CG Sequences Research Articles

Reprogramming of DNA methylation and changes of gene expression in grafted Hevea brasiliensis.

Rubber tree (Hevea brasiliensis) is reproduced by bud grafting for commercial planting, but significant intraclonal variations exist in bud-grafted clones. DNA methylation changes related to grafting may be partly responsible for intraclonal variations. In the current study, whole-genome DNA methylation profiles of grafted rubber tree plants (GPs) and their donor plants (DPs) were evaluated by whole-genome bisulfite sequencing. Data showed that DNA methylation was downregulated and DNA methylations in CG, CHG, and CHH sequences were reprogrammed in GPs, suggesting that grafting induced the reprogramming of DNA methylation. A total of 5,939 differentially methylated genes (DMGs) were identified by comparing fractional methylation levels between GPs and DPs. Transcriptional analysis revealed that there were 9,798 differentially expressed genes (DEGs) in the DP and GP comparison. A total of 1,698 overlapping genes between DEGs and DMGs were identified. These overlapping genes were markedly enriched in the metabolic pathway and biosynthesis of secondary metabolites by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Global DNA methylation and transcriptional analyses revealed that reprogramming of DNA methylation is correlated with gene expression in grafted rubber trees. The study provides a whole-genome methylome of rubber trees and an insight into the molecular mechanisms underlying the intraclonal variations existing in the commercial planting of grafted rubber trees.

Identification of Susceptibility Genes Underlying Bovine Respiratory Disease in Xinjiang Brown Cattle Based on DNA Methylation.

DNA methylation is a form of epigenetic regulation, having pivotal parts in controlling cellular expansion and expression levels within genes. Although blood DNA methylation has been studied in humans and other species, its prominence in cattle is largely unknown. This study aimed to methodically probe the genomic methylation map of Xinjiang brown (XJB) cattle suffering from bovine respiratory disease (BRD), consequently widening cattle blood methylome ranges. Genome-wide DNA methylation profiling of the XJB blood was investigated through whole-genome bisulfite sequencing (WGBS). Many differentially methylated regions (DMRs) obtained by comparing the cases and controls groups were found within the CG, CHG, and CHH (where H is A, T, or C) sequences (16,765, 7502, and 2656, respectively), encompassing 4334 differentially methylated genes (DMGs). Furthermore, GO/KEGG analyses showed that some DMGs were involved within immune response pathways. Combining WGBS-Seq data and existing RNA-Seq data, we identified 71 significantly differentially methylated (DMGs) and expressed (DEGs) genes (p < 0.05). Next, complementary analyses identified nine DMGs (LTA, STAT3, IKBKG, IRAK1, NOD2, TLR2, TNFRSF1A, and IKBKB) that might be involved in the immune response of XJB cattle infected with respiratory diseases. Although further investigations are needed to confirm their exact implication in the involved immune processes, these genes could potentially be used for a marker-assisted selection of animals resistant to BRD. This study also provides new knowledge regarding epigenetic control for the bovine respiratory immune process.

Potential Involvement of DNA Methylation in Hybrid Sterility in Hermaphroditic Argopecten Scallops.

DNA methylation is an important epigenetic modification factor in regulating fertility. Corresponding process remains poorly investigated in hermaphroditic scallops. The interspecific F1 hybrids between the hermaphroditic bay scallops (Argopecten irradians) and Peruvian scallops (Argopecten purpuratus) exhibited significant heterosis in yield, but sterility in hybrids obstructs the utilization of the genetic resources. However, the determination mechanism of hybrid sterility in the hermaphroditic Argopecten scallops is still unclear. In this study, the effect of DNA methylation in the hybrid sterility of hermaphroditic Argopecten scallops was explored. The results showed that the mean methylation level was higher in sterile hybrids than fertile hybrids, especially on chromosome 11 of the paternal parent. A total of 61,062 differentially methylated regions (DMRs) were identified, containing 3619 differentially methylated genes (DMGs) and 1165 differentially methylated promoters that are located in the DMRs of CG sequence context. The hyper-methylated genes were enriched into five KEGG pathways, including ubiquitin-mediated proteolysis, ECM-receptor interaction, non-homologous end-joining, notch signaling, and the mismatch repair pathways. The DMGs might induce hybrid sterility by inhibition of oogenesis and egg maturation, induction of apoptosis, increased ROS, and insufficient ATP supply. Our results would enrich the determination mechanism of hybrid sterility and provide new insights into the utilization of the genetic resources of the interspecific hybrids.

