RESUMO
Plant immunity is activated upon pathogen perception and often affects growth and yield when it is constitutively active. How plants fine-tune immune homeostasis in their natural habitats remains elusive. Here, we discover a conserved immune suppression network in cereals that orchestrates immune homeostasis, centering on a Ca2+-sensor, RESISTANCE OF RICE TO DISEASES1 (ROD1). ROD1 promotes reactive oxygen species (ROS) scavenging by stimulating catalase activity, and its protein stability is regulated by ubiquitination. ROD1 disruption confers resistance to multiple pathogens, whereas a natural ROD1 allele prevalent in indica rice with agroecology-specific distribution enhances resistance without yield penalty. The fungal effector AvrPiz-t structurally mimics ROD1 and activates the same ROS-scavenging cascade to suppress host immunity and promote virulence. We thus reveal a molecular framework adopted by both host and pathogen that integrates Ca2+ sensing and ROS homeostasis to suppress plant immunity, suggesting a principle for breeding disease-resistant, high-yield crops.
Assuntos
Cálcio/metabolismo , Sequestradores de Radicais Livres/metabolismo , Proteínas Fúngicas/metabolismo , Oryza/imunologia , Imunidade Vegetal , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sistemas CRISPR-Cas/genética , Membrana Celular/metabolismo , Resistência à Doença/genética , Modelos Biológicos , Oryza/genética , Doenças das Plantas/imunologia , Proteínas de Plantas/genética , Ligação Proteica , Estabilidade Proteica , Reprodução , Especificidade da Espécie , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Zea mays/imunologiaRESUMO
The discovery and application of genome editing introduced a new era of plant breeding by giving researchers efficient tools for the precise engineering of crop genomes1. Here we demonstrate the power of genome editing for engineering broad-spectrum disease resistance in rice (Oryza sativa). We first isolated a lesion mimic mutant (LMM) from a mutagenized rice population. We then demonstrated that a 29-base-pair deletion in a gene we named RESISTANCE TO BLAST1 (RBL1) caused broad-spectrum disease resistance and showed that this mutation caused an approximately 20-fold reduction in yield. RBL1 encodes a cytidine diphosphate diacylglycerol synthase that is required for phospholipid biosynthesis2. Mutation of RBL1 results in reduced levels of phosphatidylinositol and its derivative phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In rice, PtdIns(4,5)P2 is enriched in cellular structures that are specifically associated with effector secretion and fungal infection, suggesting that it has a role as a disease-susceptibility factor3. By using targeted genome editing, we obtained an allele of RBL1, named RBL1Δ12, which confers broad-spectrum disease resistance but does not decrease yield in a model rice variety, as assessed in small-scale field trials. Our study has demonstrated the benefits of editing an LMM gene, a strategy relevant to diverse LMM genes and crops.
Assuntos
Diacilglicerol Colinofosfotransferase , Resistência à Doença , Edição de Genes , Oryza , Melhoramento Vegetal , Doenças das Plantas , Resistência à Doença/genética , Edição de Genes/métodos , Genoma de Planta/genética , Oryza/enzimologia , Oryza/genética , Oryza/microbiologia , Fosfatidilinositóis/metabolismo , Melhoramento Vegetal/métodos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Alelos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Diacilglicerol Colinofosfotransferase/genética , Diacilglicerol Colinofosfotransferase/metabolismoRESUMO
TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are excised by mammalian DNA glycosylase TDG, implicating 5mC oxidation in DNA demethylation. Here, we show that the genomic locations of 5fC can be determined by coupling chemical reduction with biotin tagging. Genome-wide mapping of 5fC in mouse embryonic stem cells (mESCs) reveals that 5fC preferentially occurs at poised enhancers among other gene regulatory elements. Application to Tdg null mESCs further suggests that 5fC production coordinates with p300 in remodeling epigenetic states of enhancers. This process, which is not influenced by 5hmC, appears to be associated with further oxidation of 5hmC and commitment to demethylation through 5fC. Finally, we resolved 5fC at base resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of 5fC accumulation. Our results reveal roles of active 5mC/5hmC oxidation and TDG-mediated demethylation in epigenetic tuning at regulatory elements.
