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.

Advances in microfluidic-based DNA methylation analysis

DNA methylation has been extensively investigated in recent years,not least because of its known relationship with various diseases.Progress in analytical methods can greatly increase the relevance of DNA methylation studies to both clinical medicine and scientific research.Microflu-idic chips are excellent carriers for molecular analysis,and their use can provide improvements from multiple aspects.On-chip molecular analysis has received extensive attention owing to its advantages of portability,high throughput,low cost,and high efficiency.In recent years,the use of novel microfluidic chips for DNA methylation analysis has been widely reported and has shown obvious superiority to conventional methods.In this review,wefirst focus on DNA methylation and its applications.Then,we discuss advanced microfluidic-based methods for DNA methylation analysis and describe the great progress that has been made in recent years.Finally,we summarize the advantages that microfluidic technology brings to DNA methylation analysis and describe several challenges and perspectives for on-chip DNA methylation analysis.This review should help researchers improve their understanding and make progress in developing microfluidic-based methods for DNA methylation analysis.

Unveiling DNA methylation in Alzheimer’s disease:a review of array-based human brain studies

The intricacies of Alzheimer’s disease pathogenesis are being increasingly illuminated by the exploration of epigenetic mechanisms,particularly DNA methylation.This review comprehensively surveys recent human-centered studies that investigate whole genome DNA methylation in Alzheimer’s disease neuropathology.The examination of various brain regions reveals distinctive DNA methylation patterns that associate with the Braak stage and Alzheimer’s disease progression.The entorhinal cortex emerges as a focal point due to its early histological alterations and subsequent impact on downstream regions like the hippocampus.Notably,ANK1 hypermethylation,a protein implicated in neurofibrillary tangle formation,was recurrently identified in the entorhinal cortex.Further,the middle temporal gyrus and prefrontal cortex were shown to exhibit significant hypermethylation of genes like HOXA3,RHBDF2,and MCF2L,potentially influencing neuroinflammatory processes.The complex role of BIN1 in late-onset Alzheimer’s disease is underscored by its association with altered methylation patterns.Despite the disparities across studies,these findings highlight the intricate interplay between epigenetic modifications and Alzheimer’s disease pathology.Future research efforts should address methodological variations,incorporate diverse cohorts,and consider environmental factors to unravel the nuanced epigenetic landscape underlying Alzheimer’s disease progression.

Characterization of MyDNMT1 and Changes of Global DNA Methylation during the Embryonic Development in Mizuhopecten yessoensis

DNA methylation is a critical epigenetic mechanism that influences gene transcription, genomic stability, X-chromosome inactivation and other factors, and appropriate DNA methylation is crucial in development. DNA methyltransferase 1 （DNMT1） plays an important role in maintaining the established methylation pattern during DNA replication. Although the effect of DNA methylation on embryonic development has been well known in vertebrates, little research has been carried out in invertebrates, especially in marine bivalves. In this study, the DNMT1 gene （MyDNMT1） was firstly identified from Mizuhopecten yessoensis. The full-length cDNA of MyDNMT1 was 5 039 bp, consisted of a 5＇ untranslated region （5＇-UTR） of 79 bp, a 3＇ untranslated region （3＇-UTR） of 199 bp, and a 4 761 bp open reading frame （ORF） encoding a peptide of 1 586 amino acids without a putative signal peptide. The relative mRNA expression level of MyDNMT1 was measured during the embryonic development of M. ydssoensis using real-time PCR, which revealed that the level at stage zygote and trochophore were significantly higher than that at other stages. We further examined the global DNA methylation during development by colorimetric method. The results showed that the methylation level was increased and reached the peak at blastula stage, then dramatically decreased, and fluctuated at early D-shaped larva stage. This study provided greater insight into the DNA methylation of embryonic development, which obtained a better understanding of the relationship between the DNA methylation and the embryonic development in bivalve mollusks.

DNA methylation in poultry:a review

As an important epigenetic modification,DNA methylation is involved in many biological processes such as animal cell differentiation,embryonic development,genomic imprinting and sex chromosome inactivation.As DNA methylation sequencing becomes more sophisticated,it becomes possible to use it to solve more zoological problems.This paper reviews the characteristics of DNA methylation,with emphasis on the research and application of DNA methylation in poultry.