MBD3 Regulates Male Germ Cell Division and Sperm Fertility in Arabidopsis thaliana.

DNA methylation plays important roles through the methyl-CpG-binding domain (MBD) to realize epigenetic modifications. Thirteen AtMBD proteins have been identified from the Arabidopsis thaliana genome, but the functions of some members are unclear. AtMBD3 was found to be highly expressed in pollen and seeds and it preferably binds methylated CG, CHG, and unmethylated DNA sequences. Then, two mutant alleles at the AtMBD3 locus were obtained in order to further explore its function using CRISPR/Cas9. When compared with 92.17% mature pollen production in the wild type, significantly lower percentages of 84.31% and 78.91% were observed in the mbd3-1 and mbd3-2 mutants, respectively. About 16-21% of pollen from the mbd3 mutants suffered a collapse in reproductive transmission, whereas the other pollen was found to be normal. After pollination, about 16% and 24% of mbd3-1 and mbd3-2 mutant seeds underwent early or late abortion, respectively. Among all the late abortion seeds in mbd3-2 plants, 25% of the abnormal seeds were at the globular stage, 31.25% were at the transition stage, and 43.75% were at the heart stage. A transcriptome analysis of the seeds found 950 upregulated genes and 1128 downregulated genes between wild type and mbd3-2 mutants. Some transcriptional factors involved in embryo development were selected to be expressed, and we found significant differences between wild type and mbd3 mutants, such as WOXs, CUC1, AIB4, and RGL3. Furthermore, we found a gene that is specifically expressed in pollen, named PBL6. PBL6 was found to directly interact with AtMBD3. Our results provide insights into the function of AtMBD3 in plants, especially in sperm fertility.

Knockout of DDM1 in Physcomitrium patens disrupts DNA methylation with a minute effect on transposon regulation and development.

The Snf2 chromatin remodeler, DECREASE IN DNA METHYLATION 1 (DDM1) facilitates DNA methylation. In flowering plants, DDM1 mediates methylation in heterochromatin, which is targeted primarily by MET1 and CMT methylases and is necessary for silencing transposons and for proper development. DNA methylation mechanisms evolved throughout plant evolution, whereas the role of DDM1 in early terrestrial plants remains elusive. Here, we studied the function of DDM1 in the moss, Physcomitrium (Physcomitrella) patens, which has robust DNA methylation that suppresses transposons and is mediated by a MET1, a CMT, and a DNMT3 methylases. To elucidate the role of DDM1 in P. patens, we have generated a knockout mutant and found DNA methylation to be strongly disrupted at any of its sequence contexts. Symmetric CG and CHG sequences were affected stronger than asymmetric CHH sites. Furthermore, despite their separate targeting mechanisms, CG (MET) and CHG (CMT) methylation were similarly depleted by about 75%. CHH (DNMT3) methylation was overall reduced by about 25%, with an evident hyper-methylation activity within lowly-methylated euchromatic transposon sequences. Despite the strong hypomethylation effect, only a minute number of transposons were transcriptionally activated in Ppddm1. Finally, Ppddm1 was found to develop normally throughout the plant life cycle. These results demonstrate that DNA methylation is strongly dependent on DDM1 in a non-flowering plant; that DDM1 is required for plant-DNMT3 (CHH) methylases, though to a lower extent than for MET1 and CMT enzymes; and that distinct and separate methylation pathways (e.g. MET1-CG and CMT-CHG), can be equally regulated by the chromatin and that DDM1 plays a role in it. Finally, our data suggest that the biological significance of DDM1 in terms of transposon regulation and plant development, is species dependent.

