Dynamic Changes In DNA Methylation Research Articles

Reduced CHH methylation levels reveal a critical role of aging pathway genes in Moso bamboo flowering

Moso bamboo holds significant economic importance in China, serving various purposes, such as food, material, ornamentation, and greenery. Despite its versatility, the occurrence of flowering in Moso bamboo poses a threat to bamboo forests, resulting in substantial losses. The underlying cause of bamboo flowering remains elusive. Dynamic fluctuations in DNA methylation govern the transcriptional levels of crucial genes pivotal for plant growth and development. In this study, we conducted comprehensive DNA methylation (by whole-genome bisulfite sequencing) and transcriptome (by RNA-seq) analyses on non-flowering leaves, flowering leaves, and spikelets of Moso bamboo. Our findings revealed a notable reduction in the overall DNA methylation level, particularly CHH methylation, from leaves to spikelets, influencing the expression of differentially regulated genes. Notably, we identified DNA methylation as a regulatory mechanism for numerous flowering-related genes, including SPLs, FT, and SOC1. Specifically, the SPL3f gene, a key regulator of the aging pathway, exhibited hypomethylation and a high expression level in spikelets. Conversely, SOC1c displayed transcriptional silencing attributed to hypermethylation in the CHH context in the leaves of non-flowering plants. DNA methylation may affect the flowering mechanism of Moso bamboo by regulating the expression of key genes. In summary, our results shed light on the dynamic changes in DNA methylation between leaves and spikelets, unraveling an important epigenetic modification mechanism for flowering in Moso bamboo.

Dynamic DNA methylation modification in catechins and terpenoids biosynthesis during tea plant (Camellia sinensis) leaf development

DNA methylation plays important roles in regulating gene expression during development. However, little is known about the influence of DNA methylation on secondary metabolism during leaf development in the tea plant (Camellia sinensis). In this study, we combined the methylome, transcriptome, and metabolome to investigate the dynamic changes in DNA methylation and its potential regulatory roles in secondary metabolite biosynthesis. In this study, the level of genomic DNA methylation increased as leaf development progressed from tender to old leaf. It additionally exhibited a similar distribution across the genomic background at the two distinct developmental stages studied. Notably, integrated analysis of transcriptomic and methylomic data showed that DNA hypermethylation primarily occurred in genes of the phenylpropanoid, flavonoid, and terpenoid biosynthesis pathways. The effect of methylation on transcription of these secondary metabolite biosynthesis genes was dependent on the location of methylation (i.e., in the promoter, gene or intergenic regions) and the sequence context (i.e., CpG, CHG, or CHH). Changes in the content of catechins and terpenoids were consistent with the changes in gene transcription and the methylation state of structural genes, such as serine carboxypeptidase-like acyltransferases 1A (SCPL1A), leucoanthocyanidin reductase (LAR), and nerolidol synthase (NES). Our study provides valuable information for dissecting the effects of DNA methylation on regulation of genes involved in secondary metabolism during tea leaf development.

Uncovering the role of TET2-mediated ENPEP activation in trophoblast cell fate determination

Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Dynamic changes in DNA methylation occur during preimplantation development and are critical for cell fate determination. However, the underlying regulatory mechanism remains unclear. Recently, we derived morula-like expanded potential stem cells from human preimplantation embryos (hEPSC-em), providing a valuable tool for studying early trophoblast differentiation. Data analysis on published datasets showed differential expressions of DNA methylation enzymes during early trophoblast differentiation in human embryos and hEPSC-em derived trophoblastic spheroids. We demonstrated downregulation of DNA methyltransferase 3 members (DNMT3s) and upregulation of ten-eleven translocation methylcytosine dioxygenases (TETs) during trophoblast differentiation. While DNMT inhibitor promoted trophoblast differentiation, TET inhibitor hindered the process and reduced implantation potential of trophoblastic spheroids. Further integrative analysis identified that glutamyl aminopeptidase (ENPEP), a trophectoderm progenitor marker, was hypomethylated and highly expressed in trophoblast lineages. Concordantly, progressive loss of DNA methylation in ENPEP promoter and increased ENPEP expression were detected in trophoblast differentiation. Knockout of ENPEP in hEPSC-em compromised trophoblast differentiation potency, reduced adhesion and invasion of trophoblastic spheroids, and impeded trophoblastic stem cell (TSC) derivation. Importantly, TET2 was involved in the loss of DNA methylation and activation of ENPEP expression during trophoblast differentiation. TET2-null hEPSC-em failed to produce TSC properly. Collectively, our results illustrated the crucial roles of ENPEP and TET2 in trophoblast fate commitments and the unprecedented TET2-mediated loss of DNA methylation in ENPEP promoter.