Rice melatonin deficiency causes premature leaf senescence via DNA methylation regulation

In a study of DNA methylation changes in melatonin-deficient rice mutants,mutant plants showed premature leaf senescence during grain-filling and reduced grain yield.Melatonin deficiency led to transcriptional reprogramming,especially of genes involved in chlorophyll and carbon metabolism,redox regulation,and transcriptional regulation,during dark-induced leaf senescence.Hypomethylation of mCG and mCHG in the melatonin-deficient rice mutants was associated with the expression change of both protein-coding genes and transposable element-related genes.Changes in gene expression and DNA methylation in the melatonin-deficient mutants were compensated by exogenous application of melatonin.A decreased S-adenosyl-L-methionine level may have contributed to the DNA methylation variations in rice mutants of melatonin deficiency under dark conditions.

Advances in DNA methylation and its role in cytoplasmic male sterility in higher plants

The impact of epigenetic modifications like DNA methylation on plant phenotypes has expanded the possibilities for crop development.DNA methylation plays a part in the regulation of both the chromatin structure and gene expression,and the enzyme involved,DNA methyltransferase,executes the methylation process within the plant genome.By regulating crucial biological pathways,epigenetic changes actively contribute to the creation of the phenotype.Therefore,epigenome editing may assist in overcoming some of the drawbacks of genome editing,which can have minor off-target consequences and merely facilitate the loss of a gene’s function.These drawbacks include gene knockout,which can have such off-target effects.This review provides examples of several molecular characteristics of DNA methylation,as well as some plant physiological processes that are impacted by these epigenetic changes in the plants.We also discuss how DNA alterations might be used to improve crops and meet the demands of sustainable and environmentally-friendly farming.

Comparative DNA methylation reveals epigenetic adaptation to high altitude in snub-nosed monkeys

DNA methylation plays a crucial role in environmental adaptations.Here,using whole-genome bisulfite sequencing,we generated comprehensive genome-wide DNA methylation profiles for the high-altitude Yunnan snub-nosed monkey(Rhinopithecus bieti)and the closely related golden snub-nosed monkey(R.roxellana).Our findings indicated a slight increase in overall DNA methylation levels in golden snub-nosed monkeys compared to Yunnan snub-nosed monkeys,suggesting a higher prevalence of hypermethylated genomic regions in the former.Comparative genomic methylation analysis demonstrated that genes associated with differentially methylated regions were involved in membrane fusion,vesicular formation and trafficking,hemoglobin function,cell cycle regulation,and neuronal differentiation.These results suggest that the high-altitude-related epigenetic modifications are extensive,involving a complete adaptation process from the inhibition of single Ca^(2+)channel proteins to multiple proteins collaboratively enhancing vesicular function or inhibiting cell differentiation and proliferation.Functional assays demonstrated that overexpression or down-regulation of candidate genes,such as SNX10,TIMELESS,and CACYBP,influenced cell viability under stress conditions.Overall,this research suggests that comparing DNA methylation across closely related species can identify novel candidate genomic regions and genes associated with local adaptations,thereby deepening our understanding of the mechanisms underlying environmental adaptations.

DNA Methylation of KLRC1 and KLRC3 in Autoimmune Thyroiditis:Perspective of Different Water Iodine Exposure

Objective This study aimed to identify differentially methylated genes(DMGs) associated with natural killer cells in patients with autoimmune thyroiditis(AIT), focusing on the influence of varying water iodine exposure levels.Methods Participants were divided into categories based on median water iodine(MWI)concentrations: iodine-fortified areas(IFA, MWI < 10 μg/L), iodine-adequate areas(IAA, 40 ≤ MWI ≤ 100μg/L), and iodine-excessive areas(IEA, MWI > 300 μg/L). A total of 176 matched AIT cases and controls were recruited and divided into 89, 40, and 47 pairs for IFA, IAA, and IEA, respectively. DMGs were identified using 850K Bead Chip analysis for 10/10 paired samples. Validation of DNA methylation and m RNA expression levels of the DMGs was conducted using Methyl Target^(TM) and QRT-PCR for 176/176paired samples.Results KLRC1, KLRC3, and SH2D1B were identified as significant DMGs. Validation revealed that KLRC1 was hypomethylated and highly expressed, whereas KLRC3 was hypermethylated and highly expressed in individuals with AIT. Furthermore, KLRC1 was hypomethylated and highly expressed in both IFA and IEA.Conclusion The DNA methylation status of KLRC1 and KLRC3 may play crucial roles in AIT pathogenesis. Additionally, DNA methylation of KLRC1 seems to be influenced by different iodine concentrations in water.