Genome-wide analysis of DNA methylation reveals significant impact of two typical high temperature stress on biological functions in Apostichopus japonicus

Sea cucumber Apostichopus japonicus, an important marine benthic animal, is particularly sensitive to environmental temperature stress, which affected the growth, metabolism, and reproduction even survival. In this study, we used whole genome bisulfite sequencing (WGBS) to perform a comprehensive comparative analysis of the DNA methylation profiles of A. japonicus at three temperatures: normal (20 °C), aestivation (26 °C), and lethal (32 °C). The results showed that the total methylation level was highest in group T26 and lowest in group T32. Most of the DNA methylation occurred on the C bases of the CG type, and the degree of DNA methylation on the CG-type C bases was 13.32–14.99%, reflecting a moderate level of DNA methylation. Comparison of differentially methylated regions (DMRs) in the CG sequence context, we identified 17,174 DMRs in group T26_vs_T20 and 16,216 DMRs in group T32_vs_T26. There were more hypermethylated regions than hypomethylated regions, and this difference was more obvious in group T32_vs_T26. DMRs analysis of the promoter regions and the gene body regions identified more DMR-related genes in the gene body region than in the promoter region in both comparison groups (T26_vs_T20 and T32_vs_T26). GO and KEGG analysis showed that pathways such as protein processing in the endoplasmic reticulum, ubiquitin mediated proteolysis, and endocytosis, etc., were highly enriched in the DMR-related genes in the comparison groups. Some of these pathways that were significantly enriched in the hypermethylated genes in group T26_vs_T20 but were enriched in the hypomethylated genes in group T32_vs_T26. Enrichment analysis of genes both differentially methylated and differentially expressed showed that downregulated genes in group T32_vs_T26 that were also hypomethylated in the gene body region were significantly enriched in the metabolic and the antioxidant-related glutathione metabolism pathway. Our results suggested that DNA methylation develop a lot of variation in the regulation of the response of A. japonicus to two levels of heat stress: at aestivation temperature and at lethal temperature, and there are many differences between the two. The results of this study provide important insights into the epigenetic mechanisms underlying the response of A. japonicus to the pressure of environmental temperature.

Environmental and genealogical effects on DNA methylation in a widespread apomictic dandelion lineage.

DNA methylation in plant genomes occurs in different sequences and genomic contexts that have very different properties. DNA methylation that occurs in CG (mCG) sequence context shows transgenerational stability and high epimutation rate, and can thus provide genealogical information at short time scales. However, due to meta-stability and because mCG variants may arise due to other factors than epimutation, such as environmental stress exposure, it is not clear how well mCG captures genealogical information at micro-evolutionary time scales. Here, we analysed DNA methylation variation between accessions from a geographically widespread, apomictic common dandelion (Taraxacum officinale) lineage when grown experimentally under different light conditions. Using a reduced-representation bisulphite sequencing approach, we show that the light treatment induced differentially methylated cytosines (DMCs) in all sequence contexts, with a bias towards transposable elements. Accession differences were associated mainly with DMCs in CG context. Hierarchical clustering of samples based on total mCG profiles revealed a perfect clustering of samples by accession identity, irrespective of light conditions. Using microsatellite information as a benchmark of genetic divergence within the clonal lineage, we show that genetic divergence between accessions correlates strongly with overall mCG profiles. However, our results suggest that environmental effects that do occur in CG context may produce a heritable signal that partly dilutes the genealogical signal. Our study shows that methylation information in plants can be used to reconstruct micro-evolutionary genealogy, providing a useful tool in systems that lack genetic variation such as clonal and vegetatively propagated plants.

