SMYD5 methylation of rpL40 links ribosomal output to gastric cancer.

Dysregulated transcription due to disruption in histone lysine methylation dynamics is an established contributor to tumorigenesis1,2. However, whether analogous pathologic epigenetic mechanisms act directly on the ribosome to advance oncogenesis is unclear. Here we find that trimethylation of the core ribosomal protein L40 (rpL40) at lysine 22 (rpL40K22me3) by the lysine methyltransferase SMYD5 regulates mRNA translation output to promote malignant progression of gastric adenocarcinoma (GAC) with lethal peritoneal ascites. A biochemical-proteomics strategy identifies the monoubiquitin fusion protein partner rpL40 (ref. 3) as the principal physiological substrate of SMYD5 across diverse samples. Inhibiting the SMYD5-rpL40K22me3 axis in GAC cell lines reprogrammes protein synthesis to attenuate oncogenic gene expression signatures. SMYD5 and rpL40K22me3 are upregulated in samples from patients with GAC and negatively correlate with clinical outcomes. SMYD5 ablation in vivo in familial and sporadic mouse models of malignant GAC blocks metastatic disease, including peritoneal carcinomatosis. Suppressing SMYD5 methylation of rpL40 inhibits human cancer cell and patient-derived GAC xenograft growth and renders them hypersensitive to inhibitors of PI3K and mTOR. Finally, combining SMYD5 depletion with PI3K-mTOR inhibition and chimeric antigen receptor T cell administration cures an otherwise lethal in vivo mouse model of aggressive GAC-derived peritoneal carcinomatosis. Together, our work uncovers a ribosome-based epigenetic mechanism that facilitates the evolution of malignant GAC and proposes SMYD5 targeting as part of a potential combination therapy to treat this cancer.

RNA-methylation-dependent RNA processing controls the speed of the circadian clock.

The eukaryotic biological clock involves a negative transcription-translation feedback loop in which clock genes regulate their own transcription and that of output genes of metabolic significance. While around 10% of the liver transcriptome is rhythmic, only about a fifth is driven by de novo transcription, indicating mRNA processing is a major circadian component. Here, we report that inhibition of transmethylation reactions elongates the circadian period. RNA sequencing then reveals methylation inhibition causes widespread changes in the transcription of the RNA processing machinery, associated with m(6)A-RNA methylation. We identify m(6)A sites on many clock gene transcripts and show that specific inhibition of m(6)A methylation by silencing of the m(6)A methylase Mettl3 is sufficient to elicit circadian period elongation and RNA processing delay. Analysis of the circadian nucleocytoplasmic distribution of clock genes Per2 and Arntl then revealed an uncoupling between steady-state pre-mRNA and cytoplasmic mRNA rhythms when m(6)A methylation is inhibited.

Mitochondrial RNA modifications shape metabolic plasticity in metastasis.

Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.

One-Carbon Metabolism Supports S-Adenosylmethionine and Histone Methylation to Drive Inflammatory Macrophages.

Activated macrophages adapt their metabolic pathways to drive the pro-inflammatory phenotype, but little is known about the biochemical underpinnings of this process. Here, we find that lipopolysaccharide (LPS) activates the pentose phosphate pathway, the serine synthesis pathway, and one-carbon metabolism, the synergism of which drives epigenetic reprogramming for interleukin-1ß (IL-1ß) expression. Glucose-derived ribose and one-carbon units fed by both glucose and serine metabolism are synergistically integrated into the methionine cycle through de novo ATP synthesis and fuel the generation of S-adenosylmethionine (SAM) during LPS-induced inflammation. Impairment of these metabolic pathways that feed SAM generation lead to anti-inflammatory outcomes, implicating SAM as an essential metabolite for inflammatory macrophages. Mechanistically, SAM generation maintains a relatively high SAM:S-adenosylhomocysteine ratio to support histone H3 lysine 36 trimethylation for IL-1ß production. We therefore identify a synergistic effect of glucose and amino acid metabolism on orchestrating SAM availability that is intimately linked to the chromatin state for inflammation.