Assuntos
Citosina/análogos & derivados , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Técnicas Genéticas , Estudo de Associação Genômica Ampla , 5-Metilcitosina/metabolismo , Animais , Citosina/metabolismo , Camundongos , Elementos Reguladores de Transcrição , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Diabetes mellitus is prevalent among women of reproductive age, and many women are left undiagnosed or untreated1. Gestational diabetes has profound and enduring effects on the long-term health of the offspring2,3. However, the link between pregestational diabetes and disease risk into adulthood in the next generation has not been sufficiently investigated. Here we show that pregestational hyperglycaemia renders the offspring more vulnerable to glucose intolerance. The expression of TET3 dioxygenase, responsible for 5-methylcytosine oxidation and DNA demethylation in the zygote4, is reduced in oocytes from a mouse model of hyperglycaemia (HG mice) and humans with diabetes. Insufficient demethylation by oocyte TET3 contributes to hypermethylation at the paternal alleles of several insulin secretion genes, including the glucokinase gene (Gck), that persists from zygote to adult, promoting impaired glucose homeostasis largely owing to the defect in glucose-stimulated insulin secretion. Consistent with these findings, mouse progenies derived from the oocytes of maternal heterozygous and homozygous Tet3 deletion display glucose intolerance and epigenetic abnormalities similar to those from the oocytes of HG mice. Moreover, the expression of exogenous Tet3 mRNA in oocytes from HG mice ameliorates the maternal effect in offspring. Thus, our observations suggest an environment-sensitive window in oocyte development that confers predisposition to glucose intolerance in the next generation through TET3 insufficiency rather than through a direct perturbation of the oocyte epigenome. This finding suggests a potential benefit of pre-conception interventions in mothers to protect the health of offspring.
Assuntos
Dioxigenases , Intolerância à Glucose , Hiperglicemia , Oócitos , Adulto , Animais , Dioxigenases/metabolismo , Feminino , Glucose/metabolismo , Intolerância à Glucose/genética , Intolerância à Glucose/metabolismo , Humanos , Hiperglicemia/complicações , Hiperglicemia/genética , Hiperglicemia/metabolismo , Herança Materna , Camundongos , Oócitos/metabolismoRESUMO
Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.
Assuntos
Células-Tronco Embrionárias/citologia , Técnicas Genéticas , Camundongos Transgênicos , Animais , Blastocisto/citologia , Núcleo Celular/metabolismo , Feminino , Marcação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/citologia , Oócitos/metabolismoRESUMO
Cell-cell communication via ligand-receptor signaling is a fundamental feature of complex organs. Despite this, the global landscape of intercellular signaling in mammalian liver has not been elucidated. Here we perform single-cell RNA sequencing on non-parenchymal cells isolated from healthy and NASH mouse livers. Secretome gene analysis revealed a highly connected network of intrahepatic signaling and disruption of vascular signaling in NASH. We uncovered the emergence of NASH-associated macrophages (NAMs), which are marked by high expression of triggering receptors expressed on myeloid cells 2 (Trem2), as a feature of mouse and human NASH that is linked to disease severity and highly responsive to pharmacological and dietary interventions. Finally, hepatic stellate cells (HSCs) serve as a hub of intrahepatic signaling via HSC-derived stellakines and their responsiveness to vasoactive hormones. These results provide unprecedented insights into the landscape of intercellular crosstalk and reprogramming of liver cells in health and disease.