Developmentally dynamic changes in DNA methylation in the human pancreas

Development of the human pancreas requires the precise temporal control of gene expression via epigenetic mechanisms and the binding of key transcription factors. We quantified genome-wide patterns of DNA methylation in human fetal pancreatic samples from donors aged 6 to 21 post-conception weeks. We found dramatic changes in DNA methylation across pancreas development, with > 21% of sites characterized as developmental differentially methylated positions (dDMPs) including many annotated to genes associated with monogenic diabetes. An analysis of DNA methylation in postnatal pancreas tissue showed that the dramatic temporal changes in DNA methylation occurring in the developing pancreas are largely limited to the prenatal period. Significant differences in DNA methylation were observed between males and females at a number of autosomal sites, with a small proportion of sites showing sex-specific DNA methylation trajectories across pancreas development. Pancreas dDMPs were not distributed equally across the genome and were depleted in regulatory domains characterized by open chromatin and the binding of known pancreatic development transcription factors. Finally, we compared our pancreas dDMPs to previous findings from the human brain, identifying evidence for tissue-specific developmental changes in DNA methylation. This study represents the first systematic exploration of DNA methylation patterns during human fetal pancreas development and confirms the prenatal period as a time of major epigenomic plasticity.

DNA methyltransferase 1 (DNMT1) promotes cyst growth and epigenetic age acceleration in autosomal dominant polycystic kidney disease

Alteration of DNA methylation leads to diverse diseases, and the dynamic changes of DNA methylation (DNAm) on sets of CpG dinucleotides in mammalian genomes are termed “DNAm age” and “epigenetic clocks” that can predict chronological age. However, whether and how dysregulation of DNA methylation promotes cyst progression and epigenetic age acceleration in autosomal dominant polycystic kidney disease (ADPKD) remains elusive. Here, we show that DNA methyltransferase 1 (DNMT1) is upregulated in cystic kidney epithelial cells and tissues and that knockout of Dnmt1 and targeting DNMT1 with hydralazine, a safe demethylating agent, delays cyst growth in Pkd1 mutant kidneys and extends life span of Pkd1 conditional knockout mice. With methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq), DNMT1 chromatin immunoprecipitation (ChIP)-sequencing and RNA-sequencing analysis, we identified two novel DNMT1 targets, PTPRM and PTPN22 (members of the protein tyrosine phosphatase family). PTPRM and PTPN22 function as mediators of DNMT1 and the phosphorylation and activation of PKD associated signaling pathways, including ERK, mTOR and STAT3. With whole genome bisulfide sequencing in kidneys of patients with ADPKD versus normal individuals, we found that the methylation of epigenetic clock associated genes was dysregulated, supporting that epigenetic age is accelerated in the kidneys of patients with ADPKD. Furthermore, five epigenetic clock associated genes, including Hsd17b14, Itpkb, Mbnl1, Rassf5 and Plk2 were identified. Thus, the diverse biological roles of these five genes suggest that their methylation status may not only predict epigenetic age acceleration but also contribute to disease progression in ADPKD.

Micronutrient regulation of the DNA methylome

The formation, inheritance, and removal of DNA methylation in the genome of mammalian cells is directly regulated by two families of enzymes–DNA methyltransferases (DNMTs) and Ten-Eleven Translocation proteins (TETs). DNMTs generate and maintain the inheritance of 5-methylcytosine (5mC), which is the substrate targeted by the TET enzymes for conversion to 5-hydroxymethylcytosine (5hmC) and its downstream oxidized derivatives. The activity of DNMT and TET is dependent on the availability of micronutrients and metabolite co-factors, including essential vitamins, amino acids, and trace metals, highlighting how DNA methylation levels can be directly enhanced, suppressed, or remodeled via metabolic and nutritional perturbations. Dynamic changes in DNA methylation are required during embryonic development, lineage specification, and maintenance of somatic cell function that can be fine-tuned based on the influence of essential micronutrients. As we age, DNA methylation and hydroxymethylation levels drift in patterning, leading to epigenetic dysregulation and genomic instability that underlies the formation and progression of multiple diseases including cancer. Understanding how DNA methylation can be regulated by micronutrients will have important implications for the maintenance of normal tissue function upon aging, and in the prevention and treatment of diseases for improved health and lifespan.