Oncometabolites suppress DNA repair by disrupting local chromatin signalling.

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

Identification of differential m6A RNA methylomes and ALKBH5 as a potential prevention target in the developmental neurotoxicity induced by multiple sevoflurane exposures.

Sevoflurane, as a commonly used inhaled anesthetic for pediatric patients, has been reported that multiple sevoflurane exposures are associated with a greater risk of developing neurocognitive disorder. N6-Methyladenosine (m6A), as the most common mRNA modification in eukaryotes, has emerged as a crucial regulator of brain function in processes involving synaptic plasticity, learning and memory, and neurodevelopment. Nevertheless, the relevance of m6A RNA methylation in the multiple sevoflurane exposure-induced developmental neurotoxicity remains mostly elusive. Herein, we evaluated the genome-wide m6A RNA modification and gene expression in hippocampus of mice that received with multiple sevoflurane exposures using m6A-sequencing (m6A-seq) and RNA-sequencing (RNA-seq). We discovered 19 genes with differences in the m6A methylated modification and differential expression in the hippocampus. Among these genes, we determined that a total of nine differential expressed genes may be closely associated with the occurrence of developmental neurotoxicity induced by multiple sevoflurane exposures. We further found that the alkB homolog 5 (ALKBH5), but not methyltransferase-like 3 (METTL3) and Wilms tumor 1-associated protein (WTAP), were increased in the hippocampus of mice that received with multiple sevoflurane exposures. And the IOX1, as an inhibitor of ALKBH5, significantly improved the learning and memory defects and reduced neuronal damage in the hippocampus of mice induced by multiple sevoflurane exposures. The current study revealed the role of m6A methylated modification and m6A-related regulators in sevoflurane-induced cognitive impairment, which might provide a novel insight into identifying biomarkers and therapeutic strategies for inhaled anesthetic-induced developmental neurotoxicity.

Zhi-zi-chi decoction mitigates depression by enhancing lncRNA Six3os1 expression and promoting histone H3K4 methylation at the BDNF promoter.

Traditional Chinese medicine, particularly Zhi-zi-chi decoction (ZZCD), is gaining recognition as a potential treatment for depression. This study aimed to uncover the molecular mechanisms behind ZZCD's antidepressant effects, focusing on lncRNA Six3os1 and histone H3K4 methylation at the BDNF promoter. Network pharmacology and in vivo experiments were conducted to identify ZZCD targets and evaluate its impact on depression-related behaviours and neuron injury. The role of Six3os1 in recruiting KMT2A to the BDNF promoter and its effects on oxidative stress and neuron injury were investigated. ZZCD reduced depression-like behaviours and neuron injury in mice subjected to chronic stress. It upregulated Six3os1, which facilitated KMT2A recruitment to the BDNF promoter, leading to increased histone H3K4 methylation and enhanced BDNF expression. ZZCD also inhibited CORT-induced neuron injury, inflammatory response and oxidative stress in vitro. ZZCD's antidepressant properties involve Six3os1 upregulation, which exerts neuroprotective effects by inhibiting oxidative stress and neuron injury, thereby alleviating depressive symptoms. Targeting Six3os1 upregulation may offer a potential therapeutic intervention for depression.

Gubi decoction mitigates knee osteoarthritis via promoting chondrocyte autophagy through METTL3-mediated ATG7 m6A methylation.