Single-base resolution methylome of different ecotype from Pyrus betulaefolia reveals epigenomic changes in response to salt stress

• Drawing the cytosine methylation map across the whole genome of Pyrus betulaefolia roots. • The changes of methylomes in two ecotypes after salt exposed dependent on their salt tolerance abilities. • DNA demethylation induced higher expression levels of some Ca 2+ sensor genes in P. betulaefolia roots. • The higher K + /Na + ratio in the roots of salt-tolerance ecotype is relationship with Ca 2+ sensor genes transcription causing by hypomethylation. The root is the first organ to feel salt stress in woody fruit trees. Understanding the potential epigenetic mechanism induced by salt stress in roots of Pyrus betulaefolia is important for pear breeding using diverse genetic resources. With the help of whole-genome bisulfite sequencing, cytosine methylation was mapped at single-base resolution across the whole genome of P. betulaefolia roots. The P. betulaefolia root genome revealed nearly 11.40%, 39.93%, 22.73%, and 4.17% methylation across all sequenced C sites and in the CG, CHG, and CHH sequence contexts, respectively. Then, the changes in the methylome of the roots relating to salt stress were comparatively analysed in two ecotypes with different salt-stress tolerances. After a global methylome and transcriptome combined analysis, gene body methylation demonstrated an obvious negative correlation with transcription levels, whereas promoter-methylated genes presented lower expression levels than promoter-unmethylated genes. Furthermore, the methylation profiles of Ca 2+ sensor genes in roots of P. betulaefolia . were described, and some were demethylated under salt-stress conditions. In detail, the up-regulation of Ca 2+ sensors and their downstream genes, PbCDPK20.1, PbCDPK20.2, PbAKT1 , and PbHAK25 , resulted from salt-induced promoter methylation changes . Finally, K + accumulation was promoted, and a higher K + /Na + ratio was maintained in roots under salt-stress compared with normal conditions. Additionally, the application of the DNA methylation inhibitor 5-azacytidine in vitro induced DNA demethylation and promoted K + enrichment, whereas the accelerator methyl trifluoromethanesulfonate produced the opposite effects. In conclusion, our result revealed the relationship of DNA methylation and gene expression in P. betulaefolia during salt stress, which will increase our understanding of the epigenetic mechanisms of woody trees.

Comparison of tissue culture-induced variation in triticale regenerants obtained by androgenesis and somatic embryogenesis

Triticale is becoming an increasingly important livestock crop production. This is evidenced by increasing triticale-producing areas and by improved yields. In addition, meeting the increasing demand for cereals involves the introduction of high-yielding and stress-resistant varieties into breeding. In vitro culture techniques can accelerate the development of new varieties. Therefore, it seems extremely important to develop efficient plant regeneration methods through in vitro cultures and to understand the mechanisms involved in gaining regenerants. Obtaining regenerants of triticale through somatic embryogenesis and androgenesis may lead to tissue culture-induced variation. In the present study, we compared regenerants obtained in both regeneration systems (anther and immature zygotic embryo cultures), considering the level of genetic and epigenetic changes observed in different DNA sequence contexts for methylated cytosine (CG, CHG, CHH). The changes concerning the DNA sequence (so-called sequence variation) and the changes concerning the DNA methylation patterns, i.e., the removal of methylated cytosine (DNA demethylation) and the introduction of methylation to cytosine (de novo DNA methylation), were analyzed. We observed that regenerants derived via somatic embryogenesis and androgenesis differ notably for demethylation in the symmetrical CG sequence context and de novo methylation in the asymmetrical CHH context. These changes may be related to the reprogramming of microspore development from gametophytic to sporophytic and lack of such process in zygotic embryos.

Characterization of demethylating DNA glycosylase ROS1 from Nicotiana tabacum L.