Assuntos
Comunicação Celular/genética , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Análise de Sequência de RNA , Animais , Reprogramação Celular/genética , Modelos Animais de Doenças , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Humanos , Ligantes , Fígado/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais/genética , Análise de Célula ÚnicaRESUMO
BACKGROUND: Cell phenotype switching is increasingly being recognized in atherosclerosis. However, our understanding of the exact stimuli for such cellular transformations and their significance for human atherosclerosis is still evolving. Intraplaque hemorrhage is thought to be a major contributor to plaque progression in part by stimulating the influx of CD163+ macrophages. Here, we explored the hypothesis that CD163+ macrophages cause plaque progression through the induction of proapoptotic endothelial-to-mesenchymal transition (EndMT) within the fibrous cap. METHODS: Human coronary artery sections from CVPath's autopsy registry were selected for pathological analysis. Athero-prone ApoE-/- and ApoE-/-/CD163-/- mice were used for in vivo studies. Human peripheral blood mononuclear cell-induced macrophages and human aortic endothelial cells were used for in vitro experiments. RESULTS: In 107 lesions with acute coronary plaque rupture, 55% had pathological evidence of intraplaque hemorrhage in nonculprit vessels/lesions. Thinner fibrous cap, greater CD163+ macrophage accumulation, and a larger number of CD31/FSP-1 (fibroblast specific protein-1) double-positive cells and TUNEL (terminal deoxynucleotidyl transferase-dUTP nick end labeling) positive cells in the fibrous cap were observed in nonculprit intraplaque hemorrhage lesions, as well as in culprit rupture sections versus nonculprit fibroatheroma sections. Human aortic endothelial cells cultured with supernatants from hemoglobin/haptoglobin-exposed macrophages showed that increased mesenchymal marker proteins (transgelin and FSP-1) while endothelial markers (VE-cadherin and CD31) were reduced, suggesting EndMT induction. Activation of NF-κB (nuclear factor kappa ß) signaling by proinflammatory cytokines released from CD163+ macrophages directly regulated the expression of Snail, a critical transcription factor during EndMT induction. Western blot analysis for cleaved caspase-3 and microarray analysis of human aortic endothelial cells indicated that apoptosis was stimulated during CD163+ macrophage-induced EndMT. Additionally, CD163 deletion in athero-prone mice suggested that CD163 is required for EndMT and plaque progression. Using single-cell RNA sequencing from human carotid endarterectomy lesions, a population of EndMT was detected, which demonstrated significant upregulation of apoptosis-related genes. CONCLUSIONS: CD163+ macrophages provoke EndMT, which may promote plaque progression through fibrous cap thinning.
Assuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Macrófagos , Placa Aterosclerótica , Receptores de Superfície Celular , Humanos , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Animais , Antígenos CD/metabolismo , Antígenos CD/genética , Macrófagos/metabolismo , Macrófagos/patologia , Placa Aterosclerótica/patologia , Placa Aterosclerótica/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/genética , Camundongos , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Masculino , Camundongos Knockout para ApoE , Camundongos Endogâmicos C57BL , Apoptose , Feminino , Transição Epitelial-Mesenquimal , Vasos Coronários/patologia , Vasos Coronários/metabolismoRESUMO
Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7 and 9, play key roles in multiple important developmental and differentiation processes. Recent studies have shown that class IIa HDACs exert their transcriptional repressive function by interacting with tissue-specific transcription factors, such as members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. However, the molecular mechanism is not well understood. In this study, we determined the crystal structure of an HDAC4-MEF2A-DNA complex. This complex adopts a dumbbell-shaped overall architecture, with a 2:4:2 stoichiometry of HDAC4, MEF2A and DNA molecules. In the complex, two HDAC4 molecules form a dimer through the interaction of their glutamine-rich domain (GRD) to form the stem of the 'dumbbell'; while two MEF2A dimers and their cognate DNA molecules are bridged by the HDAC4 dimer. Our structural observations were then validated using biochemical and mutagenesis assays. Further cell-based luciferase reporter gene assays revealed that the dimerization of HDAC4 is crucial in its ability to repress the transcriptional activities of MEF2 proteins. Taken together, our findings not only provide the structural basis for the assembly of the HDAC4-MEF2A-DNA complex but also shed light on the molecular mechanism of HDAC4-mediated long-range gene regulation.
