Cytosine Methylation Alterations Research Articles

Mammalian Sex Hormones as Steroid-Structured Compounds in Wheat Seedling: Template of the Cytosine Methylation Alteration and Retrotransposon Polymorphisms with iPBS and CRED-iBPS Techniques

Phytohormones are chemical compounds found naturally in plants that have a significant effect on their growth and development. The increase in research on the occurrence of mammalian sex hormones (MSHs) in plants has prompted the need to investigate the functions performed by these hormones in plant biology. In the present study, we investigated the effects of MSHs on DNA damage and DNA methylation of wheat (Triticum aestivum L.) during the seedling growth stage, using the CRED-iPBS (coupled restriction enzyme digestion/inter primer binding site) assay and iPBS analysis to determine DNA methylation status. Exogenous treatment with four MSHs (17-β-estradiol, estrogen, progesterone, and testosterone) was carried out at four different concentrations (0, 0.05, 0.5, and 5 µM). The highest genomic template stability (GTS) value (80%) was observed for 5 µM 17-β-estradiol, 0.5 µM testosterone, and 0.05 µM estrogen, while the lowest value (70.7%) was observed for 5 µM progesterone and 0.5 µM estrogen. The results of the CRED-iPBS analysis conducted on MspI indicate that the 0.05 µM estrogen-treated group had the highest polymorphism value of 40%, while the 5 µM progesterone-treated group had the lowest value of 20%. For HpaII, treatment with 0.5 µM 17-β-estradiol had the highest polymorphism value of 33.3%, while the group treated with 0.05 µM 17-β-estradiol and 0.05 µM progesterone had the lowest value of 19.4%. In conclusion, MSH treatments altered the stability of the genomic template of wheat plants and affected the cytosine methylation status at the seedling growth stage. Upon comprehensive examination of the results, it was seen that the employed methodology successfully detected alterations in cytosine methylation of genomic DNA (gDNA), as well as changes in the pattern of genomic instability.

The Immunological Epigenetic Landscape of the Human Life Trajectory.

Adaptive immunity changes over an individual's lifetime, maturing by adulthood and diminishing with old age. Epigenetic mechanisms involving DNA and histone methylation form the molecular basis of immunological memory during lymphocyte development. Monocytes alter their function to convey immune tolerance, yet the epigenetic influences at play remain to be fully understood in the context of lifespan. This study of a healthy genetically homogenous cohort of children, adults and seniors sought to decipher the epigenetic dynamics in B-lymphocytes and monocytes. Variable global cytosine methylation within retro-transposable LINE-1 repeats was noted in monocytes compared to B-lymphocytes across age groups. The expression of the human leukocyte antigen (HLA)-DQ alpha chain gene HLA-DQA1*01 revealed significantly reduced levels in monocytes in all ages relative to B-lymphocytes, as well as between lifespan groups. High melting point analysis and bisulfite sequencing of the HLA-DQA1*01 promoter in monocytes highlighted variable cytosine methylation in children and seniors but greater stability at this locus in adults. Further epigenetic evaluation revealed higher histone lysine 27 trimethylation in monocytes from this adult group. Chromatin immunoprecipitation and RNA pulldown demonstrated association with a novel lncRNA TINA with structurally conserved similarities to the previously recognized epigenetic modifier PARTICLE. Seeking to interpret the epigenetic immunological landscape across three representative age groups, this study focused on HLA-DQA1*01 to expose cytosine and histone methylation alterations and their association with the non-coding transcriptome. Such insights unveil previously unknown complex epigenetic layers, orchestrating the strength and weakening of adaptive immunity with the progression of life.

