Delineation of metabolic gene clusters in plant genomes by chromatin signatures

Plants are a tremendous source of diverse chemicals, including many natural product-derived drugs. It has recently become apparent that the genes for the biosynthesis of numerous different types of plant natural products are organized as metabolic gene clusters, thereby unveiling a highly unusual form of plant genome architecture and offering novel avenues for discovery and exploitation of plant specialized metabolism. Here we show that these clustered pathways are characterized by distinct chromatin signatures of histone 3 lysine trimethylation (H3K27me3) and histone 2 variant H2A.Z, associated with cluster repression and activation, respectively, and represent discrete windows of co-regulation in the genome. We further demonstrate that knowledge of these chromatin signatures along with chromatin mutants can be used to mine genomes for cluster discovery. The roles of H3K27me3 and H2A.Z in repression and activation of single genes in plants are well known. However, our discovery of highly localized operon-like co-regulated regions of chromatin modification is unprecedented in plants. Our findings raise intriguing parallels with groups of physically linked multi-gene complexes in animals and with clustered pathways for specialized metabolism in filamentous fungi.     

Clustering of Pathogen‐Response Genes in the Genome of Arabidopsis thaliana

Previously, we used heterologous expressed sequence tag (EST) mapping to generate a profile of 4 935 pathogen‐response genes of Arabidopsis thaliana. In this work, we performed a computer analysis of this profile, revealing 1 594 non‐homologous clustered genes distributed among all A. thaliana chromosomes, whose co‐regulation may be related to host responses to pathogens. To supplement computer data, we arbitrarily selected two clusters and analyzed their expression levels in A. thaliana ecotypes Col‐0 and C24 during infection with the yellow strain of Cucumber mosaic virus CMV(Y). Ecotype Col‐0 is susceptible to CMV(Y), whereas C24 contains the dominant resistance gene RCY1. Upon infection with CMV(Y), all clustered genes were significantly activated in the resistant ecotype C24. In addition, we demonstrated that posttranslational histone modifications associated with trimethylation of histone H3 lysine 27 are most likely involved in regulation of several cluster genes described in this study. Overall, our experiments indicated that pathogen‐response genes in the genome of A. thaliana may be clustered and co‐regulated.     

Histone modifications associated with drought tolerance in the desert plant <Emphasis Type="Italic">Zygophyllum dumosum</Emphasis> Boiss

Zygophyllum dumosum Boiss. is a perennial Saharo-Arabian phytogeographical element and a dominant shrub on the rocky limestone southeast-facing slopes of the Negev desert. The plant is highly active during the winter, and semideciduous during the dry summer, i.e., it sheds its leaflets, while leaving the thick, fleshy petiole green and rather active during the dry season. Being resistant to extreme perennial drought, Z. dumosum appears to provide an intriguing model plant for studying epigenetic mechanisms associated with drought tolerance in natural habitats. The transition from the wet to the dry season was accompanied by a significant decrease in nuclear size and with posttranslational modifications of histone H3 N-terminal tail. Dimethylation of H3 at lysine 4 (H3K4)—a modification associated with active gene expression—was found to be high during the wet season but gradually diminished on progression to the dry season. Unexpectedly, H3K9 di- and trimethylation as well as H3K27 di- and trimethylation could not be detected in Z. dumosum; H3K9 monomethylation appears to be prominent in Z. dumosum during the wet but not during the dry season. Contrary to Z. dumosum, H3K9 dimethylation was detected in other desert plants, including Artemisia sieberi, Anabasis articulata and Haloxylon scoparium. Taken together, our results demonstrate dynamic genome organization and unique pattern of histone H3 methylation displayed by Z. dumosum, which could have an adaptive value in variable environments of the Negev desert.     