Assuntos
DNA , Histona Desacetilases , Fatores de Transcrição MEF2 , Proteínas Repressoras , DNA/química , DNA/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/metabolismo , Fatores de Regulação Miogênica/química , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Humanos , Histona Desacetilases/química , Histona Desacetilases/metabolismoRESUMO
Receptor-like kinases (RLKs) may initiate signaling pathways by perceiving and transmitting environmental signals to cellular machinery and play diverse roles in plant development and stress responses. The rice genome encodes more than one thousand RLKs, but only a small number have been characterized as receptors for phytohormones, polypeptides, elicitors, and effectors. Here, we screened the function of 11 RLKs in rice resistance to the blast fungus Magnaporthe oryzae (M. oryzae) and identified a negative regulator named BDR1 (Blast Disease Resistance 1). The expression of BDR1 was rapidly increased under M. oryzae infection, while silencing or knockout of BDR1 significantly enhanced M. oryzae resistance in two rice varieties. Protein interaction and kinase activity assays indicated that BDR1 directly interacted with and phosphorylated mitogen-activated kinase 3 (MPK3). Knockout of BDR1 compromised M. oryzae-induced MPK3 phosphorylation levels. Moreover, transcriptome analysis revealed that M. oryzae-elicited jasmonate (JA) signaling and terpenoid biosynthesis pathway were negatively regulated by BDR1 and MPK3. Mutation of JA biosynthetic (allene oxide cyclase (AOC)/signaling (MYC2) genes decreased rice resistance to M. oryzae. Besides diterpenoid, the monoterpene linalool and the sesquiterpene caryophyllene were identified as unique defensive compounds against M. oryzae, and their biosynthesis genes (TPS3 and TPS29) were transcriptionally regulated by JA signaling and suppressed by BDR1 and MPK3. These findings demonstrate the existence of a BDR1-MPK3 cascade that negatively mediates rice blast resistance by affecting JA-related defense responses.
Assuntos
Magnaporthe , Oryza , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Transdução de Sinais , Reguladores de Crescimento de Plantas/metabolismo , Oryza/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Resistência à Doença/genética , Magnaporthe/fisiologiaRESUMO
The WRINKLED1 (WRI1) and LAFL [LEAFY COTYLEDON1 (LEC1), ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEC2] transcription factors play essential roles in governing seed development and oil biosynthesis. To gain a comprehensive understanding of the transcriptional regulation of WRI1 and LAFL, we conducted genome-wide association studies for the expression profiles of WRI1 and LAFL in developing seeds at 20 and 40 days after flowering (DAF) using 302 rapeseed (Brassica napus) accessions. We identified a total of 237 expression quantitative trait nucleotides (eQTNs) and 51 expression QTN-by-environment interactions (eQEIs) associated with WRI1 and LAFL. Around these eQTNs and eQEIs, we pinpointed 41 and 8 candidate genes with known transcriptional regulations or protein interactions with their expression traits, respectively. Based on RNA-seq and ATAC-seq data, we employed the XGBoost and Basenji models which predicted 15 candidate genes potentially regulating the expression of WRI1 and LAFL. We further validated the predictions via tissue expression profile, haplotype analysis, and expression correlation analysis, and verified the transcriptional activation activity of BnaC03.MYB56 (R2R3-MYB transcription factor 56) on the expression of BnaA09.LEC1 by dual-luciferase reporter and yeast one-hybrid assays. BnaA10.AGL15 (AGAMOUS-LIKE 15), BnaC04.VAL1 (VIVIPAROUS1/ABSCISIC ACID INSENSITIVE3-LIKE 1), BnaC03.MYB56, and BnaA10.MYB56 were co-expressed with WRI1 and LAFL at 20 DAF in M35, a key module for seed development and oil biosynthesis. We further validated the positive regulation of MYB56 on seed oil accumulation using Arabidopsis (Arabidopsis thaliana) mutants. This study not only delivers a framework for future eQEI identification but also offers insights into the developmental regulation of seed oil accumulation.