82 Methyl Donor Supplementation Alters Cytosine Methylation and Biological Processes of Cells Cultured in Divergent Glucose Media Reflecting Improvements in Mitochondrial Respiration and Cell Growth Rate

Abstract We hypothesized that supplementation of one-carbon metabolites (OCM: methionine, folate, choline, and vitamin B12) to bovine embryonic tracheal fibroblasts in divergent glucose media would alter cytosine methylation, and alterations in cytosine methylation will reflect biological processes matching previously improved mitochondrial respiration, cell proliferation, and cell growth rate data. Cells were cultured with 1g/L glucose (Low) or 4.5g/L glucose (High). Control medium (CON) contained basal concentrations of folate (0.001g/L), choline (0.001g/L), vitamin B12 (4µg/L), and methionine (0.015g/L). The OCM were supplemented at 2.5 and 5 times (2.5X and 5X, respectively) the CON media, except methionine was limited to 2X across all supplemented treatments. Cells were passaged three times in their treatment media before DNA extraction. Reduced representation bisulfite sequencing was adopted to analyze and compare the genomic methylation patterns within and across treatments using edgeR. Biological processes (BP) were retrieved based on the nearest genes of differentially methylated cytosines (P < 0.01) for each comparison between treatments. In both Low and High treatments, greater OCM increased the proportion of hypomethylated vs. hypermethylated cytosines. Functional analyses pointed out positive regulation of BP related to energy metabolism, except for the contrasts within the High group. Among the BP, we can highlight positive regulation of: GTPase activity, catalytic activity, molecular function, protein modification processes, phosphorylation, protein phosphorylation, cellular protein metabolic processes, MAPK cascade, and metabolic processes. These data support previously reported results from this experiment that showed increased mitochondrial respiration, cell proliferation, and growth rates with increasing OCM levels. We interpret these data to imply that when energy and OCM requirements are met for growth and basal methylation levels, DNA methylation levels decrease which may allow for greater transcription. Thus, OCM can be utilized for other functions such as polyamine synthesis, nucleotide synthesis, energetic metabolites, and phosphatidylcholine synthesis. USDA is an equal opportunity provider and employer.

Mice Exposed to Combined Chronic Low-Dose Irradiation and Modeled Microgravity Develop Long-Term Neurological Sequelae.

Spaceflight poses many challenges for humans. Ground-based analogs typically focus on single parameters of spaceflight and their associated acute effects. This study assesses the long-term transcriptional effects following single and combination spaceflight analog conditions using the mouse model: simulated microgravity via hindlimb unloading (HLU) and/or low-dose γ-ray irradiation (LDR) for 21 days, followed by 4 months of readaptation. Changes in gene expression and epigenetic modifications in brain samples during readaptation were analyzed by whole transcriptome shotgun sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). The results showed minimal gene expression and cytosine methylation alterations at 4 months readaptation within single treatment conditions of HLU or LDR. In contrast, following combined HLU+LDR, gene expression and promoter methylation analyses showed multiple altered pathways involved in neurogenesis and neuroplasticity, the regulation of neuropeptides, and cellular signaling. In brief, neurological readaptation following combined chronic LDR and HLU is a dynamic process that involves pathways that regulate neuronal function and structure and may lead to late onset neurological sequelae.

Cadmium stress alters cytosine methylation status and expression of a select set of genes in Nicotiana benthamiana

In this paper, we evaluated the genotoxicity of cadmium (Cd) in plants by performing a methylation-sensitive amplification polymorphism (MSAP) on the model plant Nicotiana benthamiana. Among 255 loci examined, 14 genes were found to show altered cytosine methylation patterns in response to Cd stress. Four of those genes (NbMORC3, NbHGSNAT, NbMUT, and NbBG) were selected for further analysis due to their predicted roles in plant development. Cd-induced changes of cytosine methylation status in MSAP fragments of selected genes were confirmed using bisulfite sequencing polymerase chain reaction (BSP). In addition, the expression levels of these genes were found to correlate with cadmium dosage, and a knock-down of these four genes via virus-induced genes silencing (VIGS) led to abnormal development and elevated sensitivity to cadmium stress. Silencing of these four genes resulted in altered cadmium accumulation in different parts of the experimental plants. Our data indicate that cadmium exposure causes dramatic changes in the cytosine methylation status of the plant genome, thus affecting the expression of many genes that are vital for plant growth and are involved in cadmium stress response.

