylation. Here we provide the first evidence localizing Arabidopsis MORC proteins to specific regions of chromatin and find that MORC4 and MORC7 are closely co-localized with sites of RNA-directed DNA methylation (RdDM). We further show that MORC7, when tethered to DNA by an artificial zinc finger, can facilitate the establishment of RdDM. Finally, we show that MORCs are required for the efficient RdDM mediated establishment of DNA methylation and silencing of a newly integrated FWA transgene, even though morc mutations have no effect on the maintenance of preexisting methylation at the endogenous FWA gene. We propose that MORCs function as a molecular tether in RdDM complexes to reinforce RdDM activity for methylation establishment. These findings have implications for MORC protein function in a variety of other eukaryotic organisms.

MBD5 and MBD6 couple DNA methylation to gene silencing through the J-domain protein SILENZIO.

DNA methylation is associated with transcriptional repression of eukaryotic genes and transposons, but the downstream mechanism of gene silencing is largely unknown. Here we describe two Arabidopsis methyl-CpG binding domain proteins, MBD5 and MBD6, that are recruited to chromatin by recognition of CG methylation, and redundantly repress a subset of genes and transposons without affecting DNA methylation levels. These methyl-readers recruit a J-domain protein, SILENZIO, that acts as a transcriptional repressor in loss-of-function and gain-of-function experiments. J-domain proteins often serve as co-chaperones with HSP70s. Indeed, we found that SILENZIO's conserved J-domain motif was required for its interaction with HSP70s and for its silencing function. These results uncover an unprecedented role of a molecular chaperone J-domain protein in gene silencing downstream of DNA methylation.

The characterization of Mediator 12 and 13 as conditional positive gene regulators in Arabidopsis.

Mediator 12 (MED12) and MED13 are components of the Mediator multi-protein complex, that facilitates the initial steps of gene transcription. Here, in an Arabidopsis mutant screen, we identify MED12 and MED13 as positive gene regulators, both of which contribute broadly to morc1 de-repressed gene expression. Both MED12 and MED13 are preferentially required for the expression of genes depleted in active chromatin marks, a chromatin signature shared with morc1 re-activated loci. We further discover that MED12 tends to interact with genes that are responsive to environmental stimuli, including light and radiation. We demonstrate that light-induced transient gene expression depends on MED12, and is accompanied by a concomitant increase in MED12 enrichment during induction. In contrast, the steady-state expression level of these genes show little dependence on MED12, suggesting that MED12 is primarily required to aid the expression of genes in transition from less-active to more active states.

A DNA methylation reader complex that enhances gene transcription.

DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in Arabidopsis thaliana that associate with methylated DNA. Two SU(VAR)3-9 homologs, the transcriptional antisilencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes.

Mechanistic insights into plant SUVH family H3K9 methyltransferases and their binding to context-biased non-CG DNA methylation.

DNA methylation functions in gene silencing and the maintenance of genome integrity. In plants, non-CG DNA methylation is linked through a self-reinforcing loop with histone 3 lysine 9 dimethylation (H3K9me2). The plant-specific SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG (SUVH) family H3K9 methyltransferases (MTases) bind to DNA methylation marks and catalyze H3K9 methylation. Here, we analyzed the structure and function of Arabidopsis thaliana SUVH6 to understand how this class of enzyme maintains methylation patterns in the genome. We reveal that SUVH6 has a distinct 5-methyl-dC (5mC) base-flipping mechanism involving a thumb loop element. Autoinhibition of H3 substrate entry is regulated by a SET domain loop, and a conformational transition in the post-SET domain upon cofactor binding may control catalysis. In vitro DNA binding and in vivo ChIP-seq data reveal that the different SUVH family H3K9 MTases have distinct DNA binding preferences, targeting H3K9 methylation to sites with different methylated DNA sequences, explaining the context biased non-CG DNA methylation in plants.

Large-scale comparative epigenomics reveals hierarchical regulation of non-CG methylation in Arabidopsis.

Genome-wide characterization by next-generation sequencing has greatly improved our understanding of the landscape of epigenetic modifications. Since 2008, whole-genome bisulfite sequencing (WGBS) has become the gold standard for DNA methylation analysis, and a tremendous amount of WGBS data has been generated by the research community. However, the systematic comparison of DNA methylation profiles to identify regulatory mechanisms has yet to be fully explored. Here we reprocessed the raw data of over 500 publicly available Arabidopsis WGBS libraries from various mutant backgrounds, tissue types, and stress treatments and also filtered them based on sequencing depth and efficiency of bisulfite conversion. This enabled us to identify high-confidence differentially methylated regions (hcDMRs) by comparing each test library to over 50 high-quality wild-type controls. We developed statistical and quantitative measurements to analyze the overlapping of DMRs and to cluster libraries based on their effect on DNA methylation. In addition to confirming existing relationships, we revealed unanticipated connections between well-known genes. For instance, MET1 and CMT3 were found to be required for the maintenance of asymmetric CHH methylation at nonoverlapping regions of CMT2 targeted heterochromatin. Our comparative methylome approach has established a framework for extracting biological insights via large-scale comparison of methylomes and can also be adopted for other genomics datasets.

