The Tudor Domain of the PHD Finger Protein 1 Is a Dual Reader of Lysine Trimethylation at Lysine 36 of Histone H3 and Lysine 27 of Histone Variant H3t

PHF1 associates with the Polycomb repressive complex 2 and it was demonstrated to stimulate its H3K27-trimethylation activity. We studied the interaction of the PHF1 Tudor domain with modified histone peptides and found that it recognizes H3K36me3 and H3tK27me3 (on the histone variant H3t) and that it uses the same trimethyllysine binding pocket for the interaction with both peptides. Since both peptide sequences are very different, this result indicates that reading domains can have dual specificities. Sub-nuclear localization studies of full-length PHF1 in human HEK293 cells revealed that it co-localizes with K27me3, but not with K36me3, and that this co-localization depends on the trimethyllysine binding pocket indicating that K27me3 is an in vivo target for the PHF1 Tudor domain. Our data suggest that PHF1 binds to H3tK27me3 in human chromatin, and H3t has a more general role in Polycomb regulation.     

Focus on Chromatin/Epigenetics: Histone H2B Monoubiquitination Mediated by HISTONE MONOUBIQUITINATION1 and HISTONE MONOUBIQUITINATION2 Is Involved in Anther Development by Regulating Tapetum Degradation-Related Genes in Rice

Water scarcity and the increasing severity of water deficit stress are major challenges to sustaining irrigated rice (Oryza sativa) production. Despite the technologies developed to reduce the water requirement, rice growth is seriously constrained under water deficit stress compared with other dryland cereals such as wheat (Triticum aestivum). We exposed rice cultivars with contrasting responses to water deficit stress and wheat cultivars well adapted to water-limited conditions to the same moisture stress during vegetative growth to unravel the whole-plant (shoot and root morphology) and organ/tissue (root anatomy) responses. Wheat cultivars followed a water-conserving strategy by reducing specific leaf area and developing thicker roots and moderate tillering. In contrast, rice ‘IR64’ and ‘Apo’ adopted a rapid water acquisition strategy through thinner roots under water deficit stress. Root diameter, stele and xylem diameter, and xylem number were more responsive and varied with different positions along the nodal root under water deficit stress in wheat, whereas they were relatively conserved in rice cultivars. Increased metaxylem diameter and lower metaxylem number near the root tips and exactly the opposite phenomena at the root-shoot junction facilitated the efficient use of available soil moisture in wheat. Tolerant rice ‘Nagina 22’ had an advantage in root morphological and anatomical attributes over cultivars IR64 and Apo but lacked plasticity, unlike wheat cultivars exposed to water deficit stress. The key traits determining the adaptation of wheat to dryland conditions have been summarized and discussed.Among cereals, rice (Oryza sativa) and wheat (Triticum aestivum) are the most important staple food crops, and they belong to the family Poaceae. These two cereals share a common ancestor and diverged about 65 million years ago (Sorrells et al., 2003). Rice eventually developed strong adaptation potential for fully flooded conditions across tropical to temperate environments, while wheat became well adapted to aerobic conditions mostly restricted to temperate environments. Rice, with a semiaquatic behavior, consumes about 30% of the total fresh water available for agricultural crops worldwide, which equates to a 2- to 3-fold higher consumption than other cereals such as wheat and maize (Zea mays; Peng et al., 2006). Despite a significantly lower water requirement, the potential yield of wheat in a favorable environment (9 tons ha⁻¹) is comparable with the yield of fully flooded rice (9 tons ha⁻¹) in the dry season at the International Rice Research Institute (IRRI; Fischer and Edmeades, 2010). Hence, rice records very low water productivity compared with wheat and other dryland cereals. Because of growing concerns about water scarcity and the increased frequency and magnitude of water deficit stress events under current and future climates, increasing or even sustaining rice yield under fully flooded conditions is highly challenging. To minimize the total water requirement for cultivating rice, several water-saving technologies have been developed, such as direct-seeded aerobic rice cultivation (Bindraban et al., 2006). These water-saving technologies increased water productivity substantially compared with flooded conditions but were invariably associated with a yield penalty. A major challenge that water-saving technologies including aerobic rice currently face is the lack of mechanistic understanding for further genetic improvement.By virtue of its wider adaptation to a range of edaphic conditions, rice is considered to possess the diversity to adapt to upland or aerobic scenarios extending into water deficit conditions (Khush, 1997). Genetic differences in rice root biomass and rooting depth and variation in root morphology with water deficit stress exposure are well documented (Kato et al., 2006, 2007; Henry et al., 2011; Kano et al., 2011). But the underlying mechanisms differing across diverse germplasm that influence water uptake under water deficit stress are not fully understood (Gowda et al., 2011). A recent report has documented water deficit-tolerant genotypes recording a lower bleeding rate and narrow xylem diameter under stress (Henry et al., 2012). Contrastingly, a higher root hydraulic conductivity helped to maintain a higher photosynthetic rate (Adachi et al., 2010), with tolerant cultivars maintaining greater root hydraulic conductivity than susceptible cultivars (Matsuo et al., 2009). Furthermore, upland rice cultivars with deeper roots outperformed lowland cultivars possessing a shallow root system when encountered with water deficit stress (Uga et al., 2013). Additionally, major-effect grain yield metaquantitative trait loci under water deficit stress identified in rice were found to colocalize on the genomes of other dryland cereals such as wheat, maize, and pearl millet (Pennisetum glaucum; Swamy et al., 2011), indicating a possible common evolutionary pathway for water deficit adaptation across cereals. Despite these achievements and the relatedness among cereals, rice does not respond in a way similar to other dryland cereals to water deficit stress conditions. To bring in a revolutionary change in future breeding strategies for upland/aerobic rice and for water deficit tolerance in rice, there is a need for a fundamental understanding and identification of the key traits that determine the water deficit stress response in well-adapted dryland cereals. Hence, comparing whole-plant responses (shoot and root) of rice with those of other dryland cereals such as wheat is essential.A comparative study between two C₃ cereals (rice and wheat) will help identify the core adaptive mechanisms and/or a suite of traits that render wheat to grow with less water and more tolerance of water deficit stress. Such comparative analysis should target key morphological, physiological, anatomical, and agronomic traits throughout the crop growth cycle, as water deficit stress occurs at both early (vegetative stage) and late (reproductive stage) seasons in rice (Pandey et al., 2007). Extensive research efforts are currently ongoing to reduce the impact of water deficit stress during the reproductive stage in rice (Venuprasad et al., 2008; Verulkar et al., 2010; Vikram et al., 2011; Kumar et al., 2014) and in wheat (Olivares-Villegas et al., 2007; Lopes and Reynolds, 2010; Pinto et al., 2010). Therefore, our study focused on stress during the vegetative stage to identify key checkpoints that determine whole-plant responses of representative rice cultivars adapted to lowland, upland/aerobic, or water deficit conditions and of wheat cultivars with moderate to high water deficit tolerance. Cultivars from both species were exposed to moisture levels that resemble aerobic conditions and water deficit stress during the vegetative stage. Our study follows a previous report that has successfully demonstrated the approach to expose rice and wheat to the same moisture stress conditions (Praba et al., 2009) and is designed to address the following specific objectives: (1) to quantify the adaptive plasticity in shoot and root morphology and biomass partitioning among different plant parts (leaves, stem, and root); (2) to estimate the key supportive physiological mechanisms such as whole-plant water use efficiency (WUE) and leaf-level carbon isotope discrimination (Δ13C); and (3) to dissect root anatomical plasticity across different key zones in both rice and wheat roots exposed to water deficit stress. Finally, novel traits that benefit dryland adaptation in wheat compared with high water-requiring rice cultivars are highlighted.     