Methylation-sensitive amplified polymorphism analysis of resistant and susceptible interactions of cucumber with Podosphaera xanthii

Cytosine methylation alterations play key roles in gene regulation in plants interacting with biotrophic pathogens. For resistant and susceptible interactions of cucumber with the powdery mildew pathogen, Podosphaera xanthii, methylation of DNA cytosines were monitored by methylation-sensitive amplified polymorphism (MSAP) analysis from immediately prior to 96 h post inoculation (hpi). In both resistant and susceptible interactions, methylation levels significantly changed over time, but a significant association was observed for decreasing number of loci with demethylated CCGG motif in the susceptible cultivar compared to increasing numbers in the resistant cultivar. Sequencing of six demethylated MSAP fragments from the resistant cultivar and one from susceptible cultivar showed that all contained promoter and cis-acting regulatory elements (CAREs). For the resistant cultivar, three of these were in possible regulatory regions located within or near tRNA coding genes, and those three plus two others contained CAREs associated with response to biotic stress and defense hormones SA, MeJA, ABA and ET. One possible reason for demethylation in the resistant genomic tRNA genes may be that demethylation can de-suppress gene expression resulting in increasing the availability of certain amino acids for defense proteins.

Analysis of sulphur and chlorine induced DNA cytosine methylation alterations in fresh corn (Zea mays L. saccharata and rugosa) leaf tissues by methylation sensitive amplification polymorphism (MSAP) approach.

DNA (cytosine) methylation mechanism is another way through which plants respond to various cues including soil fertility amendments and abiotic stresses, and the mechanism has been used to infer some physiological, biochemical or adaptation processes. Despite numerous studies on global DNA methylation profiling in various crop species, however, researches on fresh corn (Zea mays L. saccharata or rugosa) remain largely unreported. The study aimed at investigating sulphur and chlorine induced DNA methylation changes in the fresh corn leaves of field-grown plants at the milk stage. Methylation sensitive amplification polymorphism (MSAP) technique was used to profile sulphur (S) and chlorine (Cl) induced DNA methylation patterns, levels and polymorphism alterations at the CCGG sites in fresh corn leaves of TDN21, JKN2000 and JKN928 hybrid cultivars. Twelve primer pairs used effectively detected 325 MSAP bands, exhibiting differentially methylated sites in the genomic DNA of all the three cultivars, with control showing higher (48.9-56.3%) type I bands as compared to sulphur (34.8-44.9%) and chlorine (40.9-47.4%) treatment samples. Consequently, total methylation levels were greater in S and Cl treatment samples than control; accounting for 43.7-59.7, 51.1-65.2 and 46.8-55.1% of total sites in TDN21, JKN2000 and JKN928, respectively. Full methylation of the internal cytosine was greater than hemi-methylation. Further, demethylation polymorphic loci significantly exceeded methylation polymorphic loci, being greater in S than Cl and control samples in all cultivars. Sulphur and chlorine have a profound influence on DNA methylation patterns and levels at the milk stage, principally by increasing the demethylation loci in the internal cytosine of the fresh corn genome. We speculate that these methylation alterations play an integral role in photosynthates assimilation and physiochemical pathways regulating quality parameters in kernels, as well as abiotic stress responses in fresh corn.

The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis.

Gametocidal (Gc) chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP) technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS–3C and CS–3C3C, 48.68% and 48.65%, respectively) were significantly increased when compared to common wheat CS (41.31%) and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted–repeat transposable elements), LTR-retrotransposon WIS-1p and retrotransposon Gypsy, respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.

Tris(1,3-dichloro-2-propyl)phosphate Induces Genome-Wide Hypomethylation within Early Zebrafish Embryos.