One of the main mechanisms of epigenetic regulation in higher eukaryotes is based on the methylation of cytosine at the C5 position with the formation of 5-methylcytosine (mC), which is further recognized by regulatory proteins. In mammals, methylation mainly occurs in CG dinucleotides, while in plants it targets CG, CHG, and CHH sequences (H is any base but G). Correct maintenance of the DNA methylation status is based on the balance of methylation, passive demethylation, and active demethylation. While in mammals active demethylation is based on targeted regulated damage to mC in DNA followed by the action of repair enzymes, demethylation in plants is performed by specialized DNA glycosylases that hydrolyze the N-glycosidic bond of mC nucleotides. The genome of the model plant Arabidopsis thaliana encodes four paralogous proteins, two of which, DEMETER (DME) and REPRESSOR OF SILENCING 1 (ROS1), possess 5-methylcytosine-DNA glycosylase activity and are necessary for the regulation of development, response to infections and abiotic stress and silencing of transgenes and mobile elements. Homologues of DME and ROS1 are present in all plant groups; however, outside A. thaliana, they are poorly studied. Here we report the properties of a recombinant fragment of the ROS1 protein from Nicotiana tabacum (NtROS1), which contains all main structural domains required for catalytic activity. Using homologous modeling, we have constructed a structural model of NtROS1, which revealed folding characteristic of DNA glycosylases of the helix– hairpin–helix structural superfamily. The recombinant NtROS1 protein was able to remove mC bases from DNA, and the enzyme activity was barely affected by the methylation status of CG dinucleotides in the opposite strand. The enzyme removed 5-hydroxymethylcytosine (hmC) from DNA with a lower eff iciency, showing minimal activity in the presence of mC in the opposite strand. Expression of the NtROS1 gene in cultured human cells resulted in a global decrease in the level of genomic DNA methylation. In general, it can be said that the NtROS1 protein and other homologues of DME and ROS1 represent a promising scaffold for engineering enzymes to analyze the status of epigenetic methylation and to control gene activity.

CG and CHG Methylation Contribute to the Transcriptional Control of OsPRR37-Output Genes in Rice.

Plant circadian clock coordinates endogenous transcriptional rhythms with diurnal changes of environmental cues. OsPRR37, a negative component in the rice circadian clock, reportedly regulates transcriptome rhythms, and agronomically important traits. However, the underlying regulatory mechanisms of OsPRR37-output genes remain largely unknown. In this study, whole genome bisulfite sequencing and high-throughput RNA sequencing were applied to verify the role of DNA methylation in the transcriptional control of OsPRR37-output genes. We found that the overexpression of OsPRR37 suppressed rice growth and altered cytosine methylations in CG and CHG sequence contexts in but not the CHH context (H represents A, T, or C). In total, 35 overlapping genes were identified, and 25 of them showed negative correlation between the methylation level and gene expression. The promoter of the hexokinase gene OsHXK1 was hypomethylated at both CG and CHG sites, and the expression of OsHXK1 was significantly increased. Meanwhile, the leaf starch content was consistently lower in OsPRR37 overexpression lines than in the recipient parent Guangluai 4. Further analysis with published data of time-course transcriptomes revealed that most overlapping genes showed peak expression phases from dusk to dawn. The genes involved in DNA methylation, methylation maintenance, and DNA demethylation were found to be actively expressed around dusk. A DNA glycosylase, namely ROS1A/DNG702, was probably the upstream candidate that demethylated the promoter of OsHXK1. Taken together, our results revealed that CG and CHG methylation contribute to the transcriptional regulation of OsPRR37-output genes, and hypomethylation of OsHXK1 leads to decreased starch content and reduced plant growth in rice.

The callus formation capacity of strawberry leaf explant is modulated by DNA methylation.

Shoot regeneration from leaf tissue requires the de-differentiation of cells from a highly differentiated state into an actively dividing state, but it remains unclear how this physiological transition occurs and is regulated, especially at the epigenetic level. Here, we characterized the DNA methylome represented by 5-methylcytosine (5mC) in leaf and callus tissue derived from leaf explants of woodland strawberry, Fragaria vesca. We detected an overall increase in DNA methylation and distinct 5mC enrichment patterns in the CG, CHG, and CHH sequence contexts in genes and transposable elements. Our analyses revealed an intricate relationship between DNA methylation and gene expression level in leaves or leaf-derived callus. However, when considering the genes involved in callus formation and shoot regeneration, e.g. FvePLT3/7, FveWIND3, FveWIND4, FveLOG4 and FveIAA14, their dynamic transcription levels were associated with differentially methylated regions located in the promoters or gene bodies, indicating a regulatory role of DNA methylation in the transcriptional regulation of pluripotency acquisition in strawberry. Furthermore, application of the DNA methyltransferase inhibitor 5′-azacytidine (5′-Aza) hampered both callus formation and shoot regeneration from the leaf explants. We further showed that 5′-Aza downregulated the expression of genes involved in cell wall integrity, such as expansin, pectin lyase, and pectin methylesterase genes, suggesting an essential role of cell wall metabolism during callus formation. This study reveals the contribution of DNA methylation to callus formation capacity and will provide a basis for developing a strategy to improve shoot regeneration for basic and applied research applications.