RESUMO
The Zn content in cereal seeds is an important trait for crop production as well as for human health. However, little is known about how Zn is loaded to plant seeds. Here, through a genome-wide association study (GWAS), we identify the Zn-NA (nicotianamine) transporter gene ZmYSL2 that is responsible for loading Zn to maize kernels. High promoter sequence variation in ZmYSL2 most likely drives the natural variation in Zn concentrations in maize kernels. ZmYSL2 is specifically localized on the plasma membrane facing the maternal tissue of the basal endosperm transfer cell layer (BETL) and functions in loading Zn-NA into the BETL. Overexpression of ZmYSL2 increases the Zn concentration in the kernels by 31.6%, which achieves the goal of Zn biofortification of maize. These findings resolve the mystery underlying the loading of Zn into plant seeds, providing an efficient strategy for breeding or engineering maize varieties with enriched Zn nutrition.
Assuntos
Estudo de Associação Genômica Ampla , Zea mays , Humanos , Zea mays/genética , Zea mays/metabolismo , Zinco/metabolismo , Melhoramento Vegetal , Sementes/genética , Proteínas de Membrana Transportadoras/genéticaRESUMO
BACKGROUND: Polygenic risk scores (PRSs) for coronary artery disease (CAD) potentially improve cardiovascular risk prediction. However, their relationship with histopathologic features of CAD has never been examined systematically. METHODS: From 4327 subjects referred to CVPath by the State of Maryland Office Chief Medical Examiner for sudden death between 1994 and 2015, 2455 cases were randomly selected for genotyping. We generated PRS from 291 known CAD risk loci. Detailed histopathologic examination of the coronary arteries was performed in all subjects. The primary study outcome measurements were histopathologic plaque features determining severity of atherosclerosis, including %stenosis, calcification, thin-cap fibroatheromas, and thrombotic CAD. RESULTS: After exclusion of cases with insufficient DNA sample quality or with missing data, 954 cases (mean age, 48.8±14.7 years; 75.7% men) remained in the final study cohort. Subjects in the highest PRS quintile exhibited more severe atherosclerosis compared with subjects in the lowest quintile, with greater %stenosis (80.3%±27.0% versus 50.4%±38.7%; adjusted P<0.001) and a higher frequency of calcification (69.6% versus 35.8%; adjusted P=0.004) and thin-cap fibroatheroma (26.7% versus 9.5%; adjusted P=0.007). Even after adjustment for traditional CAD risk factors, subjects within the highest PRS quintile had higher odds of severe atherosclerosis (ie, ≥75% stenosis; adjusted odds ratio, 3.77 [95% CI, 2.10-6.78]; P<0.001) and plaque rupture (adjusted odds ratio, 4.05 [95% CI, 2.26-7.24]; P<0.001). Moreover, subjects within the highest quintile had higher odds of CAD-associated cause of death, especially among those aged ≤50 years (adjusted odds ratio, 4.08 [95% CI, 2.01-8.30]; P<0.001). No statistically significant associations were observed with plaque erosion after adjusting for covariates. CONCLUSIONS: This is the first autopsy study investigating associations between PRS and atherosclerosis severity at the histopathologic level in subjects with sudden death. Our pathological analysis suggests PRS correlates with plaque burden and features of advanced atherosclerosis and may be useful as a method for CAD risk stratification, especially in younger subjects.
