Jumonji C Domain Protein JMJ705-Mediated Removal of Histone H3 Lysine 27 Trimethylation Is Involved in Defense-Related Gene Activation in Rice

Histone methylation is an important epigenetic modification in chromatin function, genome activity, and gene regulation. Dimethylated or trimethylated histone H3 lysine 27 (H3K27me2/3) marks silent or repressed genes involved in developmental processes and stress responses in plants. However, the role and the mechanism of the dynamic removal of H3K27me2/3 during gene activation remain unclear. Here, we show that the rice (Oryza sativa) Jumonji C (jmjC) protein gene JMJ705 encodes a histone lysine demethylase that specifically reverses H3K27me2/3. The expression of JMJ705 is induced by stress signals and during pathogen infection. Overexpression of the gene reduces the resting level of H3K27me2/3 resulting in preferential activation of H3K27me3-marked biotic stress-responsive genes and enhances rice resistance to the bacterial blight disease pathogen Xanthomonas oryzae pathovar oryzae. Mutation of the gene reduces plant resistance to the pathogen. Further analysis revealed that JMJ705 is involved in methyl jasmonate–induced dynamic removal of H3K27me3 and gene activation. The results suggest that JMJ705 is a biotic stress-responsive H3K27me2/3 demethylase that may remove H3K27me3 from marked defense-related genes and increase their basal and induced expression during pathogen infection.     

The trxG family histone methyltransferase SET DOMAIN GROUP 26 promotes flowering via a distinctive genetic pathway

Histone methylation is a major component in numerous processes such as determination of flowering time, which is fine‐tuned by multiple genetic pathways that integrate both endogenous and environmental signals. Previous studies identified SET DOMAIN GROUP 26 (SDG26) as a histone methyltransferase involved in the activation of flowering, as loss of function of SDG26 caused a late‐flowering phenotype in Arabidopsis thaliana. However, the SDG26 function and the underlying molecular mechanism remain largely unknown. In this study, we undertook a genetic analysis by combining the sdg26 mutant with mutants of other histone methylation enzymes, including the methyltransferase mutants Arabidopsis trithorax1 (atx1), sdg25 and curly leaf (clf), as well as the demethylase double mutant lsd1‐like1 lsd1‐like2 (ldl1 ldl2). We found that the early‐flowering mutants sdg25, atx1 and clf interact antagonistically with the late‐flowering mutant sdg26, whereas the late‐flowering mutant ldl1 ldl2 interacts synergistically with sdg26. Based on microarray analysis, we observed weak overlaps in the genes that were differentially expressed between sdg26 and the other mutants. Our analyses of the chromatin of flowering genes revealed that the SDG26 protein binds at the key flowering integrator SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1/AGAMOUS‐LIKE 20 (SOC1/AGL20), and is required for histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 36 trimethylation (H3K36me3) at this locus. Together, our results indicate that SDG26 promotes flowering time through a distinctive genetic pathway, and that loss of function of SDG26 causes a decrease in H3K4me3 and H3K36me3 at its target gene SOC1, leading to repression of this gene and the late‐flowering phenotype.     

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.     

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.     

SDG714 Regulates Specific Gene Expression and Consequently Affects Plant Growth via H3K9 Dimethylation

Histone lysine methylation is known to be involved in the epigenetic regulation of gene expression in all eukaryotes including plants. Here we show that the rice SDG714 is primarily responsible for dimethylation but not trimethylation on histone H3K9 in vivo. Overexpression of YFP-SDG714 in Arabidopsis significantly inhibits plant growth and this inhibition is associated with an enhanced level of H3K9 dimethylation. Our microarray results show that many genes essential for the plant growth and development were downregulated in transgenic Arabidopsis plants overexpressing YFP-SDG714. By chromatin immunoprecipitation analysis, we show that YFP-SDG714 is targeted to specific chromatin regions and dimethylate the H3K9, which is linked with heterochromatinization and the downregulation of genes. Most interestingly, when YFP-SDG714 production is stopped, the inhibited plants can partially restore their growth, suggesting that the perturbation of gene expression caused by YFP-SDG714 is revertible. Taken together, our results point to an important role of SDG714 in H3K9 dimethylation, suppression of gene expression and plant growth, and provide a potential method to regulate gene expression and plant development by an on-off switch of SDG714 expression.     

