Association of DNA methylation/demethylation with the functional outcome of stroke in a hyperinflammatory state

Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.

Punicalagin prevents obesity-related cardiac dysfunction through promoting DNA demethylation in mice

The aim of this study was to investigate whether punicalagin(PU)could prevent obesity-related cardiac dysfunction by promoting DNA demethy lation,and to explore its possible mechanism.C57BL/6J mice were fed with standard diet,high-fat diet(HFD),HFD supplemented with resveratrol,low-dose PU(LPU)and high-dose PU(HPU)for 8 weeks.Compared with HFD group,body weight was significantly lower in PU treatment groups,number of cardionwocytes and the protein level of myosin heavy chain 7B were significantly higher in PU treatment groups.Levels of 5-hydroxymethylcytosine and 5-formylcytosine were significantly lower in HFD group than in other groups.Compared with the HFD group,the protein level of ten-eleven translocation enzyme(TET)2 was significantly higher in PU treatment groups,p-AMP-activated protein kinase(AMPK)was significantly higher in LPU group.Levels of total antioxidant capacity and the protein levels of complexesⅡ/Ⅲ/Ⅴ,oxoglutarate dehydrogenase,succinate dehydrogenase B and fumarate hdrolase were significantly lower in HFD group than PU treatment group.The ratio of(succinic acid+fumaric acid)/a-ketoglutarate was significantly higher in HFD group than other groups.In conclusion,PU up-regulated TETs enzyme activities and TET2 protein stability through alleviating mitochondrial dysfunction and activating AMPK,so as to promote DNA demethylation,thus preventing obesity-related cardiac dysfunction.

Observation on the Clinical Effect of Applying Venetoclax Combined with Demethylation Drug Therapy in Patients with Acute Myeloid Leukemia

Objective: To investigate the therapeutic effect of applying venetoclax combined with demethylating drugs in treating patients with acute myeloid leukemia (AML). Methods: Eighty cases of AML patients treated with venetoclax combined with demethylating drugs in our hospital were selected from March 2021 to March 2024, including 40 cases of primary treatment patients and 40 cases of relapsed and refractory patients. The efficacy and safety of the combined drug therapy was analyzed. Results: The primary treatment group was presented with a complete remission (CR) rate of 40.5%, partial remission (PR) rate of 47.50%, no response (NR) rate of 12.50%, and a remission rate of 87.50%. The relapsed- refractory group was presented with a CR rate of 37.50%, PR rate of 42.50%, NR rate of 17.50%, and a remission rate of 87.50%. There was no statistical significance between the groups (P > 0.05). The hematological adverse reactions of the combined treatment for AML were leukopenia and the non-hematological adverse reactions were mainly infections, with an incidence rate of 87.50%. Conclusion: The efficacy of venetoclax combined with demethylating drugs in AML was remarkable and the treatment regimen can be adjusted according to the treatment-resistant response.

Dynamic changes in DNA demethylation in the tree shrew (Tupaia belangeri chinensis) brain during postnatal development and aging

Brain development and aging are associated with alterations in multiple epigenetic systems, including DNA methylation and demethylation patterns. Here, we observed that the levels of the 5- hydroxymethylcytosine （5hmC） ten-eleven transtocation （TET） enzyme-mediated active DNA demethylation products were dynamically changed and involved in postnatal brain development and aging in tree shrews （Tupaia belangeri chinensis）. The levels of 5hmC in multiple anatomic structures showed a gradual increase throughout postnatal development, whereas a significant decrease in 5hmC was found in several brain regions in aged tree shrews, including in the prefrontal cortex and hippocampus, but not the cerebellum. Active changes in Tet mRNA levels indicated that TET2 and TET3 predominantly contributed to the changes in 5hmC levels. Our findings provide new insight into the dynamic changes in 5hmC levels in tree shrew brains during postnatal development and aging processes.