Insight Between the Epigenetics and Transcription Responding of Cotton Hypocotyl Cellular Elongation Under Salt-Alkaline Stress.

Gossypium barbadense is a cultivated cotton not only known for producing superior fiber but also for its salt and alkaline resistance. Here, we used Whole Genome Bisulfite Sequencing (WGBS) technology to map the cytosine methylation of the whole genome of the G. barbadense hypocotyl at single base resolution. The methylation sequencing results showed that the mapping rates of the three samples were 75.32, 77.54, and 77.94%, respectively. In addition, the Bisulfite Sequence (BS) conversion rate was 99.78%. Approximately 71.03, 53.87, and 6.26% of the cytosine were methylated at CG, CHG, and CHH sequence contexts, respectively. A comprehensive analysis of DNA methylation and transcriptome data showed that the methylation level of the promoter region was a positive correlation in the CHH context. Saline-alkaline stress was related to the methylation changes of many genes, transcription factors (TFs) and transposable elements (TEs), respectively. We explored the regulatory mechanism of DNA methylation in response to salt and alkaline stress during cotton hypocotyl elongation. Our data shed light into the relationship of methylation regulation at the germination stage of G. barbadense hypocotyl cell elongation and salt-alkali treatment. The results of this research help understand the early growth regulation mechanism of G. barbadense in response to abiotic stress.

Deciphering the synergistic and redundant roles of CG and non-CG DNA methylation in plant development and transposable element silencing.

Summary DNA methylation plays key roles in transposable element (TE) silencing and gene expression regulation. DNA methylation occurs at CG, CHG and CHH sequence contexts in plants. However, the synergistic and redundant roles of CG and non‐CG methylation are poorly understood.By introducing CRISPR/Cas9‐induced met1 mutation into the ddcc (drm1 drm2 cmt2 cmt3) mutant, we attempted to knock out all five DNA methyltransferases in Arabidopsis and then investigate the synergistic and redundant roles of CG and non‐CG DNA methylation.We found that the homozygous ddcc met1 quintuple mutants are embryonically lethal, although met1 and ddcc mutants only display some developmental abnormalities. Unexpectedly, the ddcc met1 quintuple mutations only reduce transmission through the male gametophytes. The ddcc met1+/− mutants show apparent size divergence, which is not associated with difference in DNA methylation patterns, but associated with the difference in the levels of DNA damage. Finally, we show that a group of TEs are specifically activated in the ddcc met1+/− mutants.This work reveals that CG and non‐CG DNA methylation synergistically and redundantly regulate plant reproductive development, vegetative development and TE silencing in Arabidopsis. Our findings provide insights into the roles of DNA methylation in plant development.

Processing and Bypass of a Site-Specific DNA Adduct of the Cytotoxic Platinum-Acridinylthiourea Conjugate by Polymerases Involved in DNA Repair: Biochemical and Thermodynamic Aspects.