The lemon genome and DNA methylome unveil epigenetic regulation of citric acid biosynthesis during fruit development.

Citric acid gives lemons their unique flavor, which impacts their sensory traits and market value. However, the intricate process of citric acid accumulation during lemon fruit growth remains incompletely understood. Here, we achieved a chromosomal-level genome assembly for the 'Xiangshui' lemon variety, spanning 364.85Mb across nine chromosomes. This assembly revealed 27 945 genes and 51.37% repetitive sequences, tracing the divergence from citron 2.85 million years ago. DNA methylome analysis of lemon fruits across different developmental stages revealed significant variations in DNA methylation. We observed decreased CG and CHG methylation but increased CHH methylation. Notably, the expression of RdDM pathway-related genes increased with fruit development, suggesting a connection with elevated CHH methylation, which is potentially influenced by the canonical RdDM pathway. Furthermore, we observed that elevated CHH DNA methylation within promoters significantly influenced the expression of key genes, critically contributing to vital biological processes, such as citric acid accumulation. In particular, the pivotal gene phosphoenolpyruvate carboxykinase (ClPEPCK), which regulates the tricarboxylic acid cycle, was strikingly upregulated during fruit development, concomitant with increased CHH methylation in its promoter region. Other essential genes associated with citric acid accumulation, such as the MYB transcription factor (ClPH1/4/5) and ANTHOCYANIN 1 (ClAN1), were strongly correlated with DNA methylation levels. These results strongly indicate that DNA methylation crucially orchestrates the metabolic synthesis of citric acid. In conclusion, our study revealed dynamic changes in DNA methylation during lemon fruit development, underscoring the significant role of DNA methylation in controlling the citric acid metabolic pathway.

Abstract 25: Long-Term Trends of BMI During Childhood and Adolescence Modify the Association of DNA Methylation Change at TXNIP With Glucose Change During Midlife

Background: Thioredoxin-interacting protein (TXNIP) is a central master regulator of whole-body glucose homeostasis in humans; and DNA methylation (DNAm) at TXNIP has been identified to be related to diabetes. It remains unknown whether dynamic changes in DNAm at TXNIP are associated with glycemic changes; and whether early-life adiposity changes modify such association. Hypothesis: We hypothesized that dynamic changes in DNAm at TXNIP are associated with glycemic changes in midlife, and early-life adiposity changes may modify such association. Methods: A total of 594 Bogalusa Heart Study participants who had DNAm measurements at two time points in midlife were included in this study. Of them, 353 participants had at least 4 examinations of BMI during childhood and adolescence (aged 4–19 years). The area under the curve (AUC) was calculated as a measure of long-term burden (total AUC) and trends (incremental AUC) of BMI during childhood and adolescence. Results: We found 25.3% of participants showed greater than 5% DNAm change at TXNIP during a mean of 6.2 years follow-up. Increase in DNAm at TXNIP was significantly associated with decrease in FPG independent of cell-type composition and other covariates ( P <0.001). Moreover, we found the increasing trend of BMI during childhood and adolescence significantly modified the above dynamic association. Each 1% increase in DNAm at TXNIP was associated with 2.90 (0.77) mg/dl decrease in FPG among participants with the highest tertile of incremental AUC of BMI, 0.96 (0.38) mg/dl decrease among those with middle tertile, whereas no significant association was observed among participants with the lowest tertile ( P -interaction=0.003). Conclusions: Our results indicate that changes in DNAm at TXNIP are significantly associated with dynamics in FPG in midlife, and such association is modified by BMI changes during childhood and adolescence.