DNA methylation and demethylation link the properties of mesenchymal stem cells: Regeneration and immunomodulation

Mesenchymal stem cells(MSCs)are a heterogeneous population that can be isolated from various tissues,including bone marrow,adipose tissue,umbilical cord blood,and craniofacial tissue.MSCs have attracted increasingly more attention over the years due to their regenerative capacity and function in immunomodulation.The foundation of tissue regeneration is the potential of cells to differentiate into multiple cell lineages and give rise to multiple tissue types.In addition,the immunoregulatory function of MSCs has provided insights into therapeutic treatments for immune-mediated diseases.DNA methylation and demethylation are important epigenetic mechanisms that have been shown to modulate embryonic stem cell maintenance,proliferation,differentiation and apoptosis by activating or suppressing a number of genes.In most studies,DNA hypermethylation is associated with gene suppression,while hypomethylation or demethylation is associated with gene activation.The dynamic balance of DNA methylation and demethylation is required for normal mammalian development and inhibits the onset of abnormal phenotypes.However,the exact role of DNA methylation and demethylation in MSC-based tissue regeneration and immunomodulation requires further investigation.In this review,we discuss how DNA methylation and demethylation function in multi-lineage cell differentiation and immunomodulation of MSCs based on previously published work.Furthermore,we discuss the implications of the role of DNA methylation and demethylation in MSCs for the treatment of metabolic or immune-related diseases.

Impact of GFRA1 gene reactivation by DNA demethylation on prognosis of patients with metastatic colon cancer

BACKGROUND The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers,which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways.Several therapeutic anti-GFRA1 antibody-drug conjugates are under development.Demethylation(or hypomethylation)of GFRA1 CpG islands(dmGFRA1)is associated with increased gene expression and metastasis risk of gastric cancer.However,it is unknown whether dmGFRA1 affects the metastasis of other cancers,including colon cancer(CC).AIM To study whether dmGFRA1 is a driver for CC metastasis and GFRA1 is a potential therapeutic target.METHODS CC and paired surgical margin tissue samples from 144 inpatients and normal colon mucosal biopsies from 21 noncancer patients were included in this study.The methylation status of GFRA1 islands was determined by MethyLight and denaturing high-performance liquid chromatography and bisulfite-sequencing.Kaplan-Meier analysis was used to explore the effect of dmGFRA1 on the survival of CC patients.Impacts of GFRA1 on CC cell proliferation and migration were evaluated by a battery of biological assays in vitro and in vivo.The phosphorylation of AKT and ERK proteins was examined by Western blot analysis.RESULTS The proportion of dmGFRA1 in CC,surgical margin,and normal colon tissues by MethyLight was 68.4%,73.4%,and 35.9%(median;nonparametric test,P=0.001 and<0.001),respectively.Using the median value of dmGFRA1 peak area proportion as the cutoff,the proportion of dmGFRA1-high samples was much higher in poorly differentiated CC samples than in moderately or welldifferentiated samples(92.3%%vs 55.8%,Chi-square test,P=0.002)and significantly higher in CC samples with distant metastasis than in samples without(77.8%vs 46.0%,P=0.021).The overall survival of patients with dmGFRA1-low CC was significantly longer than that of patients with dmGFRA1-high CC(adjusted hazard ratio=0.49,95%confidence interval:0.24-0.98),especially for 89 CC patients with metastatic CC(adjusted hazard ratio=0.41,95%confidence interval:0.18-0.91).These data were confirmed by the mining results from TCGA datasets.Furthermore,GFRA1 overexpression significantly promoted the proliferation/invasion of RKO and HCT116 cells and the growth of RKO cells in nude mice but did not affect their migration.GFRA1 overexpression markedly increased the phosphorylation levels of AKT and ERK proteins,two key molecules in two classic GFRA1 downstream pathways.CONCLUSION GFRA1 expression is frequently reactivated by DNA demethylation in CC tissues and is significantly associated with a poor prognosis in patients with CC,especially those with metastatic CC.GFRA1 can promote the proliferation/growth of CC cells,probably by the activation of AKT and ERK pathways.GFRA1 might be a therapeutic target for CC patients,especially those with metastatic potential.

Mechanoresponsive Gene Upregulation by Force Depends on H3K9Demethylation

All living cells in a human body are made of the same DNA molecule but cells in different tissues express different genes and proteins.How the transcription process is controlled and regulated is largely unknown.Specifically,mechanical forces are increasingly recognized to play critical roles in cell and tissue functions.However,what controls force-induced gene transcription is elusive.Recently we have reported that a local surface force transfers from integrins to the cytoskeleton and the link of nucleoskeleton and the cytoskeleton(LINC)into the nucleus and deforms chromatin directly to induce rapid activation of transgene DHFR.Here we show that endogenous mechanoresponsive genes egr-1 and Cav1 are rapidly upregulated and their upregulation depends on stress angles relative to the cell long axis,suggesting direct impact of these genes by force.Demethylation of histone 3 at lysine 9(H3K9)trimethylation(H3K9me3)at nuclear interiors(euchromatin)is necessary for force-induced transcription upregulation.Our findings suggest that force-rapid upregulation of mechanoresponsive genes by force depends on H3K9me3 demethylation.