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.     

RBS1, an RNA Binding Protein,Interacts with SPIN1 and Is Involved in Flowering Time Control in Rice

The rice U-box/ARM E3 ubiquitin ligase SPL11 negatively regulates programmed cell death (PCD) and disease resistance, and controls flowering time through interacting with the novel RNA/DNA binding KH domain protein SPIN1. Overexpression of Spin1 causes late flowering in transgenic rice under short-day (SD) and long-day (LD) conditions. In this study, we characterized the function of the RNA-binding and SPIN1-interacting 1 (RBS1) protein in flowering time regulation. Rbs1was identified in a yeast-two-hybrid screen using the full-length Spin1 cDNA as a bait and encodes an RNA binding protein with three RNA recognition motifs. The protein binds RNA in vitro and interacts with SPIN1 in the nucleus. Rbs1 overexpression causes delayed flowering under SD and LD conditions in rice. Expression analyses of flowering marker genes show that Rbs1 overexpression represses the expression of Hd3a under SD and LD conditions. Rbs1 is upregulated in both Spin1 overexpression plants and in the spl11 mutant. Interestingly, Spin1 expression is increased but Spl11 expression is repressed in the Rbs1 overexpression plants. Western blot analysis revealed that the SPIN1 protein level is increased in the Rbs1 overexpression plants and that the RBS1 protein level is also up-regulated in the Spin1 overexpression plants. These results suggest that RBS1 is a new negative regulator of flowering time that itself is positively regulated by SPIN1 but negatively regulated by SPL11 in rice.     