Differential expression of Tet family genes and their potential role in regulating skeletal muscle development of Siniperca chuatsi

DNA methylation and demethylation are crucial epigenetic modification and regulation for animal development, and their dynamic changes may affect skeletal muscle development. The ten-eleven translocation (Tet) family proteins are demethylases which are involved in the dynamic changes of DNA methylation. However, the expression pattern of Tet family genes and their role in myogenesis in fish remains unclear. In this study, the temporal and spatial expression profiles of Tet1, Tet2 and Tet3 were assayed with RT-qPCR techniques in Chinese perch, Siniperca chuatsi. The obtained data showed that three Tet family genes were differentially expressed at different development stages. Tet1 was expressed low at blastula stage, but highly expressed at gastrula stage, then remained low until hatching. The expressions of Tet2 and Tet3 were significantly increased at late gastrula and kept high expression before hatching stage. At the spatial level, the Tet1 expression was highest in gill tissue, moderate level in brain and slow muscle. Tet2 was similar to that of Tet1 except that it was expressed at a lower level in slow muscle, and Tet3 exhibited a higher expression level in gill and brain, a moderate level in fast muscle. Cosinor analysis turned out that the expression of Tet1 and Tet2 displayed a significant daily rhythm in fast muscle, but Tet3 did not show daily rhythmicity. Inhibiting the activity of Tet1/2 proteins by injecting Bobcat339 significantly reduced the expression of MyoD and MRF4, but not MyoG and Myf5, by which leads to the increase of the number of satellite cells and proliferating myoblasts. Together, the results suggest that Tet1/2 may target to MyoD and MRF4 resulted in DNA demethylation and promoting their expression, thus stimulating myoblast differentiation.

DNA hypomethylation mediates flower opening and senescence in sweet osmanthus through auxin and ethylene responsive pathways

Recent studies have shown that DNA methylation is critical for fruit ripening and leaf senescence, but its effect on flowering is unclear. This study investigated the dynamic changes in DNA methylation and its regulatory role on flower opening and senescence of Osmanthus fragrans ‘Liuyejingui’ by whole-genome bisulfite sequencing and transcriptome sequencing at six developmental stages (unopened floral bud to late flower senescence, S1 to S6). In total 3222 differentially methylated regions (DMRs) were identified in senescent flowers, of which 89% were hypomethylated (hypo-DMRs), and the number increased form S1 to S6, suggesting that DNA demethylation is associated with the progression of flowering. Analysis of hypo-DMR-associated up-regulated differentially expressed genes revealed that small auxin up-regulated RNAs (OfSAURs) were the most highly significantly enriched from S3 to S5, and ethylene-responsive transcription factors (OfERFs) were strongly activated in senescent flowers. These results suggested that DNA hypomethylation was important for flower expansion and flower senescence through the auxin and ethylene response pathways. Spray application of a DNA methylation inhibitor to unopened floral buds triggered a premature opening and senescence phenotype by promoting endogenous ethylene production and carotenoid accumulation, and indicated that DNA demethylation mediated flower opening and senescence by activating the expression of OfSAURs and OfERFs. An epigenetic regulatory three-component is proposed, in which flower opening and senescence are regulated by DNA demethylation, mediating the expression of specific gene families, together with the plant hormones auxin and ethylene, to transition the flower into an opening- and senescence-competent state. Data availabilityThe genome and transcriptome sequencing data of S1-S6 in O. fragrans ‘Liuyejingui’ were under the public database of the National Center of Biotechnology Information (NCBI) BioProject under the accession number of PRJNA679852 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA679852). Whole Genome Bisulfate Sequencing and transcriptome sequencing data in DNA methylation inhibitor 5-azacytidine and ddH2O treatment have been deposited into the public database of NCBI BioProject under the accession number of PRJNA798828 and PRJNA798559.

DNA methylation dynamics during germline development

DNA methylation plays essential homeostatic functions in eukaryotic genomes. In animals, DNA methylation is also developmentally regulated and, in turn, regulates development. In the past two decades, huge research effort has endorsed the understanding that DNA methylation plays a similar role in plant development, especially during sexual reproduction. The power of whole-genome sequencing and cell isolation techniques, as well as bioinformatics tools, have enabled recent studies to reveal dynamic changes in DNA methylation during germline development. Furthermore, the combination of these technological advances with genetics, developmental biology and cell biology tools has revealed functional methylation reprogramming events that control gene and transposon activities in flowering plant germlines. In this review, we discuss the major advances in our knowledge of DNA methylation dynamics during male and female germline development in flowering plants.

Dynamic DNA methylation changes reveal tissue-specific gene expression in sugarcane.