Synergistic repression of the embryonic programme by SET DOMAIN GROUP 8 and EMBRYONIC FLOWER 2 in Arabidopsis seedlings

The seed maturation programme occurs only during the late phase of embryo development, and repression of the maturation genes is pivotal for seedling development. However, mechanisms that repress the expression of this programme in vegetative tissues are not well understood. A genetic screen was performed for mutants that express maturation genes in leaves. Here, it is shown that mutations affecting SDG8 (SET DOMAIN GROUP 8), a putative histone methyltransferase, cause ectopic expression of a subset of maturation genes in leaves. Further, to investigate the relationship between SDG8 and the Polycomb Group (PcG) proteins, which are known to repress many developmentally important genes including seed maturation genes, double mutants were made and formation of somatic embryos was observed on mutant seedlings with mutations in both SDG8 and EMF2 (EMBRYONIC FLOWER 2). Analysis of histone methylation status at the chromatin sites of a number of maturation loci revealed a synergistic effect of emf2 and sdg8 on the deposition of the active histone mark which is the trimethylation of Lys4 on histone 3 (H3K4me3). This is consistent with high expression of these genes and formation of somatic embryos in the emf2 sdg8 double mutants. Interestingly, a double mutant of sdg8 and vrn2 (vernalization2), a paralogue of EMF2, grew and developed normally to maturity. These observations demonstrate a functional cooperative interplay between SDG8 and an EMF2-containing PcG complex in maintaining vegetative cell identity by repressing seed genes to promote seedling development. The work also indicates the functional specificities of PcG complexes in Arabidopsis.     

Arabidopsis Trithorax histone methyltransferases are redundant in regulating development and DNA methylation

Although the Trithorax histone methyltransferases ATX1–5 are known to regulate development and stress responses by catalyzing histone H3K4 methylation in Arabidopsis thaliana, it is unknown whether and how these histone methyltransferases affect DNA methylation. Here, we found that the redundant ATX1–5 proteins are not only required for plant development and viability but also for the regulation of DNA methylation. The expression and H3K4me3 levels of both RNA-directed DNA methylation (RdDM) genes (NRPE1, DCL3, IDN2, and IDP2) and active DNA demethylation genes (ROS1, DML2, and DML3) were downregulated in the atx1/2/4/5 mutant. Consistent with the facts that the active DNA demethylation pathway mediates DNA demethylationmainly at CG and CHG sites, and that the RdDM pathway mediates DNA methylation mainly at CHH sites, whole-genome DNA methylation analyses showed that hyper-CG and CHG DMRs in atx1/2/4/5 significantly overlapped with those in the DNA demethylation pathway mutant ros1 dml2 dml3 (rdd), and that hypo-CHH DMRs in atx1/2/4/5 significantly overlapped with those in the RdDM mutant nrpe1, suggesting that the ATX paralogues function redundantly to regulate DNA methylation by promoting H3K4me3 levels and expression levels of both RdDM genes and active DNA demethylation genes. Given that the ATX proteins function as catalytic subunits of COMPASS histone methyltransferase complexes, we also demonstrated that the COMPASS complex components function as a whole to regulate DNA methylation. This study reveals a previously uncharacterized mechanism underlying the regulation of DNA methylation.     

G9α‐dependent histone H3K9me3 hypomethylation promotes overexpression of cardiomyogenesis‐related genes in foetal mice

Alcohol consumption during pregnancy can cause foetal alcohol syndrome and congenital heart disease. Nonetheless, the underlying mechanism of alcohol‐induced cardiac dysplasia remains unknown. We previously reported that alcohol exposure during pregnancy can cause abnormal expression of cardiomyogenesis‐related genes, and histone H3K9me3 hypomethylation was observed in alcohol‐treated foetal mouse heart. Hence, an imbalance in histone methylation may be involved in alcohol‐induced cardiac dysplasia. In this study, we investigated the involvement of G9α histone methyltransferase in alcohol‐induced cardiac dysplasia in vivo and in vitro using heart tissues of foetal mice and primary cardiomyocytes of neonatal mice. Western blotting revealed that alcohol caused histone H3K9me3 hypomethylation by altering G9α histone methyltransferase expression in cardiomyocytes. Moreover, overexpression of cardiomyogenesis‐related genes (MEF2C, Cx43, ANP and β‐MHC) was observed in alcohol‐exposed foetal mouse heart. Additionally, we demonstrated that G9α histone methyltransferase directly interacted with histone H3K9me3 and altered its methylation. Notably, alcohol did not down‐regulate H3K9me3 methylation after G9α suppression by short hairpin RNA in primary mouse cardiomyocytes, preventing MEF2C, Cx43, ANP and β‐MHC overexpression. These findings suggest that G9α histone methyltransferase‐mediated imbalance in histone H3K9me3 methylation plays a critical role in alcohol‐induced abnormal expression cardiomyogenesis‐related genes during pregnancy. Therefore, G9α histone methyltransferase may be an intervention target for congenital heart disease.