Knee osteoarthritis (KOA) is a chronic joint disease that significantly affects the health of the elderly. As an herbal remedy, Gubi decoction (GBD) has been traditionally used for the treatment of osteoarthritis-related syndromes. However, the anti-KOA efficacy and mechanism of GBD remain unclear. This study aimed to experimentally investigate the anti-KOA efficacy and the underlying mechanism of GBD. The medial meniscus (DMM) mice model and IL-1ß-stimulated chondrocytes were, respectively, constructed as in vivo and in vitro models of KOA to evaluate the osteoprotective effect and molecular mechanism of GBD. The UPLC-MS/MS analysis showed that GBD mainly contained pinoresinol diglucoside, rehmannioside D, hesperidin, liquiritin, baohuoside I, glycyrrhizic acid, kaempferol and tangeretin. Animal experiment showed that GBD could alleviate articular cartilage destruction and recover histopathological alterations in DMM mice. In addition, GBD inhibited chondrocyte apoptosis and restored DMM-induced dysregulated autophagy evidenced by the upregulation of ATG7 and LC3 II/LC3 I but decreased P62 level. Mechanistically, METTL3-mediated m6A modification decreased the expression of ATG7 in DMM mice, as it could be significantly attenuated by GBD. METTL3 overexpression significantly counteracted the protective effect of GBD on chondrocyte autophagy. Further research showed that GBD promoted proteasome-mediated ubiquitination degradation of METLL3. Our findings suggest that GBD could act as a protective agent against KOA. The protective effect of GBD may result from its promotion on chondrocyte autophagy by suppressing METTL3-dependent ATG7 m6A methylation.

The role of N6-methyladenosine RNA methylation in the crosstalk of circadian clock and neuroinflammation in rodent suprachiasmatic nuclei.

N6-methyladenosine (m6A) is the most abundant epitranscriptomic mark that regulates the fate of RNA molecules. Recent studies have revealed a bidirectional interaction between m6A modification and the circadian clock. However, the precise temporal dynamics of m6A global enrichment in the central circadian pacemaker have not been fully elucidated. Our study investigates the relationship between FTO demethylase and molecular clocks in primary cells of the suprachiasmatic nucleus (SCN). In addition, we examined the effects of lipopolysaccharide (LPS) on Fto expression and the role of FTO in LPS-induced reactive oxygen species (ROS) production in primary SCN cell culture. We observed circadian rhythmicity in the global m6A levels, which mirrored the rhythmic expression of the Fto demethylase. Silencing FTO using siRNA reduced the mesor of Per2 rhythmicity in SCN primary cells and extended the period of the PER2 rhythm in SCN primary cell cultures from PER2::LUC mice. When examining the immune response, we discovered that exposure to LPS upregulated global m6A levels while downregulating Fto expression in SCN primary cell cultures. Interestingly, we found a loss of circadian rhythmicity in Fto expression following LPS treatment, indicating that the decrease of FTO levels may contribute to m6A upregulation without directly regulating its circadian rhythm. To explore potential protective mechanisms against neurotoxic inflammation, we examined ROS production following LPS treatment in SCN primary cell cultures pretreated with FTO siRNA. We observed a time-dependent pattern of ROS induction, with significant peak at 32 h but not at 20 h after synchronization. Silencing the FTO demethylase abolished ROS induction following LPS exposure, supporting the hypothesis that FTO downregulation serves as a protective mechanism during LPS-induced neuroinflammation in SCN primary cell cultures.

RNA m6 A methylation regulates sorafenib resistance in liver cancer through FOXO3-mediated autophagy.

N6-methyladenosine (m6 A) is an abundant nucleotide modification in mRNA, known to regulate mRNA stability, splicing, and translation, but it is unclear whether it is also has a physiological role in the intratumoral microenvironment and cancer drug resistance. Here, we find that METTL3, a primary m6 A methyltransferase, is significantly down-regulated in human sorafenib-resistant hepatocellular carcinoma (HCC). Depletion of METTL3 under hypoxia promotes sorafenib resistance and expression of angiogenesis genes in cultured HCC cells and activates autophagy-associated pathways. Mechanistically, we have identified FOXO3 as a key downstream target of METTL3, with m6 A modification of the FOXO3 mRNA 3'-untranslated region increasing its stability through a YTHDF1-dependent mechanism. Analysis of clinical samples furthermore showed that METTL3 and FOXO3 levels are tightly correlated in HCC patients. In mouse xenograft models, METTL3 depletion significantly enhances sorafenib resistance of HCC by abolishing the identified METTL3-mediated FOXO3 mRNA stabilization, and overexpression of FOXO3 restores m6 A-dependent sorafenib sensitivity. Collectively, our work reveals a critical function for METTL3-mediated m6 A modification in the hypoxic tumor microenvironment and identifies FOXO3 as an important target of m6 A modification in the resistance of HCC to sorafenib therapy.