DNA methylation is an important mechanism for the dynamic regulation of gene expression and silencing of transposons during plant developmental processes. Here, we analyzed genome-wide methylation patterns in sugarcane (Saccharum officinarum) leaves, roots, rinds, and piths at single-base resolution. DNA methylation patterns were similar among the different sugarcane tissues, whereas DNA methylation levels differed. We also found that DNA methylation in different genic regions or sequence contexts plays different roles in gene expression. Differences in methylation among tissues resulted in many differentially methylated regions (DMRs) between tissues, particularly CHH DMRs. Genes overlapping with DMRs tended to be differentially expressed (DEGs) between tissues, and these DMR-associated DEGs were enriched in biological pathways related to tissue function, such as photosynthesis, sucrose synthesis, stress response, transport, and metabolism. Moreover, we observed many DNA methylation valleys (DMVs), which always overlapped with transcription factors (TFs) and sucrose-related genes, such as WRKY, bZIP, WOX, SPS, and FBPase. Collectively, these findings provide significant insights into the complicated interplay between DNA methylation and gene expression and shed light on the epigenetic regulation of sucrose-related genes in sugarcane.

CRISPR/dCas9-Dnmt3a-mediated targeted DNA methylation of APP rescues brain pathology in a mouse model of Alzheimer’s disease

BackgroundAberrant DNA methylation patterns have been observed in neurodegenerative diseases, including Alzheimer's disease (AD), and dynamic changes in DNA methylation are closely associated with the onset and progression of these diseases. Particularly, hypomethylation of the amyloid precursor protein gene (APP) has been reported in patients with AD.MethodsIn this study, we used catalytically inactivated Cas9 (dCas9) fused with Dnmt3a for targeted DNA methylation of APP, and showed that the CRISPR/dCas9-Dnmt3a-mediated DNA methylation system could efficiently induce targeted DNA methylation of APP both in vivo and in vitro.ResultsWe hypothesized that the targeted methylation of the APP promoter might rescue AD-related neuronal cell death by reducing APP mRNA expression. The cultured APP-KI mouse primary neurons exhibited an altered DNA-methylation pattern on the APP promoter after dCas9-Dnmt3a treatment. Likewise, the APP mRNA level was significantly reduced in the dCas9-Dnmt3a-treated wild-type and APP-KI mouse primary neurons. We also observed decreased amyloid-beta (Aβ) peptide level and Aβ42/40 ratio in the dCas9-Dnmt3a-treated APP-KI mouse neurons compared to the control APP-KI mouse neurons. In addition, neuronal cell death was significantly decreased in the dCas9-Dnmt3a-treated APP-KI mouse neurons. Furthermore, the in vivo methylation of APP in the brain via dCas9-Dnmt3a treatment altered Aβ plaques and attenuated cognitive and behavioral impairments in the APP-KI mouse model.ConclusionsThese results suggest that the targeted methylation of APP via dCas9-Dnmt3a treatment can be a potential therapeutic strategy for AD.

Epigenetic Dynamics and Regulation of Plant Male Reproduction.

Flowering plant male germlines develop within anthers and undergo epigenetic reprogramming with dynamic changes in DNA methylation, chromatin modifications, and small RNAs. Profiling the epigenetic status using different technologies has substantially accumulated information on specific types of cells at different stages of male reproduction. Many epigenetically related genes involved in plant gametophyte development have been identified, and the mutation of these genes often leads to male sterility. Here, we review the recent progress on dynamic epigenetic changes during pollen mother cell differentiation, microsporogenesis, microgametogenesis, and tapetal cell development. The reported epigenetic variations between male fertile and sterile lines are summarized. We also summarize the epigenetic regulation-associated male sterility genes and discuss how epigenetic mechanisms in plant male reproduction can be further revealed.

P-759 Comparison of DNA methylation profiles of human embryo cultured in either uninterrupted or interrupted incubators