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is a high-production volume organophosphate-based plasticizer and flame retardant widely used within the United States. Using zebrafish as a model, the objectives of this study were to determine whether (1) TDCIPP inhibits DNA methyltransferase (DNMT) within embryonic nuclear extracts; (2) uptake of TDCIPP from 0.75 h postfertilization (hpf, 2-cell) to 2 hpf (64-cell) or 6 hpf (shield stage) leads to impacts on the early embryonic DNA methylome; and (3) TDCIPP-induced impacts on cytosine methylation are localized to CpG islands within intergenic regions. Within this study, 5-azacytidine (5-azaC, a DNMT inhibitor) was used as a positive control. Although 5-azaC significantly inhibited zebrafish DNMT, TDCIPP did not affect DNMT activity in vitro at concentrations as high as 500 μM. However, rapid embryonic uptake of 5-azaC and TDCIPP from 0.75 to 2 hpf resulted in chemical- and chromosome-specific alterations in cytosine methylation at 2 hpf. Moreover, TDCIPP exposure predominantly resulted in hypomethylation of positions outside of CpG islands and within intragenic (exon) regions of the zebrafish genome. Overall, these findings provide the foundation for monitoring DNA methylation dynamics within zebrafish as well as identifying potential associations among TDCIPP exposure, adverse health outcomes, and DNA methylation status within human populations.

Interpopulation hybridization generates meiotically stable rDNA epigenetic variants in allotetraploid Tragopogon mirus.

Uniparental silencing of 35S rRNA genes (rDNA), known as nucleolar dominance (ND), is common in interspecific hybrids. Allotetraploid Tragopogon mirus composed of Tragopogon dubius (d) and Tragopogon porrifolius (p) genomes shows highly variable ND. To examine the molecular basis of such variation, we studied the genetic and epigenetic features of rDNA homeologs in several lines derived from recently and independently formed natural populations. Inbred lines derived from T. mirus with a dominant d-rDNA homeolog transmitted this expression pattern over generations, which may explain why it is prevalent among natural populations. In contrast, lines derived from the p-rDNA dominant progenitor were meiotically unstable, frequently switching to co-dominance. Interpopulation crosses between progenitors displaying reciprocal ND resulted in d-rDNA dominance, indicating immediate suppression of p-homeologs in F1 hybrids. Original p-rDNA dominance was not restored in later generations, even in those segregants that inherited the corresponding parental rDNA genotype, thus indicating the generation of additional p-rDNA and d-rDNA epigenetic variants. Despite preserved intergenic spacer (IGS) structure, they showed altered cytosine methylation and chromatin condensation patterns, and a correlation between expression, hypomethylation of RNA Pol I promoters and chromatin decondensation was apparent. Reversion of such epigenetic variants occurred rarely, resulting in co-dominance maintained in individuals with distinct genotypes. Generally, interpopulation crosses may generate epialleles that are not present in natural populations, underlying epigenetic dynamics in young allopolyploids. We hypothesize that highly expressed variants with distinct IGS features may induce heritable epigenetic reprogramming of the partner rDNA arrays, harmonizing the expression of thousands of genes in allopolyploids.

MTR-07GLIOBLASTOMA ADAPTATION TO SUSTAINED cdk4/6 INHIBITION INVOLVES SUPPRESSION OF Rb EXPRESSION THROUGH HISTONE H3K4 MODIFICATION