Assuntos
Aterosclerose , Doença da Artéria Coronariana , Placa Aterosclerótica , Masculino , Humanos , Adulto , Pessoa de Meia-Idade , Feminino , Estratificação de Risco Genético , Constrição Patológica , Fatores de Risco , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Morte Súbita , AutopsiaRESUMO
SF3B1 is the most commonly mutated RNA splicing factor in cancer1-4, but the mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here we integrated pan-cancer splicing analyses with a positive-enrichment CRISPR screen to prioritize splicing alterations that promote tumorigenesis. We report that diverse SF3B1 mutations converge on repression of BRD9, which is a core component of the recently described non-canonical BAF chromatin-remodelling complex that also contains GLTSCR1 and GLTSCR1L5-7. Mutant SF3B1 recognizes an aberrant, deep intronic branchpoint within BRD9 and thereby induces the inclusion of a poison exon that is derived from an endogenous retroviral element and subsequent degradation of BRD9 mRNA. Depletion of BRD9 causes the loss of non-canonical BAF at CTCF-associated loci and promotes melanomagenesis. BRD9 is a potent tumour suppressor in uveal melanoma, such that correcting mis-splicing of BRD9 in SF3B1-mutant cells using antisense oligonucleotides or CRISPR-directed mutagenesis suppresses tumour growth. Our results implicate the disruption of non-canonical BAF in the diverse cancer types that carry SF3B1 mutations and suggest a mechanism-based therapeutic approach for treating these malignancies.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Neoplasias/genética , Splicing de RNA , Spliceossomos/metabolismo , Animais , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias/patologia , Fosfoproteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Spliceossomos/genética , Fatores de Transcrição/metabolismoRESUMO
Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by ten-eleven translocation (TET) dioxygenases results in a cascade of additional epigenetic marks and promotes demethylation of DNA in mammals1,2. However, the enzymatic activity and function of TET homologues in other eukaryotes remains largely unexplored. Here we show that the green alga Chlamydomonas reinhardtii contains a 5mC-modifying enzyme (CMD1) that is a TET homologue and catalyses the conjugation of a glyceryl moiety to the methyl group of 5mC through a carbon-carbon bond, resulting in two stereoisomeric nucleobase products. The catalytic activity of CMD1 requires Fe(II) and the integrity of its binding motif His-X-Asp, which is conserved in Fe-dependent dioxygenases3. However, unlike previously described TET enzymes, which use 2-oxoglutarate as a co-substrate4, CMD1 uses L-ascorbic acid (vitamin C) as an essential co-substrate. Vitamin C donates the glyceryl moiety to 5mC with concurrent formation of glyoxylic acid and CO2. The vitamin-C-derived DNA modification is present in the genome of wild-type C. reinhardtii but at a substantially lower level in a CMD1 mutant strain. The fitness of CMD1 mutant cells during exposure to high light levels is reduced. LHCSR3, a gene that is critical for the protection of C. reinhardtii from photo-oxidative damage under high light conditions, is hypermethylated and downregulated in CMD1 mutant cells compared to wild-type cells, causing a reduced capacity for photoprotective non-photochemical quenching. Our study thus identifies a eukaryotic DNA base modification that is catalysed by a divergent TET homologue and unexpectedly derived from vitamin C, and describes its role as a potential epigenetic mark that may counteract DNA methylation in the regulation of photosynthesis.
Assuntos
5-Metilcitosina/metabolismo , Proteínas de Algas/metabolismo , Ácido Ascórbico/metabolismo , Biocatálise , Chlamydomonas reinhardtii/enzimologia , DNA/química , DNA/metabolismo , 5-Metilcitosina/química , Dióxido de Carbono/metabolismo , Metilação de DNA , Glioxilatos/metabolismo , Nucleosídeos/química , Nucleosídeos/metabolismo , FotossínteseRESUMO
The stability of mRNAs is regulated by signals within their sequences, but a systematic and predictive understanding of the underlying sequence rules remains elusive. Here we introduce UTR-seq, a combination of massively parallel reporter assays and regression models, to survey the dynamics of tens of thousands of 3' UTR sequences during early zebrafish embryogenesis. UTR-seq revealed two temporal degradation programs: a maternally encoded early-onset program and a late-onset program that accelerated degradation after zygotic genome activation. Three signals regulated early-onset rates: stabilizing poly-U and UUAG sequences and destabilizing GC-rich signals. Three signals explained late-onset degradation: miR-430 seeds, AU-rich sequences, and Pumilio recognition sites. Sequence-based regression models translated 3' UTRs into their unique decay patterns and predicted the in vivo effect of sequence signals on mRNA stability. Their application led to the successful design of artificial 3' UTRs that conferred specific mRNA dynamics. UTR-seq provides a general strategy to uncover the rules of RNA cis regulation.