Effectiveness of fecal DNA syndecan-2 methylation testing for detection of colorectal cancer in a high-risk Chinese population

BACKGROUND Colorectal cancer(CRC)is among the most prevalent and life-threatening malignancies worldwide.Syndecan-2 methylation(mSDC2)testing has emerged as a widely used biomarker for early detection of CRC in stool and serum samples.Cancer(CRC)is among the most prevalent and life-threatening malignancies worldwide.mSDC2 testing has emerged as a widely used biomarker for early detection of CRC in stool and serum samples.AIM To validate the effectiveness of fecal DNA mSDC2 testing in the detection of CRC among a high-risk Chinese population to provide evidence-based data for the development of diagnostic and/or screening guidelines for CRC in China.METHODS A high-risk Chinese cohort consisting of 1130 individuals aged 40-79 years was selected for evaluation via fecal mSDC2 testing.Sensitivity and specificity for CRC,advanced adenoma(AA)and advanced colorectal neoplasia(ACN)were determined.High-risk factors for the incidence of colorectal lesions were determined and a logistic regression model was constructed to reflect the efficacy of the test.RESULTS A total of 1035 high-risk individuals were included in this study according to established criteria.Among them,16 suffered from CRC(1.55%),65 from AA(6.28%)and 189 from non-AAs(18.26%);150 patients were diagnosed with polyps(14.49%).Diagnoses were established based upon colonoscopic and pathological examinations.Sensitivities of the mSDC2 test for CRC and AA were 87.50%and 40.00%,respectively;specificities were 95.61%for other groups.Positive predictive values of the mSDC2 test for CRC,AA and ACN were 16.09%,29.89%and 45.98%,respectively;the negative predictive value for CRC was 99.79%.After adjusting for other high-risk covariates,mSDC2 test positivity was found to be a significant risk factor for the occurrence of ACN(P<0.001).CONCLUSION Our findings confirmed that offering fecal mSDC2 testing and colonoscopy in combination for CRC screening is effective for earlier detection of malignant colorectal lesions in a high-risk Chinese population.

Eucaryotic DNA Methylation and Gene Mutation

5-methylcytosine (m5C) as a rare base exists in eucaryotic genomes, it is a normal constituent of many eucaryotic DNA, whose existence is a character of eucaryotic DNA. In the regular physiological conditions, cytosine residue of eucaryotic DNA is methylated to be popular. Up to the present, many people consider that the m5C may be mutation hotspots by the m5C deamination leading to gene mutation. Our theoretical investigations indicated that the spontaneous mutation caused by the transition of G - C-A - T, in eukaryotic DNA, may be a result caused by the tautomer changing base pairs and may also be caused by other factor actions, however it could not be caused by the deamination of m5C.

Revolutionizing Non-Invasive Biomarker Discoveries: The Power of Methylation Screening Analysis in Cell-Free DNA Liquid Biopsy

Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylation patterns for both hypermethylation and hypomethylation lead the way in discovery of novel diagnosis and treatment targets. Many different approaches are present to detect the level of methylation in whole genome (whole genome bisulfite sequencing, microarray) as well as at specific loci (methylation specific PCR). Cell-free DNA (cf-DNA) found in body fluids like blood provides information about DNA methylation and serves as a less invasive approach for genetic screening. Cell-free DNA and methylation screening technologies, when combined, have the potential to transform the way we approach genetic screening and personalized therapy. These technologies can help enhance disease diagnostic accuracy and inform the development of targeted therapeutics by providing a non-invasive way for acquiring genomic information and identifying disease-associated methylation patterns. We highlight the clinical benefits of using cell-free DNA (cf-DNA) liquid biopsy analysis and available methylation screening technologies that have been crucial in identifying biomarkers for disease from patients using a non-invasive way. Powering such biomarker discoveries are various methods of cf-DNA methylation analysis such as Bisulfite Sequencing and most recently, Methylation-Specific Restriction Enzyme (MSRE-seq) Analysis, paving the way for novel epigenetic biomarker discoveries for more robust diagnosis such as early disease detection, prognosis, monitoring of disease progression and treatment response as well as discovery of novel drug targets.

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.

Effects of histone acetylation and DNA methylation on p21^(WAF1)regulation

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.

Plasma DNA methylation detection for early screening,diagnosis,and monitoring of esophageal adenocarcinoma and squamous cell carcinoma

BACKGROUND The early diagnosis rate of esophageal cancer(EC),one of the most prevalent digestive tract cancers worldwide,remains low.AIM To investigate the utility of plasma SHOX2,SEPTIN9,EPO,and RNF180 methylation in the clinical diagnosis and monitoring of EC.Plasma samples were collected from 210 patients at Hubei Cancer Hospital,and TaqMan polymerase chain reaction was employed to detect plasma SHOX2,SEPTIN9,RNF180,and EPO methylation.The area under the curve was used to estimate their diagnostic value for EC.Cox and logistic regression analyses were used to estimate the independent screening risk factors for patients with EC.RESULTS The sensitivity and specificity of combined assessment of plasma SHOX2,SEPTIN9,RNF180,and EPO methylation for adenocarcinoma,squamous cell carcinoma(SCC),and EC detection were 66.67%and 86.27%,77.40%and 85.29%,and 76.19%and 86.27%,respectively;the area under the curve values for diagnosing adenocarcinoma,SCC,and EC were 0.737[95%confidence interval(CI):0.584–0.89],0.824(95%CI:0.775–0.891),and 0.864(95%CI:0.809–0.92),respectively.CONCLUSION According to our findings,plasma SHOX2,SEPTIN9,RNF180,and EPO methylation exhibits appreciated sensitivity for diagnosing EC.The precise measurement of plasma SHOX2,SEPTIN9,RNF180,and EPO methylation can improve EC diagnosis and therapy efficacy monitoring.

DNA Methylation Variation Is Identified in Monozygotic Twins Discordant for Congenital Heart Diseases

Aims:Multiple genes and environmental factors are known to be involved in congenital heart disease(CHD),but epigenetic variation has received little attention.Monozygotic(MZ)twins with CHD provide a unique model for exploring this phenomenon.In order to investigate the potential role of Deoxyribonucleic Acid(DNA)methyla-tion in CHD pathogenesis,the present study examined DNA methylation variation in MZ twins discordant for CHD,especially ventricular septal defect(VSD).Methods and Results:Using genome-wide DNA methylation profiles,we identified 4004 differentially methylated regions(DMRs)in 18 MZ twin pairs discordant for CHD,and 2826 genes were identified.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed a list of CHD-associated pathways.To further investigate the role of DNA methylation in VSD,data from 7 pairs of MZ twins with VSD were analyzed.We identified 1614 DMRs corresponding to 1443 genes associated with arrhythmogenic right ventricular cardiomyopathy,cyclic guanosine monopho-sphate-protein kinase G(cGMP-PKG)signaling pathway by KEGG analysis,and cell-cell adhesion,calcium ion transmembrane transport by GO analysis.A proportion of DMR-associated genes were involved in calcium signaling pathways.The methylation changes of calcium signaling genes might be related to VSD pathogenesis.Conclusion:CHD is associated with differential DNA methylation in MZ twins.CHD may be etiologically linked to DNA methylation,and methylation of calcium signaling genes may be involved in the development of VSD.