Abstract Study question Are there are differences in the DNA methylation profiles of human embryos cultured in Time-lapse imaging (TLI) and standard incubators (SI)? Summary answer The genome-wide DNA methylation landscape was globally similar between the SI and TLI groups. What is known already Early embryonic development is a special biological process, along with dynamic changes of DNA methylation. In vitro culturing ensured the successful development of human embryo, and is the most important step of ART cycle. TLI is one type of newly developed ART embryo culture systems, which allows for continuous assessment of embryonic development. Our previous study performed transcriptome analysis to compare SI and TLI and found that the global transcriptomic profiles were similar between the two groups. However, whether there are differences in the DNA methylation profiles of human embryos cultured in TLI and SI still unknown. Study design, size, duration This study was designed to explore the influence of SI and TLI incubator culturing on genome-wide DNA methylation of human eight-cell embryos. A total of 9 women who received IVF treatment, ≤ 30 years old (range: 20–30 years), without a history of genetic diseases or smoking were included in this study. Participants/materials, setting, methods Transvaginal oocyte retrieval was performed 36 h after HCG injection. Cumulus-enclosed oocytes were collected in 2.5 mL IVF medium and incubated in 5% O2, 6% CO2, and 37 °C incubators for insemination. The fertilized oocytes were transferred into pre-equilibrated Embryoslides. Then the embryoslides were cultured in either TLI or SI at 37 °C with 5% O2 and 6% CO2 until embryo transfer on Day 3. Samples were sequenced by the Illumina HiSeq 4000 with a 150-bp paired-end. Main results and the role of chance The genome-wide methylation patterns and CpG methylation levels in transposable elements and imprinting control regions of TLI-cultured embryos were similar to those of the SI-cultured embryos. However, a small number of differentially methylated regions (DMRs) were detected, and these DMRs mainly occurred in exons other than promoters. Functional annotation revealed that the genes in DMRs tended to execute functions such as cell cycle, DNA damage stimulus, histone modification, mitochondrial, glucose import, and MAPK signaling pathway. Limitations, reasons for caution Small sample size. Wider implications of the findings Evaluated the safety of TLI culture system from the perspective of DNA methylation at single-cell level, and provided an important reference for understanding the association between embryo culture condition and epigenetic regulation. Trial registration number not applicable

DNA hypermethylation modification promotes the development of hepatocellular carcinoma by depressing the tumor suppressor gene ZNF334

DNA methylation plays a pivotal role in the development and progression of tumors. However, studies focused on the dynamic changes of DNA methylation in the development of hepatocellular carcinoma (HCC) are rare. To systematically illustrate the dynamic DNA methylation alternation from premalignant to early-stage liver cancer with the same genetic background, this study enrolled 5 HBV-related patients preceded with liver cirrhosis, pathologically identified as early-stage HCC with dysplastic nodules. Liver fibrosis tissues, dysplastic nodules and early HCC tissues from these patients were used to measure DNA methylation. Here, we report significant differences in the DNA methylation spectrum among the three types of tissues. In the early stage of HCC, DNA hypermethylation of tumor suppressor genes is predominant. Additionally, DNA hypermethylation in the early stage of HCC changes the binding ability of transcription factor TP53 to the promoter of tumor suppressor gene ZNF334, and inhibits the expression of ZNF334 at the transcription level. Furthermore, through a series of in vivo and in vitro experiments, we have clarified the exacerbation effect of tumor suppressor gene ZNF334 deletion in the occurrence of HCC. Combined with clinical data, we found that the overall survival and relapse-free survival of patients with high ZNF334 expression are significantly longer. Thus, we partly elucidated a sequential alternation of DNA methylation modification during the occurrence of HCC, and clarified the biological function and regulatory mechanism of the tumor suppressor gene ZNF334, which is regulated by related DNA methylation sites. Our study provides a new target and clinical evidence for the early diagnosis and sheds light on the precise treatment of liver cancer.

Stochastic Variation in DNA Methylation Modulates Nucleosome Occupancy and Alternative Splicing in Arabidopsis thaliana.

Plants use complex gene regulatory mechanisms to overcome diverse environmental challenges. For instance, cold stress induces rapid and massive transcriptome changes via alternative splicing (AS) to confer cold tolerance in plants. In mammals, mounting evidence suggests chromatin structure can regulate co-transcriptional AS. Recent evidence also supports co-transcriptional regulation of AS in plants, but how dynamic changes in DNA methylation and the chromatin structure influence the AS process upon cold stress remains poorly understood. In this study, we used the DNA methylation inhibitor 5-Aza-2′-Deoxycytidine (5-aza-dC) to investigate the role of stochastic variations in DNA methylation and nucleosome occupancy in modulating cold-induced AS, in Arabidopsis thaliana (Arabidopsis). Our results demonstrate that 5-aza-dC derived stochastic hypomethylation modulates nucleosome occupancy and AS profiles of genes implicated in RNA metabolism, plant hormone signal transduction, and of cold-related genes in response to cold stress. We also demonstrate that cold-induced remodelling of DNA methylation regulates genes involved in amino acid metabolism. Collectively, we demonstrate that sudden changes in DNA methylation via drug treatment can influence nucleosome occupancy levels and modulate AS in a temperature-dependent manner to regulate plant metabolism and physiological stress adaptation.