Assuntos
Regiões 3' não Traduzidas , Embrião não Mamífero/metabolismo , Genes Reporter , Estabilidade de RNA , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Zigoto/metabolismo , Animais , Embrião não Mamífero/citologia , Regulação da Expressão Gênica , MicroRNAs , RNA Mensageiro , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Zigoto/crescimento & desenvolvimentoRESUMO
Genomic imprinting is an allelic gene expression phenomenon primarily controlled by allele-specific DNA methylation at the imprinting control region (ICR), but the underlying mechanism remains largely unclear. N-α-acetyltransferase 10 protein (Naa10p) catalyzes N-α-acetylation of nascent proteins, and mutation of human Naa10p is linked to severe developmental delays. Here we report that Naa10-null mice display partial embryonic lethality, growth retardation, brain disorders, and maternal effect lethality, phenotypes commonly observed in defective genomic imprinting. Genome-wide analyses further revealed global DNA hypomethylation and enriched dysregulation of imprinted genes in Naa10p-knockout embryos and embryonic stem cells. Mechanistically, Naa10p facilitates binding of DNA methyltransferase 1 (Dnmt1) to DNA substrates, including the ICRs of the imprinted allele during S phase. Moreover, the lethal Ogden syndrome-associated mutation of human Naa10p disrupts its binding to the ICR of H19 and Dnmt1 recruitment. Our study thus links Naa10p mutation-associated Ogden syndrome to defective DNA methylation and genomic imprinting.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Deficiências do Desenvolvimento/genética , Epigênese Genética , Impressão Genômica , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , RNA Longo não Codificante/genética , Animais , DNA/genética , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Deleção de Genes , Genes Letais , Estudo de Associação Genômica Ampla , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/patologia , Acetiltransferase N-Terminal A/deficiência , Acetiltransferase N-Terminal E/deficiência , Ligação Proteica , RNA Longo não Codificante/metabolismo , Fase S/genéticaRESUMO
Oncogenic imbalance of DNA methylation is well recognized in cancer development. The ten-eleven translocation (TET) family of dioxygenases, which facilitates DNA demethylation, is frequently dysregulated in cancers. How such dysregulation contributes to tumorigenesis remains poorly understood, especially in solid tumors which present infrequent mutational incidence of TET genes. Here, we identify loss-of-function mutations of TET in 7.4% of human lung adenocarcinoma (LUAD), which frequently co-occur with oncogenic KRAS mutations, and this co-occurrence is predictive of poor survival in LUAD patients. Using an autochthonous mouse model of KrasG12D -driven LUAD, we show that individual or combinational loss of Tet genes markedly promotes tumor development. In this Kras-mutant and Tet-deficient model, the premalignant lung epithelium undergoes neoplastic reprogramming of DNA methylation and transcription, with a particular impact on Wnt signaling. Among the Wnt-associated components that undergo reprogramming, multiple canonical Wnt antagonizing genes present impaired expression arising from elevated DNA methylation, triggering aberrant activation of Wnt signaling. These impairments can be largely reversed upon the restoration of TET activity. Correspondingly, genetic depletion of ß-catenin, the transcriptional effector of Wnt signaling, substantially reverts the malignant progression of Tet-deficient LUAD. These findings reveal TET enzymes as critical epigenetic barriers against lung tumorigenesis and highlight the therapeutic vulnerability of TET-mutant lung cancer through targeting Wnt signaling.
Assuntos
Adenocarcinoma de Pulmão/metabolismo , Metilação de DNA , DNA de Neoplasias/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Experimentais/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Via de Sinalização Wnt , Adenocarcinoma de Pulmão/genética , Animais , DNA de Neoplasias/genética , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Cytidine diphosphate diacylglycerol (CDP-DAG), an important intermediate for glycerolipid biosynthesis, is synthesized under the catalytic activity of CDP-DAG synthase (CDS) to produce anionic phosphoglycerolipids such as phosphatidylglycerol (PG) and cardiolipin (CL). Previous studies showed that Arabidopsis CDSs are encoded by a small gene family, termed CDS1-CDS5, the members of which are integral membrane proteins in endoplasmic reticulum (ER) and in plastids. However, the details on how CDP-DAG is provided for mitochondrial membrane-specific phosphoglycerolipids are missing. Here we present the identification of a mitochondrion-specific CDS, designated CDS6. Enzymatic activity of CDS6 was demonstrated by the complementation of CL synthesis in the yeast CDS-deficient tam41Δ mutant. The Arabidopsis cds6 mutant lacking CDS6 activity showed decreased mitochondrial PG and CL biosynthesis capacity, a severe growth deficiency finally leading to plant death. These defects were rescued partly by complementation with CDS6 or supplementation with PG and CL. The ultrastructure of mitochondria in cds6 was abnormal, missing the structures of cristae. The degradation of triacylglycerol (TAG) in lipid droplets and starch in chloroplasts in the cds6 mutant was impaired. The expression of most differentially expressed genes involved in the mitochondrial electron transport chain was upregulated, suggesting an energy-demanding stage in cds6. Furthermore, the contents of polar glycerolipids in cds6 were dramatically altered. In addition, cds6 seedlings lost the capacity for cell proliferation and showed a higher oxidase activity. Thus, CDS6 is indispensable for the biosynthesis of PG and CL in mitochondria, which is critical for establishing mitochondrial structure, TAG degradation, energy production and seedling development.