Improved Denaturation of Small RNA Duplexes and Its Application for Northern Blotting.

Small RNAs (sRNAs) are short (18-30 nucleotide) noncoding RNA molecules, which control gene expression and pathogen response in eukaryotes. They are associated with and guide nucleases to target nucleic acids by nucleotide base pairing. We found that current techniques for small RNA detection are adversely affected by the presence of complementary RNA. Thus we established FDF-PAGE (fully denaturing formaldehyde polyacrylamide gel electrophoresis), which dramatically improves denaturation efficiency and subsequently the detection of sequestered sRNAs.

Arabidopsis AtMORC4 and AtMORC7 Form Nuclear Bodies and Repress a Large Number of Protein-Coding Genes.

The MORC family of GHKL ATPases are an enigmatic class of proteins with diverse chromatin related functions. In Arabidopsis, AtMORC1, AtMORC2, and AtMORC6 act together in heterodimeric complexes to mediate transcriptional silencing of methylated DNA elements. Here, we studied Arabidopsis AtMORC4 and AtMORC7. We found that, in contrast to AtMORC1,2,6, they act to suppress a wide set of non-methylated protein-coding genes that are enriched for those involved in pathogen response. Furthermore, atmorc4 atmorc7 double mutants show a pathogen response phenotype. We found that AtMORC4 and AtMORC7 form homomeric complexes in vivo and are concentrated in discrete nuclear bodies adjacent to chromocenters. Analysis of an atmorc1,2,4,5,6,7 hextuple mutant demonstrates that transcriptional de-repression is largely uncoupled from changes in DNA methylation in plants devoid of MORC function. However, we also uncover a requirement for MORC in both DNA methylation and silencing at a small but distinct subset of RNA-directed DNA methylation target loci. These regions are characterized by poised transcriptional potential and a low density of sites for symmetric cytosine methylation. These results provide insight into the biological function of MORC proteins in higher eukaryotes.

FDF-PAGE: a powerful technique revealing previously undetected small RNAs sequestered by complementary transcripts.

Small RNAs, between 18nt and 30nt in length, are a diverse class of non-coding RNAs that mediate a range of cellular processes, from gene regulation to pathogen defense. They guide ribonucleoprotein complexes to their target nucleic acids by Watson-Crick base pairing. We report here that current techniques for small RNA detection and library generation are biased by formation of RNA duplexes. To address this problem, we established FDF-PAGE (fully-denaturing formaldehyde polyacrylamide gel electrophoresis) to prevent annealing of sRNAs to their complement. By applying FDF-PAGE, we provide evidence that both strands of viral small RNA are present in near equimolar ratios, indicating that the predominant precursor is a long double-stranded RNA. Comparing non-denaturing conditions to FDF-PAGE uncovered extensive sequestration of miRNAs in model organisms and allowed us to identify candidate small RNAs under the control of competing endogenous RNAs (ceRNAs). By revealing the full repertoire of small RNAs, we can begin to create a better understanding of small RNA mediated interactions.

Chlorophyll Content Assay to Quantify the Level of Necrosis Induced by Different R Gene/Elicitor Combinations after Transient Expression.

This assay can be used to rapidly and accurately quantify levels of leaf necrosis induced after transient expression of R genes and elicitor combinations (Harris et al., 2013). It is based on the inverse correlation between level of necrosis and chlorophyll content in leaf tissue. It is adapted from the calculations described by (Strain et al., 1971).

Stepwise artificial evolution of a plant disease resistance gene.

Genes encoding plant nucleotide-binding leucine-rich repeat (NB-LRR) proteins confer dominant resistance to diverse pathogens. The wild-type potato NB-LRR protein Rx confers resistance against a single strain of potato virus X (PVX), whereas LRR mutants protect against both a second PVX strain and the distantly related poplar mosaic virus (PopMV). In one of the Rx mutants there was a cost to the broad-spectrum resistance because the response to PopMV was transformed from a mild disease on plants carrying wild-type Rx to a trailing necrosis that killed the plant. To explore the use of secondary mutagenesis to eliminate this cost of broad-spectrum resistance, we performed random mutagenesis of the N-terminal domains of this broad-recognition version of Rx and isolated four mutants with a stronger response against the PopMV coat protein due to enhanced activation sensitivity. These mutations are located close to the nucleotide-binding pocket, a highly conserved structure that likely controls the "switch" between active and inactive NB-LRR conformations. Stable transgenic plants expressing one of these versions of Rx are resistant to the strains of PVX and the PopMV that previously caused trailing necrosis. We conclude from this work that artificial evolution of NB-LRR disease resistance genes in crops can be enhanced by modification of both activation and recognition phases, to both accentuate the positive and eliminate the negative aspects of disease resistance.