MBD protein recognizes flower control genes regulated by DNA methylation in <i>Chrysanthemum lavandulifolium</i>

Dynamic changes in DNA methylation regulate the expression of genes and play important roles especially in the flowering processes of higher plants. Methyl-CpG-binding domain protein could specifically recognize hypermethylated regions in the genome, thus MBD sequencing technology and CpG islands analysis of the sequences were used to identify candidate genes that were regulated by DNA methylation, in particular the flowering induction stage of <em>Chrysanthemum lavandulifolium</em>. MBD-seq identified 89 candidate genes which included 49 genes exhibiting changes in DNA methylation status during floral induction. Based on CpG islands analysis of the sequences, 27 candidate genes were selected that may be regulated by DNA methylation. The expression levels of 30 candidate genes and nine key genes were determined by RT-PCR and qRT-PCR during floral induction (7D), four genes (<em>ClFT</em>, <em>ClMET</em>, <em>DFL</em> and <em>ClWRKY21</em>) were similarly up-regulated. Methylation-specific PCR analysis also indicated that there were changes in the DNA methylation status in the <em>DFL</em> and <em>ClWRKY21</em>. The changes in the DNA methylation status during the induction phase of flowering may lead to changes in gene expression. In this study, a set of genes were identified that are proposed to be involved in floral induction and two key genes were identified (<em>DFL, ClWRKY21</em>) that were regulated by DNA methylation during the flowering process of <em>C. lavandulifolium</em>.

Dynamic Changes of DNA Methylation During Wild Strawberry (Fragaria nilgerrensis) Tissue Culture.

Tissue culture is an important tool for asexual propagation and genetic transformation of strawberry plants. In plant tissue culture, variation of DNA methylation is a potential source of phenotypic variation in regenerated plants. However, the genome wide dynamic methylation patterns of strawberry tissue culture remain unclear. In this study, we used whole-genome bisulfite sequencing (WGBS) to study genomic DNA methylation changes of a wild strawberry Fragaria nilgerrensis at six stages: from explants of shoot tips to outplanting and acclimation. Global methylation levels showed that CG sites exhibited the highest methylation level in all stages with an average of 49.5%, followed by CHG (33.2%) and CHH (12.4%). Although CHH accounted for the lowest proportion of total cytosine methylation, it showed the most obvious methylation change and the most of these changes occurred in the transposable element regions. The overall methylation levels alternately decreased and increased during the entire tissue culture process and the distribution of DNA methylation was non-uniform among different genetic regions. Furthermore, much more differentially methylated regions (DMRs) were detected in dedifferentiation and redifferentiation stages and most of them were transposable elements, suggesting these processes involved activating or silencing of amounts of transposons. The functional enrichment of the DMR-related genes indicated that genes involved in hormone metabolic processes, plant development and the stress response changed methylation throughout the tissue culture process. Finally, the quantitative real-time PCR (qRT-PCR) was conducted to examine the association of methylation and gene expression of a set of different methylated genes. Our findings give deeper insight into the epigenetic regulation of gene expression during the plant tissue cultures process, which will be useful in the efficient control of somaclonal variations and in crop improvement.

Dynamic DNA Methylation Changes in the COMT Gene Promoter Region in Response to Mental Stress and Its Modulation by Transcranial Direct Current Stimulation.

Changes in epigenetic modifications present a mechanism how environmental factors, such as the experience of stress, can alter gene regulation. While stress-related disorders have consistently been associated with differential DNA methylation, little is known about the time scale in which these alterations emerge. We investigated dynamic DNA methylation changes in whole blood of 42 healthy male individuals in response to a stressful cognitive task, its association with concentration changes in cortisol, and its modulation by transcranial direct current stimulation (tDCS). We observed a continuous increase in COMT promotor DNA methylation which correlated with higher saliva cortisol levels and was still detectable one week later. However, this lasting effect was suppressed by concurrent activity-enhancing anodal tDCS to the dorsolateral prefrontal cortex. Our findings support the significance of gene-specific DNA methylation in whole blood as potential biomarkers for stress-related effects. Moreover, they suggest alternative molecular mechanisms possibly involved in lasting behavioral effects of tDCS.