Stress Treatments and DNA Methylation Affected the Somatic Embryogenesis of Citrus Callus

The evaluation on the callus embryogenesis capacity of 15 genotypes of citrus showed that stress treatments were conducive to somatic embryogenesis and could enhance the recovery of the missed capacity of embryogenesis for some genotypes. Randomly amplified polymorphic DNA (RAPD) and methylation sensitive amplified polymorphism (MSAP) analysis indicated that there existed significant differences in DNA methylation status between the callus capable of producing somatic embryoids and that which missed the embryogenesis capacity of the same genotype Newhall navel orange ( Citrus sinensis Osb. cv. Newhall). The DNA methylation level of the former was lower than that of the latter. However, RAPD profiles did not show any difference between these two kinds of callus.

Effects of DNA Methylation and Histone Modification on Differentiation-associated Gene Expression in ES,NIH3T3,and NIT-1

The effects of epigenetic modification on the differentiation of islet cells and the expression of associated genes（Pdx-1,Pax4,MafA,and Nkx6.1,etc） were investigated.The promoter methylation status of islet differentiation-associated genes（Pdx-1,Pax4,MafA and Nkx6.1）,Oct4 and MLH1 genes of mouse embryonic stem cells,NIH3T3 cells and NIT-1 cells were profiled by methylated DNA immunoprecipitation,real-time quantitative PCR（MeDIP-qPCR） techniques.The histone modification status of these genes promoter region in different cell types was also measured by using chromatin immunoprecipitation real-time quantitative PCR methods.The expression of these genes in these cells was detected by using real-time quantitative PCR.The relationship between the epigenetic modification（DNA methylation,H3 acetylation,H3K4m3 and H3K9m3） of these genes and their expression was analyzed.The results showed that：（1） the transcription-initiation-sites of Pdx-1,MafA and Nkx6.1 were highly methylated in NIH3T3 cells; （2） NIH3T3 cells showed a significantly higher level of DNA methylation modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIT-1 cells（P〈0.05）; （3） NIT-1 cells had a significantly higher level of H3K4m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIH3T3 cells（P〈0.05）,with significantly increased level of gene expression; （4） NIH3T3 cell had a significantly higher level of H3K9m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and with NIT-1 cell（P〈0.05）,with no detectable mRNA expression of these genes.It was concluded that histone modification（H3K4m3 and H3K9m3） and DNA methylation might have an intimate communication between each other in the differentiation process from embryonic stem cells into islet cells.

DNA Methylation and Birth Weight: a Genome-wide Analysis

The study illustrate the inner correlation between global DNA methylation variation and different birth weights. Infant birth weight was used to identify cases and controls. Cord blood and placentas were collected. We performed DNA methylation profiling of bisulphite‐converted DNA. We have identified many differentially methylated Cp G sites in experimental groups; these sites involved in hundreds of signalings. Among these, more than ten pathways were referred to the glucose and lipid metabolism. Methylation changes in the insulin‐signaling pathway（ISP）, adipocytokine signaling pathway（ASP） and MAPK signaling pathway are involved in the fetal programming of diabetes.

Expression of the C-Terminal Domain of Mammalian TET3 DNA Dioxygenase in Arabidopsis thaliana Induces Heritable Methylation Changes at rDNA Loci

In plants, demethylation of 5-methylcytosine (5 mC) residues is controlled by DNA glycosylases, while in mammals it requires oxidation of 5 mC by TET proteins, a group of Fe(II)/2-oxoglutaratedependent dioxygenases. We analysed the effects of expressing the C-terminal catalytic domain of the human TET3 gene (TET3c) in Arabidopsis thaliana, using an rDNA region as a methylation reporter. In TET3c transformants, epialleles with hypomethylation or hypermethylation patterns can be induced, which is each stably retained in progeny lines even after removal of the TET3c transgene. In TET3c transformants, 5-hydroxymethylcytosine (5 hmC) marks are detected, indicative of the oxidative activity of the transgenic enzyme. 5-formylcytosine (5 fC) is only detectable in TET3c transformants with a DNA glycosylase mutant background suggesting further oxidation of 5 hmC residues to 5 fC by TET3c, and efficient recognition and removal of 5 fC by plant glycosylases. The results suggest that TET3c can be employed to induce heritable locus-specific changes in DNA methylation, and that accumulation of 5 hmC can be used as a marker for TET3c target regions.