Cornel iridoid glycoside inhibits PP2Ac demethylation by regulationg PME-1

OBJECTIVE PP2Ac demethyl⁃ation is regulated by LCMT(a specific leucine carboxyl methyltransferase catalyzing methyla⁃tion of PP2A)and PME(a specific methylester⁃ase catalyzing demethylation of PP2A.This study was to investigate the mechanism of Cor⁃nel iridoid glycoside(CIG)on PP2A catalytic sub⁃unit C(PP2Ac).METHODS Recombined lentivi⁃rus vector was used to deliver PME-1 genetic materials into N2a cells or transfected LCMT-1 siRNA into N2a cells to block the expression of LCMT-1.Twenty-four hours later,cells were rinsed twice with cold PBS(pH 7.4)and CIG at different concentrations(50,100 and 200 g·L^(-1),respectively)were added for 24 h.Western blotting was used to PP2Ac,demethylaion/methylation PP2Ac,LCMT-1 and PME-1.The ac⁃tivity of PP2A was detected by a biochemical as⁃say.RESULTS①Lentivirus transferred PME-1 was expressed at high level in the N2a cells after transduction.Correspondingly,the demethylation of PP2Ac was increasing and PP2A activity was decreasing after transduction.Treatment with CIG for 24 h reversed the increase of PME-1 and demethylation of PP2Ac without influencing LCMT-1 expression.PP2A activity was also sig⁃nificantly enhanced in CIG treatment group,compared with the cells after PME-1 transduc⁃tion.②LCMT-1 siRNA significantly decreased LCMT-1 expression.CIG did not affect LCMT-1expression.however,demethylation of PP2Ac is increased in siRNA-transfected cells and CIG could reversed the high demethylation of PP2Ac and PP2A activity.CONLUSION CIG increases methylation of PP2A subunit C by inhibiting PME-1.

Demethylation of FANCF gene may be a potential treatment through inhibiting the proliferation of cervical cancer

Objective: The aim of the study was to explore the effect of demethylating agent 5-Aza-2'-deoxycytidine (5-ADC) on expression of Fanconi anemia complementation group F (FANCF) gene and the proliferation of cervical cancer cells, to observe cell's sensitivity to chemotherapeutic drug taxol, and to explore the antitumor effect of 5-ADC as well as the new treatment of cervical cancer. Methods: Cervical cancer cell lines SiHa (FANCF gene full-methylated) and Hela (unmethylated) were treated with 5-ADC. We used the methylation-specific PCR (MSP), reverse transcription-polymerase chain reaction (RT-PCR) and Western blot to detect the FANCF methylation, mRNA and protein respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferation of cells. The cytotoxicity of taxol was measured by flow cytometer. The nude mice bearing SiHa was used to observe the effect of 5-ADC in vivo. Results: Inhibition of DNA promoter methylation by 5-ADC reactivated the expression of FANCF mRNA and protein in SiHa cells, consistent with decreased growth speed and increased taxol resistance. These results were proven in experiments in vivo. Conclusion: The 5-ADC probably become a potential treatment drug through inhibiting the proliferation of cervical cancer cells in taxol-resistant patients.

Recent Advances in Deciphering the Mechanisms and Biological Functions of DNA Demethylation

5-Methylcytosine (5mC) is a dynamic and reversible epigenetic modification in genomic DNA of higher eukaryotes.It has been well-established that the demethylation of 5mC occurs through the ten-eleven translocation (TET)-mediated oxidation of 5mC followed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER).Recent findings also have identified an alternative pathway of DNA demethylation.In this pathway,TET enzymes directly oxidize 5mC to form 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC).These modified bases can undergo direct deformylation or decarboxylation,respectively.Additionally,DNA demethylation can also occur through the deamination of 5mC and 5hmC,resulting in the production of thymine and 5-hydroxymethyluracil (5hmU),respectively.Various DNA demethylation pathways possess critical functional implications and roles in biological processes.This Recent Advances article will focus on the studies of mechanisms and biological functions of DNA demethylation,shedding light on the reversible nature of the epigenetic modification of 5mC.