DNA-dependent DNA and RNA polymerases are important modulators of biological functions such as replication, transcription, recombination, or repair. In this work performed in cell-free media, we studied the ability of selected DNA polymerases to overcome a monofunctional adduct of the cytotoxic/antitumor platinum–acridinylthiourea conjugate [PtCl(en)(L)](NO3)2 (en = ethane-1,2-diamine, L = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) (ACR) in its favored 5′-CG sequence. We focused on how a single site-specific ACR adduct with intercalation potency affects the processivity and fidelity of DNA-dependent DNA polymerases involved in translesion synthesis (TLS) and repair. The ability of the G(N7) hybrid ACR adduct formed in the 5′-TCGT sequence of a 24-mer DNA template to inhibit the synthesis of a complementary DNA strand by the exonuclease-deficient Klenow fragment of DNA polymerase I (KFexo−) and human polymerases eta, kappa, and iota was supplemented by thermodynamic analysis of the polymerization process. Thermodynamic parameters of a simulated translesion synthesis across the ACR adduct were obtained by using microscale thermophoresis (MST). Our results show a strong inhibitory effect of an ACR adduct on enzymatic TLS: there was only small synthesis of a full-length product (less than 10%) except polymerase eta (~20%). Polymerase eta was able to most efficiently bypass the ACR hybrid adduct. Incorporation of a correct dCMP opposite the modified G residue is preferred by all the four polymerases tested. On the other hand, the frequency of misinsertions increased. The relative efficiency of misinsertions is higher than that of matched cytidine monophosphate but still lower than for the nonmodified control duplex. Thermodynamic inspection of the simulated TLS revealed a significant stabilization of successively extended primer/template duplexes containing an ACR adduct. Moreover, no significant decrease of dissociation enthalpy change behind the position of the modification can contribute to the enzymatic TLS observed with the DNA-dependent, repair-involved polymerases. This TLS could lead to a higher tolerance of cancer cells to the ACR conjugate compared to its enhanced analog, where thiourea is replaced by an amidine group: [PtCl(en)(L)](NO3)2 (complex AMD, en = ethane-1,2-diamine, L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine).

Effect of the Base-Pair Sequence on B-DNA Thermal Conductivity.

The thermal conductivity of double-stranded (ds) B-DNA was systematically investigated using classical molecular dynamics (MD) simulations. The effect of changing the base pair (bp) on the thermal conductivity of dsDNA needed investigation at a molecular level. Hence, four sequences, viz., poly(A), poly(G), poly(CG), and poly(AT), were initially analyzed in this work. First, the length of these sequences was varied from 4 to 40 bp at 300 K, and the respective thermal conductivity (κ) was computed. Second, the temperature-dependent thermal conductivities between 100 and 400 K were obtained in 50 K steps at 28 bp length. The Müller-Plathe reverse nonequilibrium molecular dynamics (RNEMD) was employed to set a thermal gradient and obtain all thermal conductivities in this work. Moreover, mixed sequences using AT and CG sequences, namely, A(CG)nT (n = 3-7), ACGC(AT)mGCGT (m = 0-5), and ACGC(AT)nAGCGT (n = 1-4), were investigated based on the hypothesis that these sequences could be better thermoelectrics. One-dimensional lattices are said to have diverging thermal conductivities at longer lengths, which violate the Fourier law. These follow the power law, where κ ∝ Lβ. At longer lengths, the exponent β needs to satisfy the condition β > 1/3 for divergent thermal conductivity. We find no such significant Fourier law violation through divergence of thermal conductivities at 80 bp lengths or 40 bp lengths. Also, in the case of the second study, the presence of short (m ≤ 2) encapsulated AT sequences within CG sequences shows an increasing trend. These results are important for engineering DNA-based thermal devices.

Evolution of CG Methylation Maintenance Machinery in Plants.

Cytosine methylation is an epigenetic mark present in most eukaryotic genomes that contributes to the regulation of gene expression and the maintenance of genome stability. DNA methylation mostly occurs at CG sequences, where it is initially deposited by de novo DNA methyltransferases and propagated by maintenance DNA methyltransferases (DNMT) during DNA replication. In this review, we first summarize the mechanisms maintaining CG methylation in mammals that involve the DNA Methyltransferase 1 (DNMT1) enzyme and its cofactor, UHRF1 (Ubiquitin-like with PHD and RING Finger domain 1). We then discuss the evolutionary conservation and diversification of these two core factors in the plant kingdom and speculate on potential functions of novel homologues typically observed in land plants but not in mammals.

Title: Hypermethylation of miRNA Genes During Nodule Development.