Histone modification and signalling cascade of the dormancy‐associated MADS‐box gene,PpMADS13‐1, in Japanese pear (Pyrus pyrifolia) during endodormancy

Dormancy‐associated MADS‐box (DAM) genes play an important role in endodormancy phase transition. We investigated histone modification in the DAM homolog (PpMADS13‐1) from Japanese pear, via chromatin immunoprecipitation–quantitative PCR, to understand the mechanism behind the reduced expression of the PpMADS13‐1 gene towards endodormancy release. Our results indicated that the reduction in the active histone mark by trimethylation of the histone H3 tail at lysine 4 contributed to the reduction of PpMADS13‐1 expression towards endodormancy release. In contrast, the inactive histone mark by trimethylation of the histone H3 tail at lysine 27 in PpMADS13‐1 locus was quite low, and these levels were more similar to a negative control [normal mouse immunoglobulin G (IgG)] than to a positive control (AGAMOUS) in endodormancy phase transition. The loss of histone variant H2A.Z also coincided with the down‐regulation of PpMADS13‐1. Subsequently, we investigated the PpMADS13‐1 signalling cascade and found that PpCBF2, a pear C‐repeated binding factor, regulated PpMADS13‐1 expression via interaction of PpCBF2 with the 5′‐upstream region of PpMADS13‐1 by transient reporter assay. Furthermore, transient reporter assay confirmed no interaction between the PpMADS13‐1 protein and the pear FLOWERING LOCUS T genes. Taken together, our results enhance understanding of the molecular mechanisms underlying endodormancy phase transition in Japanese pear.     

Computational characterization of substrate and product specificities,and functionality of S‐adenosylmethionine binding pocket in histone lysine methyltransferases from Arabidopsis,rice and maize

Histone lysine methylation by histone lysine methyltransferases (HKMTs) has been implicated in regulation of gene expression. While significant progress has been made to understand the roles and mechanisms of animal HKMT functions, only a few plant HKMTs are functionally characterized. To unravel histone substrate specificity, degree of methylation and catalytic activity, we analyzed Arabidopsis Trithorax‐like protein (ATX), Su (var)3‐9 h omologs protein (SUVH), Su(var)3‐9 related protein (SUVR), ATXR5, ATXR6, and E(Z) HKMTs of Arabidopsis, maize and rice through sequence and structure comparison. We show that ATXs may exhibit methyltransferase specificity toward histone 3 lysine 4 (H3K4) and might catalyse the trimethylation. Our analyses also indicate that most SUVH proteins of Arabidopsis may bind histone H3 lysine 9 (H3K9). We also predict that SUVH7, SUVH8, SUVR1, SUVR3, ZmSET20 and ZmSET22 catalyse monomethylation or dimethylation of H3K9. Except for SDG728, which may trimethylate H3K9, all SUVH paralogs in rice may catalyse monomethylation or dimethylation. ZmSET11, ZmSET31, SDG713, SDG715, and SDG726 proteins are predicted to be catalytically inactive because of an incomplete S‐adenosylmethionine (SAM) binding pocket and a post‐SET domain. E(Z) homologs can trimethylate H3K27 substrate, which is similar to the Enhancer of Zeste homolog 2 of humans. Our comparative sequence analyses reveal that ATXR5 and ATXR6 lack motifs/domains required for protein‐protein interaction and polycomb repressive complex 2 complex formation. We propose that subtle variations of key residues at substrate or SAM binding pocket, around the catalytic pocket, or presence of pre‐SET and post‐SET domains in HKMTs of the aforementioned plant species lead to variations in class‐specific HKMT functions and further determine their substrate specificity, the degree of methylation and catalytic activity.     