A mild iodocyclohexane demethylation for highly enhancing antioxidant activity of lignin

Lignin,as a natural antioxidant,shows great potential in food engineering and medicine.How-ever,the inherent macromolecular structure,high polydispersity,and few phenolic hydroxy seri-ously limit its antioxidant activity.In this work,a mild iodocyclohexane demethylation for highly improving the antioxidant activity of lignin was proposed.The results showed-OCH 3 content exhibited an almost linear decrease as a function of treating time,and the demethylation and cleavage of𝛽-aryl ether bonds prompt an obvious increase in phenolic hydroxyl content(4.01 mmol/g)and a significant decline in aliphatic hydroxyl(∼0.03 mmol/g).Meanwhile,attributing to the fragmentation of𝛽β-O-4,β-β,and𝛽β-5 substructures,the polydispersity of lignin molecular weight decreases from 2.7 to 2.2.As a result,the formed catechol-typed lignin showed an out-standing antioxidant activity,with the radical(DPPH·)scavenging index(inverse of concentration for 50%of maximal effect(EC 50)value)over 2000 mL/mg,much superior to the commercial antioxidants(<500 mL/mg).Further structure-activity relationship analysis implied that the Ph-OH/-OCH 3 ratio might act as a key factor influencing the antioxidant activity of lignin.This mild demethylation demonstrates a facile and effective method for highly enhancing the antioxidant activity of lignin and makes the catechol-typed lignin a green and promising product for practical use in food,medicine,and pharmacy.

RNA-directed DNA methylation and demethylation in plants

RNA-directed DNA methylation (RdDM) is a nuclear process in which small interfering RNAs (siRNAs) direct the cytosine methylation of DNA sequences that are complementary to the siRNAs. In plants, double stranded-RNAs (dsRNAs) generated by RNA-dependent RNA polymerase 2 (RDR2) serve as precursors for Dicer-like 3 dependent biogenesis of 24-nt siRNAs. Plant specific RNA polymerase IV (Pol IV) is presumed to generate the initial RNA transcripts that are substrates for RDR2. siRNAs are loaded onto an argonaute4-containing RISC (RNA-induced silencing complex) that targets the de novo DNA methyltransferase DRM2 to RdDM target loci. Nascent RNA transcripts from the target loci are generated by another plant-specific RNA polymerase, Pol V, and these transcripts help recruit com- plementary siRNAs and the associated RdDM effector complex to the target loci in a transcrip- tion-coupled DNA methylation process. Small RNA binding proteins such as ROS3 may direct tar- get-specific DNA demethylation by the ROS1 family of DNA demethylases. Chromatin remodeling en- zymes and histone modifying enzymes also participate in DNA methylation and possibly demethylation. One of the well studied functions of RdDM is transposon silencing and genome stability. In addition, RdDM is important for paramutation, imprinting, gene regulation, and plant development. Lo- cus-specific DNA methylation and demethylation, and transposon activation under abiotic stresses suggest that RdDM is also important in stress responses of plants. Further studies will help illuminate the functions of RdDM in the dynamic control of epigenomes during development and environmental stress responses.

SIZ1-Mediated SUMOylation of ROS1 Enhances Its Stability and Positively Regulates Active DNA Demethylation in Arabidopsis

The 5-methylcytosine DNA glycosylase/lyase REPRESSOR OF SILENCING 1(ROS1)-mediated active DNA demethylation is critical for shaping the genomic DNA methylation landscape in Arabidopsis.Whether and how the stability of ROS1 may be regulated by post-translational modifications is unknown.Using a methylation-sensitive PCR(CHOP-PCR)-based forward genetic screen forArabidopsis DNA hyper-methyl-ation mutants,we identified the SUMO E3 ligase SIZ1 as a critical regulator of active DNA demethylation.Dysfunction of SIZ1 leads to hyper-methylation at approximately 1000 genomic regions.SIZ1 physically in-teracts with ROS1 and mediates the SUMOylation of ROS1.The SUMOylation of ROS1 is reduced in siz1 mutant plants.Compared with that in wild-type plants,the protein level of ROS1 is significantly decreased,whereas there is an increased level of ROS1 transcripts in siz1 mutant plants.Our results suggest that SIZ1-mediated SUMOylation of ROS1 promotes its stability and positively regulates active DNA demethylation.