Assuntos
Arabidopsis , Arabidopsis/metabolismo , Glicogênio Sintase/metabolismo , Cistina Difosfato/metabolismo , Diglicerídeos/metabolismo , Diacilglicerol Colinofosfotransferase/metabolismo , Mitocôndrias/metabolismo , Fosfatidilgliceróis/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Precise gene-editing using CRISPR/Cas9 technology remains a long-standing challenge, especially for genes with low expression and no selectable phenotypes in Chlamydomonas reinhardtii, a classic model for photosynthesis and cilia research. Here, we developed a multi-type and precise genetic manipulation method in which a DNA break was generated by Cas9 nuclease and the repair was mediated using a homologous DNA template. The efficacy of this method was demonstrated for several types of gene editing, including inactivation of two low-expression genes (CrTET1 and CrKU80), the introduction of a FLAG-HA epitope tag into VIPP1, IFT46, CrTET1 and CrKU80 genes, and placing a YFP tag into VIPP1 and IFT46 for live-cell imaging. We also successfully performed a single amino acid substitution for the FLA3, FLA10 and FTSY genes, and documented the attainment of the anticipated phenotypes. Lastly, we demonstrated that precise fragment deletion from the 3'-UTR of MAA7 and VIPP1 resulted in a stable knock-down effect. Overall, our study has established efficient methods for multiple types of precise gene editing in Chlamydomonas, enabling substitution, insertion and deletion at the base resolution, thus improving the potential of this alga in both basic research and industrial applications.
Assuntos
Chlamydomonas reinhardtii , Chlamydomonas , Sistemas CRISPR-Cas , Chlamydomonas/genética , Edição de Genes/métodos , Chlamydomonas reinhardtii/genéticaRESUMO
Ophthalmic manifestations have recently been observed in acute and post-acute complications of COVID-19 caused by SARS-CoV-2 infection. Our precious study has shown that host RNA editing is linked to RNA viral infection, yet ocular adenosine to inosine (A-to-I) RNA editing during SARS-CoV-2 infection remains uninvestigated in COVID-19. Herein we used an epitranscriptomic pipeline to analyze 37 samples and investigate A-to-I editing associated with SARS-CoV-2 infection, in five ocular tissue types including the conjunctiva, limbus, cornea, sclera, and retinal organoids. Our results revealed dramatically altered A-to-I RNA editing across the five ocular tissues. Notably, the transcriptome-wide average level of RNA editing was increased in the cornea but generally decreased in the other four ocular tissues. Functional enrichment analysis showed that differential RNA editing (DRE) was mainly in genes related to ubiquitin-dependent protein catabolic process, transcriptional regulation, and RNA splicing. In addition to tissue-specific RNA editing found in each tissue, common RNA editing was observed across different tissues, especially in the innate antiviral immune gene MAVS and the E3 ubiquitin-protein ligase MDM2. Analysis in retinal organoids further revealed highly dynamic RNA editing alterations over time during SARS-CoV-2 infection. Our study thus suggested the potential role played by RNA editing in ophthalmic manifestations of COVID-19, and highlighted its potential transcriptome impact, especially on innate immunity.