Full methylation of H3K27 by PRC2 is dispensable for initial embryoid body formation but required to maintain differentiated cell identity.

Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 on lysine 27 and is required for normal development of complex eukaryotes. The nature of that requirement is not clear. H3K27me3 is associated with repressed genes, but the modification is not sufficient to induce repression and, in some instances, is not required. We blocked full methylation of H3K27 with both a small molecule inhibitor, GSK343, and by introducing a point mutation into EZH2, the catalytic subunit of PRC2, in the mouse CJ7 cell line. Cells with substantively decreased H3K27 methylation differentiate into embryoid bodies, which contrasts with EZH2 null cells. PRC2 targets had varied requirements for H3K27me3, with a subset that maintained normal levels of repression in the absence of methylation. The primary cellular phenotype of blocked H3K27 methylation was an inability of altered cells to maintain a differentiated state when challenged. This phenotype was determined by H3K27 methylation in embryonic stem cells through the first 4 days of differentiation. Full H3K27 methylation therefore was not necessary for formation of differentiated cell states during embryoid body formation but was required to maintain a stable differentiated state.

Deoxynivalenol induces m6A-mediated upregulation of p21 and growth arrest of mouse hippocampal neuron cells in vitro.

Hippocampal neurons maintain the ability of proliferation throughout life to support neurogenesis. Deoxynivalenol (DON) is a mycotoxin that exhibits brain toxicity, yet whether and how DON affects hippocampal neurogenesis remains unknown. Here, we use mouse hippocampal neuron cells (HT-22) as a model to illustrate the effects of DON on neuron proliferation and to explore underlying mechanisms. DON exposure significantly inhibits the proliferation of HT-22 cells, which is associated with an up-regulation of cell cycle inhibitor p21 at both mRNA and protein levels. Global and site-specific m6A methylation levels on the 3'UTR of p21 mRNA are significantly increased in response to DON treatment, whereas inhibition of m6A hypermethylation significantly alleviates DON-induced cell cycle arrest. Further mechanistic studies indicate that the m6A readers YTHDF1 and IGF2BP1 are responsible for m6A-mediated increase in p21 mRNA stability. Meanwhile, 3'UTR of E3 ubiquitin ligase TRIM21 mRNA is also m6A hypermethylated, and another m6A reader YTHDF2 binds to the m6A sites, leading to decreased TRIM21 mRNA stability. Consequently, TRIM21 suppression impairs ubiquitin-mediated p21 protein degradation. Taken together, m6A-mediated upregulation of p21, at both post-transcriptional and post-translational levels, contributes to DON-induced inhibition of hippocampal neuron proliferation. These results may provide new insights for epigenetic therapy of neurodegenerative diseases.

Mitochondrial translation requires folate-dependent tRNA methylation.

Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine. Antifolates are important immunosuppressive and anticancer agents. In proliferating lymphocytes and human cancers, mitochondrial folate enzymes are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of the catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not of other folate enzymes, leads to defective oxidative phosphorylation in human cells due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors to produce the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling in SHMT2-knockout human cells reveals that the lack of this modified base causes defective translation, with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in the impaired expression of respiratory chain enzymes. Stalling at these specific codons also occurs in certain inborn errors of mitochondrial metabolism. Disruption of whole-cell folate metabolism, by either folate deficiency or antifolate treatment, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for mitochondrial translation and thus oxidative phosphorylation.