Retinoic acid inhibits white adipogenesis by disrupting GADD45A-mediated Zfp423 DNA demethylation

Retinoic acid （RA）, a bioactive metaboUte of vitamin A, is a critical mediator of cell differentiation. RA blocks adipogenesis, but mechanisms remain to be established. ZFP423 is a key transcription factor maintaining white adipose identity. We found that RA inhibits Zfp423 expression and adipogenesis via blocking DNA demethylation in the promoter of Zfp423, a process mediated by growth arrest and DNA-damage-inducible protein alpha （GADD45A）. RA induces the partnering between retinoic acid receptor （PAR） and tumor suppressor inhibitor of growth protein 1 （ING1）, which prevents the formation of GADD45A and ING1 complex necessary for locus-specific Zfp423 DNA demethylation. In vivo, vitamin A supplementation prevents obesity, downregulates Godd45a expression, and reduces GADD45A binding and DNA demethylation in the Zfp423 promoter. Inhibition of Zfp423 expres- sion due to PA contributes to the enhanced brown adipogenesis. In summary, PA inhibits white adipogenesis by inducing PAR and ING1 interaction and inhibiting Godd45a expression, which prevents GADD45A-mediated DNA demethylation.

A novel protein complex that regulates active DNA demethylation in Arabidopsis

Active DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression.The 5-methylcytosine DNA glycosylase/lyase ROS1 initiates a base-excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at 1000 s of genomic regions in Arabidopsis.How ROS1 is regulated and targeted to specific genomic regions is not well understood.Here,we report the discovery of an Arabidopsis protein complex that contains ROS1,regulates ROS1 gene expression,and likely targets the ROS1 protein to specific genomic regions.ROS1 physically interacts with a WD40 domain protein(RWD40),which in turn interacts with a methyl-DNA binding protein(RMB1)as well as with a zinc finger and homeobox domain protein(RHD1).RMB1 binds to DNA that is methylated in any sequence context,and this binding is necessary for its function in vivo.Loss-of-function mutations in RWD40,RMB1,or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1.Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter,plants mutated in RWD40,RMB1,or RHD1 show increased ROS1 expression.Importantly,ROS1 binding to the ROS1 promoter requires RWD40,RMB1,and RHD1,suggesting that this complex dictates ROS1 targeting to this locus.Our results demonstrate that ROS1 forms a protein complex with RWD40,RMB1,and RHD1,and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.

Molecules and mechanisms controlling the active DNA demethylation of the mammalian zygotic genome

The active DNA demethylation in early embryos is essential for subsequent development. Although the zygotic genome is globally demethylated, the DNA methylation of imprinted regions, part of repeat sequences and some gamete-specific regions are maintained. Recent evidence has shown that multiple proteins and biological pathways participate in the regulation of active DNA demethylation, such as TET proteins, DNA repair pathways and DNA methyltransferases. Here we review the recent understanding regarding proteins associated with active DNA demethylation and the regulatory networks controlling the active DNA demethylation in early embryos.

Transformation of 5-Carboxylcytosine to Cytosine Through C-C Bond Cleavage in Human Cells Constitutes a Novel Pathway for DNA Demethylation

Active demethylation of 5-methylcytosine(5mC)can be realized through ten-eleven translocation(TET)dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC),and 5-carboxylcytosine(5caC),followed by thymine DNA glycosylase(TDG)-initiated base excision repair(BER).The TDG-BER pathwaymay lead to the generation of DNA strand breaks,potentially compromising genome integrity.Alternatively,direct decarboxylation of TET-produced 5caC is highly attractive because this mechanism allows for conversion of 5mC to cytosine without the formation of DNA strand breaks.However,cleavage of the C–C bond in 5caC in human cells remains an open question.We examined this reaction in cell extract and live cells using 5caC-carrying hairpin DNA substrate.After incubation with whole-cell protein extract or transfection into human cells,we monitored the transformation of 5caC to cytosine through direct decarboxylation or BER using liquid chromatography–tandem mass spectrometry(LCMS/MS)analyses at both the mononucleotide and oligodeoxynucleotide levels.Our results clearly showed the direct conversion of 5caC to cytosine in human cells,providing evidence to support a novel pathway for active DNA demethylation.