Histone H2B Ubiquitylation Is Not Required for Histone H3 Methylation at Lysine 4 in Tetrahymena

Ubiquitylation of histone H2B and/or a component of the system that ubiquitylates H2B is required for methylation of histone H3 at lysine 4 (H3K4) in yeasts and probably in humans. In this study, the single ubiquitylation site was mapped to conserved lysine 115 of the C-terminal region of histone H2B in the single-cell model organism Tetrahymena thermophila. In strains lacking H2B ubiquitylation, H3K4 methylation was not detectably affected. As in other organisms, the E2 ubiquitin-conjugating enzyme Ubc2 and the E3 ubiquitin ligase Bre1 were required for H2B ubiquitylation. However, neither enzyme was required for H3K4 methylation. These studies argue that, in T. thermophila, the histone ubiquitylation mechanism is not required for H3K4 methylation, demonstrating that different organisms can speak different languages in the “cross-talk” among post-translational modifications on different histones.     

Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

Histone lysine methylation is an important epigenetic modification in regulating chromatin structure and gene expression. Histone H3 lysine 4 methylation (H3K4me), which can be in a mono-, di-, or trimethylated state, has been shown to play an important role in gene expression involved in plant developmental control and stress adaptation. However, the resetting mechanism of this epigenetic modification is not yet fully understood. In this work, we identified a JmjC domain-containing protein, JMJ703, as a histone lysine demethylase that specifically reverses all three forms of H3K4me in rice. Loss-of-function mutation of the gene affected stem elongation and plant growth, which may be related to increased expression of cytokinin oxidase genes in the mutant. Analysis of crystal structure of the catalytic core domain (c-JMJ703) of the protein revealed a general structural similarity with mammalian and yeast JMJD2 proteins that are H3K9 and H3K36 demethylases. However, several specific features were observed in the structure of c-JMJ703. Key residues that interact with cofactors Fe(II) and N-oxalylglycine and the methylated H3K4 substrate peptide were identified and were shown to be essential for the demethylase activity in vivo. Several key residues are specifically conserved in known H3K4 demethylases, suggesting that they may be involved in the specificity for H3K4 demethylation.     

Folate Polyglutamylation Is Involved in Chromatin Silencing by Maintaining Global DNA Methylation and Histone H3K9 Dimethylation in Arabidopsis

DNA methylation and repressive histone Histone3 Lysine9 (H3K9) dimethylation correlate with chromatin silencing in plants and mammals. To identify factors required for DNA methylation and H3K9 dimethylation, we screened for suppressors of the repressor of silencing1 (ros1) mutation, which causes silencing of the expression of the RD29A (RESPONSE TO DESSICATION 29A) promoter-driven luciferase transgene (RD29A-LUC) and the 35S promoter-driven NPTII (NEOMYCIN PHOSPHOTRANSFERASE II) transgene (35S-NPTII). We identified the folylpolyglutamate synthetase FPGS1 and the known factor DECREASED DNA METHYLATION1 (DDM1). The fpgs1 and ddm1 mutations release the silencing of both RD29A-LUC and 35S-NPTII. Genome-wide analysis indicated that the fpgs1 mutation reduces DNA methylation and releases chromatin silencing at a genome-wide scale. The effect of fpgs1 on chromatin silencing is correlated with reduced levels of DNA methylation and H3K9 dimethylation. Supplementation of fpgs1 mutants with 5-formyltetrahydrofolate, a stable form of folate, rescues the defects in DNA methylation, histone H3K9 dimethylation, and chromatin silencing. The competitive inhibitor of methyltransferases, S-adenosylhomocysteine, is markedly upregulated in fpgs1, by which fpgs1 reduces S-adenosylmethionine accessibility to methyltransferases and accordingly affects DNA and histone methylation. These results suggest that FPGS1-mediated folate polyglutamylation is required for DNA methylation and H3K9 dimethylation through its function in one-carbon metabolism. Our study makes an important contribution to understanding the complex interplay among metabolism, development, and epigenetic regulation.     

The Histone H4 Lysine 20 Monomethyl Mark,Set by PR-Set7 and Stabilized by L(3)mbt,Is Necessary for Proper Interphase Chromatin Organization

Drosophila PR-Set7 or SET8 is a histone methyltransferase that specifically monomethylates histone H4 lysine 20 (H4K20). L(3)MBT has been identified as a reader of methylated H4K20. It contains several conserved domains including three MBT repeats binding mono- and dimethylated H4K20 peptides. We find that the depletion of PR-Set7 blocks de novo H4K20me1 resulting in the immediate activation of the DNA damage checkpoint, an increase in the size of interphase nuclei, and drastic reduction of cell viability. L(3)mbt on the other hand stabilizes the monomethyl mark, as L(3)mbt-depleted S2 cells show a reduction of more than 60% of bulk monomethylated H4K20 (H4K20me1) while viability is barely affected. Ploidy and basic chromatin structure show only small changes in PR-Set7-depleted cells, but higher order interphase chromatin organization is significantly affected presumably resulting in the activation of the DNA damage checkpoint. In the absence of any other known functions of PR-Set7, the setting of the de novo monomethyl mark appears essential for cell viability in the presence or absence of the DNA damage checkpoint, but once newly assembled chromatin is established the monomethyl mark, protected by L(3)mbt, is dispensable.