DNA methylation has recently emerged as a powerful regulatory mechanism controlling the expression of key regulators of various developmental processes, including nodulation. However, the functional role of DNA methylation in regulating the expression of microRNA (miRNA) genes during the formation and development of nitrogen-fixing nodules remains largely unknown. In this study, we profiled DNA methylation patterns of miRNA genes during nodule formation, development, and early senescence stages in soybean (Glycine max) through the analysis of methylC—seq data. Absolute DNA methylation levels in the CG, CHH, and CHH sequence contexts over the promoter and primary transcript regions of miRNA genes were significantly higher in the nodules compared with the corresponding root tissues at these three distinct nodule developmental stages. We identified a total of 82 differentially methylated miRNAs in the nodules compared with roots. Differential DNA methylation of these 82 miRNAs was detected only in the promoter (69), primary transcript region (3), and both in the promoter and primary transcript regions (10). The large majority of these differentially methylated miRNAs were hypermethylated in nodules compared with the corresponding root tissues and were found mainly in the CHH context and showed stage-specific methylation patterns. Differentially methylated regions in the promoters of 25 miRNAs overlapped with transposable elements, a finding that may explain the vulnerability of miRNAs to DNA methylation changes during nodule development. Gene expression analysis of a set of promoter-differentially methylated miRNAs pointed to a negative association between DNA methylation and miRNA expression. Gene Ontology and pathways analyses indicate that changes in DNA methylation of miRNA genes are reprogrammed and contribute to nodule development through indirect regulation of genes involved in cellular processes and pathways with well-established roles in nodulation.

Parental variation in CHG methylation is associated with allelic-specific expression in elite hybrid rice.

Heterosis refers to the superior performance of hybrid lines over inbred parental lines. Besides genetic variation, epigenetic differences between parental lines are suggested to contribute to heterosis. However, the precise nature and extent of differences between the parental epigenomes and the reprograming in hybrids that govern heterotic gene expression remain unclear. In this work, we analyzed DNA methylomes and transcriptomes of the widely cultivated and genetically studied elite hybrid rice (Oryza sativa) SY63, the reciprocal hybrid, and the parental varieties ZS97 and MH63, for which high-quality reference genomic sequences are available. We showed that the parental varieties displayed substantial variation in genic methylation at CG and CHG (H = A, C, or T) sequences. Compared with their parents, the hybrids displayed dynamic methylation variation during development. However, many parental differentially methylated regions (DMRs) at CG and CHG sites were maintained in the hybrid. Only a small fraction of the DMRs displayed non-additive DNA methylation variation, which, however, showed no overall correlation relationship with gene expression variation. In contrast, most of the allelic-specific expression (ASE) genes in the hybrid were associated with DNA methylation, and the ASE negatively associated with allelic-specific methylation (ASM) at CHG. These results revealed a specific DNA methylation reprogramming pattern in the hybrid rice and pointed to a role for parental CHG methylation divergence in ASE, which is associated with phenotype variation and hybrid vigor in several plant species.

Copper Ions Induce DNA Sequence Variation in Zygotic Embryo Culture-Derived Barley Regenerants.

In vitro tissue culture could be exploited to study cellular mechanisms that induce sequence variation. Altering the metal ion composition of tissue culture medium affects biochemical pathways involved in tissue culture-induced variation. Copper ions are involved in the mitochondrial respiratory chain and Yang cycle. Copper ions may participate in oxidative mutations, which may contribute to DNA sequence variation. Silver ions compete with copper ions to bind to the complex IV subunit of the respiratory chain, thus affecting the Yang cycle and DNA methylation. The mechanisms underlying somaclonal variation are unknown. In this study, we evaluated embryo-derived barley regenerants obtained from a single double-haploid plant via embryo culture under varying copper and silver ion concentrations and different durations of in vitro culture. Morphological variation among regenerants and the donor plant was not evaluated. Methylation-sensitive Amplified Fragment Length Polymorphism analysis of DNA samples showed DNA methylation pattern variation in CG and CHG (H = A, C, or T) sequence contexts. Furthermore, modification of in vitro culture conditions explained DNA sequence variation, demethylation, and de novo methylation in the CHG context, as indicated by analysis of variance. Linear regression indicated that DNA sequence variation was related to de novo DNA methylation in the CHG context. Mediation analysis showed the role of copper ions as a mediator of sequence variation in the CHG context. No other contexts showed a significant sequence variation in mediation analysis. Silver ions did not act as a mediator between any methylation contexts and sequence variation. Thus, incorporating copper ions in the induction medium should be treated with caution.