Properties of Bark Particleboard Bonded with Demethylated Lignin Adhesives Derived from Leucaena leucocephala Bark

Lignin extraction from bark can maximize the utilization of biomass waste,offer cost-effectiveness,and promote environmental friendliness when employed as an adhesive material in bark particleboard production.Particles of fine(0.2 to 1.0 mm),medium(1.0 to 2.5 mm),and coarse(2.5 to 12.0 mm)sizes,derived from the bark of Leucaena leucocephala,were hot-pressed using a heating plate at 175℃for 7 min to create single-layer particleboards measuring 320 mm×320 mm×10 mm,targeting a density of 700 kg/m^(3).Subsequently,the samples were trimmed and conditioned at 20℃and 65%relative humidity.In this study,we compared bark particleboard bonded with urea formaldehyde(UF)adhesive to fine-sized particleboard bonded with demethylated lignin adhesive.The results indicated that bark particleboards utilizing demethylated lignin and UF adhesives exhibited similar qualities.Coarse particleboard showed differences in modulus of elasticity(MOE)and modulus of rupture(MOR),while medium-sized particles exhibited significant variations in moisture content(MC)and water absorption(WA).Furthermore,the thickness swelling of coarse and medium-sized particles under wet and oven-dried conditions exhibited notable distinctions.Overall,the demethylated lignin adhesive extracted from L.leucocephala bark demonstrated similar quality to UF adhesive,with particle size correlating inversely to the strength of the bark particleboard.

DEMETHYLATION REGULATOR 1 regulates DNA demethylation of the nuclear and mitochondrial genomes

Active DNA demethylation effectively modulates gene expression during plant development and in response to stress.However,little is known about the upstream regulatory factors that regulate DNA demethylation.We determined that the demethylation regulator 1(demr1)mutant exhibits a distinct DNA methylation profile at selected loci queried by methylation-sensitive polymerase chain reaction and globally based on whole-genome bisulfite sequencing.Notably,the transcript levels of the DNA demethylase gene REPRESSOR OF SILENCING 1(ROS1)were lower in the demr1 mutant.We established that DEMR1 directly binds to the ROS1 promoter in vivo and in vitro,and the methylation level in the DNA methylation monitoring sequence of ROS1 promoter decreased by 60%in the demr1 mutant.About 40%of the hyper-differentially methylated regions(DMRs)in the demr1 mutant were shared with the ros1-4 mutant.Genetic analysis indicated that DEMR1 acts upstream of ROS1 to positively regulate abscisic acid(ABA)signaling during seed germination and seedling establishment stages.Surprisingly,the loss of DEMR1 function also caused a rise in methylation levels of the mitochondrial genome,impaired mitochondrial structure and an early flowering phenotype.Together,our results show that DEMR1 is a novel regulator of DNA demethylation of both the nuclear and mitochondrial genomes in response to ABA and plant development in Arabidopsis.

Active DNA demethylation regulates MAMP-triggered immune priming in Arabidopsis

Plants recognize microbe-associated molecular patterns(MAMPs)to activate immune responses and defense priming to defend against pathogen infections.Transcriptional regulation of gene expression is crucial for plant immunity and is mediated by multiple factors,including DNA methylation.However,it remains unknown whether and how DNA demethylation contributes to immune responses in MAMPtriggered immunity.Here,we report that active DNA demethylation is required for MAMP-triggered immunity to bacterial pathogens.The rdd-2 triple mutant carrying mutations in ROS1,DML2,and DML3 that encode DNA glycosylases,which are key DNA demethylation enzymes,exhibits compromised immune responses triggered by the MAMPs fig22 and elf18.Genome-wide methylome analysis reveals that fig22 induces rapid and specific DNA demethylation in an RDD-dependent manner.The expression levels of salicylic acid signaling-related and phytoalexin biosynthesis-related genes are tightly associated with the fig22-induced promoter demethylation.The compromised accumulation of priming compounds and antimicrobial metabolites ultimately leads to a defense priming defect in the rdd-2 mutant.Our results reveal the critical role of active DNA demethylation in the MAMP-triggered immune response and provide unique insight into the molecular mechanism of fig22-modulated DNA demethylation.