M6A RNA Methylation-Mediated Dysregulation of AGAP2-AS1 Promotes Trastuzumab Resistance of Breast Cancer.

INTRODUCTION: Trastuzumab is commonly used to treat human epidermal growth factor receptor-2-positive (HER2+) breast cancer, but its efficacy is often limited by chemotherapy resistance. Recent studies have indicated that long non-coding RNAs (lncRNAs) play important roles in tumor progression and response to therapy. However, the regulatory mechanisms associating lncRNAs and trastuzumab resistance remain unknown. METHODS: Quantitative polymerase chain reaction was performed to detect the expression of related genes. Western blot and immunofluorescence assays were used to evaluate protein expression levels. A series of gain- or loss-of-function assays confirmed the function of AGAP2-AS1 in trastuzumab resistance, both in vitro and in vivo. RNA immunoprecipitation and pull-down analyses were conducted to verify the interaction between METTL3/YTHDF2 and lncRNA AGAP2-AS1. RESULTS: AGAP2-AS1 was upregulated in trastuzumab-resistant cells and SKBR-3R-generated xenografts in nude mice. Silencing AGAP2-AS1 significantly decreased trastuzumab-induced cytotoxicity both in vitro and in vivo. Furthermore, m6A methylation of AGAP2-AS1 was reduced in trastuzumab-resistant cells compared to that in parental cells. In addition, METTL3 increased m6A methylation of AGAP2-AS1, which finally induced the suppressed AGAP2-AS1 expression. Moreover, YTHDF2 was essential for METTL3-mediated m6A methylation of AGAP2-AS1. Functionally, AGAP2-AS1 regulated trastuzumab resistance by inducing autophagy and increasing ATG5 expression. CONCLUSION: we demonstrated that METTL3/YTHDF2-mediated m6A methylation increased the expression of AGAP2-AS1, which could promote trastuzumab resistance in breast cancer. AGAP2-AS1 regulates trastuzumab resistance by inducing autophagy. Therefore, AGAP2-AS1 may be a promising predictive biomarker and therapeutic target in patients with breast cancer.

Depletion of H3K36me2 recapitulates epigenomic and phenotypic changes induced by the H3.3K36M oncohistone mutation.

Hotspot histone H3 mutations have emerged as drivers of oncogenesis in cancers of multiple lineages. Specifically, H3 lysine 36 to methionine (H3K36M) mutations are recurrently identified in chondroblastomas, undifferentiated sarcomas, and head and neck cancers. While the mutation reduces global levels of both H3K36 dimethylation (H3K36me2) and trimethylation (H3K36me3) by dominantly inhibiting their respective specific methyltransferases, the relative contribution of these methylation states to the chromatin and phenotypic changes associated with H3K36M remains unclear. Here, we specifically deplete H3K36me2 or H3K36me3 in mesenchymal cells, using CRISPR-Cas9 to separately knock out the corresponding methyltransferases NSD1/2 or SETD2. By profiling and comparing the epigenomic and transcriptomic landscapes of these cells with cells expressing the H3.3K36M oncohistone, we find that the loss of H3K36me2 could largely recapitulate H3.3K36M's effect on redistribution of H3K27 trimethylation (H3K27me3) and gene expression. Consistently, knockout of Nsd1/2, but not Setd2, phenocopies the differentiation blockade and hypersensitivity to the DNA-hypomethylating agent induced by H3K36M. Together, our results support a functional divergence between H3K36me2 and H3K36me3 and their nonredundant roles in H3K36M-driven oncogenesis.

The critical role of SETDB1-mediated CCND1/PI3K/AKT pathway via p53-RS di-methylation at K370 in the proliferation of WRL68 cells induced by nicotine.

