Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair.

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

Gcn5- and Bre1-mediated Set2 degradation promotes chronological aging of Saccharomyces cerevisiae.

Loss of transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3) contributes to shorter lifespans in eukaryotes. However, the molecular mechanism of the decline of H3K36me3 during aging remains poorly understood. Here, we report that the degradation of the methyltransferase Set2 is the cause of decreased H3K36me3 levels during chronological aging in budding yeast. We show that Set2 protein degradation during cellular senescence and chronological aging is mainly mediated by the ubiquitin-conjugating E2 enzyme Ubc3 and the E3 ligase Bre1. Lack of Bre1 or abolishment of the ubiquitination stabilizes Set2 protein, sustains H3K36me3 levels at the aging-related gene loci, and upregulates their gene expression, thus leading to extended chronological lifespan. We further illustrate that Gcn5-mediated Set2 acetylation is a prerequisite for Bre1-catalyzed Set2 polyubiquitination and proteolysis during aging. We propose that two sequential post-translational modifications regulate Set2 homeostasis, suggesting a potential strategy to target the Gcn5-Bre1-Set2 axis for intervention of longevity.

From promoting aggregation to enhancing obstruction: A negative feedback regulatory mechanism of alleviation of trivalent chromium toxicity by silicon in rice.

Trivalent chromium [Cr(III)] is a threat to the environment and crop production. Silicon (Si) has been shown to be effective in mitigating Cr(III) toxicity in rice. However, the mechanisms by which Si reduces Cr(III) uptake in rice are unclear. Herein, we hypothesized that the ability of Si to obstruct Cr(III) diffusion via apoplastic bypass is related to silicic acid polymerization, which may be affected by Cr(III) in rice roots. To test this hypothesis, we employed hydroponics experiments on rice (Oryza sativa L.) and utilized apoplastic bypass tracer techniques, as well as model simulations, to investigate 1) the effect of Si on Cr(III) toxicity and its obstruction capacity via apoplastic bypass, 2) the effect of Cr(III) on silicic acid polymerization, and 3) the relationship between the degree of silicic acid polymerization and its Cr(III) obstruction capacity. We found that Si reversed the damage caused by Cr(III) stress in rice. Si exerted an obstruction effect in the apoplast, significantly decreasing the share of Cr(III) uptake via the apoplastic bypass from 18% to 11%. Moreover, Cr(III) reduced silica particles' radii and increased Si concentration in roots. Modeling revealed that a 5-fold reduction in their radii decreased the diffusion of Cr(III) in apoplast by approximately 17%. We revealed that Cr(III) promoted silicic acid polymerization, resulting in the formation of a higher number of Si particles with a smaller radius in roots, which in turn increased the ability of Si to obstruct Cr(III) diffusion. This negative feedback regulatory mechanism is novel and crucially important for maintaining homeostasis in rice, unveiling the unique role of Si under Cr(III) ion stress and providing a theoretical basis for promoting the use of Si fertilizer in the field.

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.

Set2-mediated H3K36 methylation states redundantly repress the production of antisense transcripts: role in transcription regulation.

Methyltransferase Set2-mediated methylation of histone H3 lysine 36 (H3K36), which involves the addition of up to three methyl groups at this site, has been demonstrated to function in many chromatin-coupled events. The methylation of H3K36 is known to recruit different chromatin effector proteins, affecting transcription, mRNA splicing and DNA repair. In this study, we engineered two yeast set2 mutants that lack H3K36 mono/dimethylation (H3K36me1/2) and trimethylation (H3K36me3), respectively, and characterized their roles in the production of antisense transcripts under nutrient-rich conditions. Using our new bioinformatics identification pipeline analysis, we are able to identify a larger number of antisense transcripts in set2∆ cells than has been published previously. We further show that H3K36me1/2 or H3K36me3 redundantly repressed the production of antisense transcripts. Moreover, gene ontology (GO) analysis implies that H3K36me3-mediated antisense transcription might play a role in DNA replication and DNA damage repair, which is independent of regulation of the corresponding sense gene expression. Overall, our results validate a coregulatory mechanism of different H3K36 methylation states, particularly in the repression of antisense transcription.

Effect of organosilicone and mineral silicon fertilizers on chemical forms of cadmium and lead in soil and their accumulation in rice.

Cadmium (Cd) and lead (Pb) pollution in soil and their accumulation in edible parts possess a worldwide eco-environmental and health risk, especially in developing countries. Recently, organosilicone fertilizer (OSiF) has been reported to reduce uptake of heavy metals, but the effectiveness has not been verified and its associated mechanisms are not fully understood. This work investigated whether and how OSiF and mineral silicon fertilizer (MSiF) affect mitigation of Cd and Pb stress in rice (Oryza sativa). Both soil incubation and pot experiments were conducted to assess the effect of OSiF and MSiF on bioavailability of Cd and Pb in soil and their accumulation in rice. Additionally, a hydroponic experiment was conducted to study whether Si in rice can alleviate Cd stress. We found that both Si fertilizers could increase soil pH, induce the transformation of the acid soluble and reducible fractions of Cd and Pb to the oxidizable and residual fractions in soil, decreasing their bioavailability and the uptake of Cd and Pb in rice. However, Si in OSiF was not phyto-available, but Si in MSiF was available since available Si in soil and Si in plant increased in MSiF treatments but not in OSiF treatments. Meanwhile, rice grain yields significantly increased and the Cd and Pb content of brown rice reduced in MSiF treatments but not in OSiF treatments. In addition, Si was found to be able to alleviate Cd stress by improving the antioxidant capacity of rice. These results suggested that the decreased Cd and Pb accumulation in OSiF-treated rice was due to Cd and Pb immobilization in soil simply with pH increase, but in MSiF-treated rice Cd and Pb immobilization in soil (ex planta effect) and Si-conferred inhibitory effect of root-to-shoot Cd and Pb transport (in planta effect) contribute to the lower accumulation in rice.

A methylation-phosphorylation switch determines Plk1 kinase activity and function in DNA damage repair.

Polo-like kinase 1 (Plk1) is a crucial regulator of cell cycle progression; but the mechanism of regulation of Plk1 activity is not well understood. We present evidence that Plk1 activity is controlled by a balanced methylation and phosphorylation switch. The methyltransferase G9a monomethylates Plk1 at Lys209, which antagonizes phosphorylation of T210 to inhibit Plk1 activity. We found that the methyl-deficient Plk1 mutant K209A affects DNA replication, whereas the methyl-mimetic Plk1 mutant K209M prolongs metaphase-to-anaphase duration through the inability of sister chromatids separation. We detected accumulation of Plk1 K209me1 when cells were challenged with DNA damage stresses. Ablation of K209me1 delays the timely removal of RPA2 and RAD51 from DNA damage sites, indicating the critical role of K209me1 in guiding the machinery of DNA damage repair. Thus, our study highlights the importance of a methylation-phosphorylation switch of Plk1 in determining its kinase activity and functioning in DNA damage repair.