Mutants in the imprinted PICKLE RELATED 2 gene suppress seed abortion of fertilization independent seed class mutants and paternal excess interploidy crosses in Arabidopsis

Endosperm cellularization is essential for embryo development and viable seed formation. Loss of function of the FERTILIZATION INDEPENDENT SEED (FIS) class Polycomb genes, which mediate trimethylation of histone H3 lysine27 (H3K27me3), as well as imbalanced contributions of parental genomes interrupt this process. The causes of the failure of cellularization are poorly understood. In this study we identified PICKLE RELATED 2 (PKR2) mutations which suppress seed abortion in fis1/mea by restoring endosperm cellularization. PKR2, a paternally expressed imprinted gene (PEG), encodes a CHD3 chromatin remodeler. PKR2 is specifically expressed in syncytial endosperm and its maternal copy is repressed by FIS1. Seed abortion in a paternal genome excess interploidy cross was also partly suppressed by pkr2. Simultaneous mutations in PKR2 and another PEG, ADMETOS (ADM), additively rescue the seed abortion in fis1 and in the interploidy cross, suggesting that PKR2 and ADM modulate endosperm cellularization independently and reproductive isolation between plants of different ploidy is established by imprinted genes. Genes upregulated in fis1 and downregulated in the presence of pkr2 are enriched in glycosyl‐hydrolyzing activity, while genes downregulated in fis1 and upregulated in the presence of pkr2 are enriched with microtubule motor activity, consistent with the cellularization patterns in fis1 and the suppressor line. The antagonistic functions of FIS1 and PKR2 in modulating endosperm development are similar to those of PICKLE (PKL) and CURLY LEAF (CLF), which antagonistically regulate root meristem activity. Our results provide further insights into the function of imprinted genes in endosperm development and reproductive isolation.     

H2A.Z and H2B.Z double‐variant nucleosomes define intergenic regions and dynamically occupy var gene promoters in the malaria parasite Plasmodium falciparum

Histone variants are important components of eukaryotic chromatin and can alter chromatin structure to confer specialized functions. H2B variant histones are rare in nature but have evolved independently in the phyla Apicomplexa and Trypanasomatida. Here, we investigate the apicomplexan‐specific Plasmodium falciparum histone variant Pf H2B.Z and show that within nucleosomes Pf H2B.Z dimerizes with the H2A variant Pf H2A.Z and that Pf H2B.Z and Pf H2A.Z occupancy correlates in the subset of genes examined. These double‐variant nucleosomes also carry common markers of euchromatin like H3K4me3 and histone acetylation. Pf H2B.Z levels are elevated in intergenic regions across the genome, except in the var multigene family, where Pf H2A.Z/Pf H2B.Z double‐variant nucleosomes are only enriched in the promoter of the single active var copy and this enrichment is developmentally regulated. Importantly, this pattern seems to be specific for var genes and does not apply to other heterochromatic gene families involved in red blood cell invasion which are also subject to clonal expression. Thus, Pf H2A.Z/Pf H2B.Z double‐variant nucleosomes appear to have a highly specific function in the regulation of P. falciparum virulence.     

Drought‐inducible changes in the histone modification H3K9ac are associated with drought‐responsive gene expression in Brachypodium distachyon

• H3K9ac, an epigenetic marker, is widely distributed in plant genomes. H3K9ac enhances gene expression, which is highly conserved in eukaryotes. However, genome‐wide studies of H3K9ac in monocot species are limited, and the changes in H3K9ac under drought stress for individual genes are still not clear.
• We analysed changes in the H3K9ac level of Brachypodium distachyon under 20% PEG‐6000‐simulated drought stress conditions. We also performed chromatin immunoprecipitation, followed by next generation sequencing (ChIP‐seq) on H3K9ac to reveal changes in H3K9ac for individual genes at the genome‐wide level.
• Our study showed that H3K9ac was mainly enriched in gene exon regions. Drought increased or decreased the H3K9ac level at specific genomic loci. We identified 40 genes associated with increased H3K9ac levels and 36 genes associated with decreased H3K9ac levels under drought stress. Further, RT‐qPCR analyses showed that H3K9ac was positively associated with gene expression of those drought‐responsive genes.
• We conclude that H3K9ac enhances the expression level of a large number of drought‐responsive genes under drought stress in B. distachyon. The data presented here will help to reveal the correlation of some specific drought‐responsive genes and their enriched H3K9ac levels in the model plant B. distachyon.