Constituents of cigarette smoke are known to be carcinogens. Additionally, there is mounting evidence that the liver is an organ susceptible to tobacco carcinogenicity. Nicotine, the primary constituent of tobacco, plays a role in cancer progression. In our previous study, it was found that nicotine enhances the proliferation of a human normal fetal hepatic (WRL68) cell due to the activation of p53 mutation at Ser249 (p53-RS)/STAT1/CCND1 signaling pathway. Here, we further elucidated the mechanism of regulating this pathway. Firstly, dose-dependent increase of SETDB1 protein level in WRL68 cells upon exposure to nicotine (1.25, 2.5, and 5 µM), significantly enhanced cellular proliferation. In addition, the upregulation of SETDB1 protein was necessary for the nuclear translocation of p53-RS to establish a ternary complex with STAT1 and SETDB1, which facilitated p53-RS di-methylation at K370 (p53-RS/K370me2). After that, the activation of CCND1/PI3K/AKT pathway was initiated when STAT1 stability was enhanced by p53-RS/K370me2, ultimately resulting in cell proliferation. Altogether, the study revealed that the increase in SETDB1 expression could potentially have a significant impact on the activation of CCND1/PI3K/AKT pathway through p53-RS/K370me2, leading to the proliferation of WRL68 cells induced by nicotine, which could contribute to hepatocellular carcinoma for smokers. Besides, the results of this study provided a foundation for the development of anticancer therapies for cancers associated with tobacco use.

RLB (RICE LATERAL BRANCH) recruits PRC2-mediated H3K27 tri-methylation on OsCKX4 to regulate lateral branching.

Lateral branches such as shoot and panicle are determining factors and target traits for rice (Oryza sativa L.) yield improvement. Cytokinin promotes rice lateral branching; however, the mechanism underlying the fine-tuning of cytokinin homeostasis in rice branching remains largely unknown. Here, we report the map-based cloning of RICE LATERAL BRANCH (RLB) encoding a nuclear-localized, KNOX-type homeobox protein from a rice cytokinin-deficient mutant showing more tillers, sparser panicles, defected floret morphology as well as attenuated shoot regeneration from callus. RLB directly binds to the promoter and represses the transcription of OsCKX4, a cytokinin oxidase gene with high abundance in panicle branch meristem. OsCKX4 over-expression lines phenocopied rlb, which showed upregulated OsCKX4 levels. Meanwhile, RLB physically binds to Polycomb repressive complex 2 (PRC2) components OsEMF2b and co-localized with H3K27me3, a suppressing histone modification mediated by PRC2, in the OsCKX4 promoter. We proposed that RLB recruits PRC2 to the OsCKX4 promoter to epigenetically repress its transcription, which suppresses the catabolism of cytokinin, thereby promoting rice lateral branching. Moreover, antisense inhibition of OsCKX4 under the LOG promoter successfully increased panicle size and spikelet number per plant without affecting other major agronomic traits. This study provides insight into cytokinin homeostasis, lateral branching in plants, and also promising target genes for rice genetic improvement.

Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription.

Heterochromatic DNA domains have important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. From fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway. Typically, sRNAs, originating from long noncoding RNAs, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and trimethylation (H3K9me2 and H3K9me3, respectively) and the formation of heterochromatin. H3K9me is in turn required for the recruitment of RNAi to chromatin to promote the amplification of sRNA. Yet, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast Schizosaccharomyces pombe homologue of mammalian SUV39H H3K9 methyltransferases, we design active-site mutations that block H3K9me3, but allow H3K9me2 catalysis. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing at pericentromeric DNA repeats. Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, the transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct chromatin states and uncover a mechanism for the formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in sRNA-mediated genome defence.

Post-translational modification of RNA m6A demethylase ALKBH5 regulates ROS-induced DNA damage response.

Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N6-methyladenosine (m6A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m6A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m6A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m6A levels that protect genomic integrity of cells in response to ROS.