We have previously shown that pharmacologic inhibition of cdk4/6, in preclinical, Rb proficient models of glioblastoma (GBM), slows tumor growth, and extends the survival of animal subjects with intracranial GBM xenografts. In the current study we examined molecular characteristics of two distinct GBM xenograft models that developed resistance to cdk4/6 targeted therapy when subjected to sustained inhibitor treatment. For each model, mice with intracranial GBM xenograft, established from G55 and U87 cells, received daily administration of inhibitor, which slowed tumor growth, as indicated by bioluminescence imaging, and significantly extended animal subject survival (p < 0.001). However, tumor bioluminescence slowly increased during the course of treatment, and all treatment group mice eventually succumbed to tumor burden. Intracranial tumor, from mice subjected to continuous daily inhibitor treatment, was dissected from mouse brain, then minced and injected subcutaneously into new athymic host mice, with host mice continuing to receive daily administration of cdk4/6 inhibitor until engrafted subcutaneous tumors showed growth rates comparable to corresponding subcutaneous tumors that were left untreated. Subcutaneous tumors exposed to continuous daily inhibitor treatment were used as cell sources for intracranial therapy response experiments, with results showing that tumors subjected to continuous and extended daily administration of inhibitor no longer responded to inhibitor treatment, as indicated by bioluminescence imaging and survival analysis. Immunoblot analysis revealed that resistant tumors showed greatly reduced or a complete absence of Rb protein expression. RB sequence analysis of DNA from resistant tumors failed to reveal any sequence alterations, and there was no indication of altered cytosine methylation in RB regulatory sequences. In contrast, chromatin immunoprecipitation revealed 11x (U87) and 7x (G55) reductions in RB promoter sequence association with histone H3K4 trimethyl (H3K4me3) antibody, indicating suppression of RB expression, in GBM subjected to sustained cdk4/6 inhibition, through a histone modification associated with gene silencing.

Chilling- and Freezing-Induced Alterations in Cytosine Methylation and Its Association with the Cold Tolerance of an Alpine Subnival Plant, Chorispora bungeana.

Chilling (0–18°C) and freezing (<0°C) are two distinct types of cold stresses. Epigenetic regulation can play an important role in plant adaptation to abiotic stresses. However, it is not yet clear whether and how epigenetic modification (i.e., DNA methylation) mediates the adaptation to cold stresses in nature (e.g., in alpine regions). Especially, whether the adaptation to chilling and freezing is involved in differential epigenetic regulations in plants is largely unknown. Chorispora bungeana is an alpine subnival plant that is distributed in the freeze-thaw tundra in Asia, where chilling and freezing frequently fluctuate daily (24 h). To disentangle how C. bungeana copes with these intricate cold stresses through epigenetic modifications, plants of C. bungeana were treated at 4°C (chilling) and -4°C (freezing) over five periods of time (0–24 h). Methylation-sensitive amplified fragment-length polymorphism markers were used to investigate the variation in DNA methylation of C. bungeana in response to chilling and freezing. It was found that the alterations in DNA methylation of C. bungeana largely occurred over the period of chilling and freezing. Moreover, chilling and freezing appeared to gradually induce distinct DNA methylation variations, as the treatment went on (e.g., after 12 h). Forty-three cold-induced polymorphic fragments were randomly selected and further analyzed, and three of the cloned fragments were homologous to genes encoding alcohol dehydrogenase, UDP-glucosyltransferase and polygalacturonase-inhibiting protein. These candidate genes verified the existence of different expressive patterns between chilling and freezing. Our results showed that C. bungeana responded to cold stresses rapidly through the alterations of DNA methylation, and that chilling and freezing induced different DNA methylation changes. Therefore, we conclude that epigenetic modifications can potentially serve as a rapid and flexible mechanism for C. bungeana to adapt to the intricate cold stresses in the alpine areas.

DNA methylation changes in fusarium wilt resistant and sensitive chickpea genotypes (Cicer arietinum L.)

DNA methylation plays an important role in the regulation of gene expression in biotic and abiotic stresses. In the present study, a methylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in seven resistant and sensitive chickpea genotypes following inoculation with Fusarium oxysporum f. sp. ciceris. In all, 27468 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using nine selective primer pairs. DNA methylation was evaluated in leaves, stems and roots in control and inoculated plants. Extensive cytosine methylation alterations were found in the pathogen-treated genotypes compared with the corresponding control, including hypermethylation and demethylation as well as the potential conversion of methylation types. For all genotypes, the percentage of demethylated sites were more than methylated sites in infected plants compared with the corresponding control. No significant differences were observed for banding patterns in infected and control leaf tissues, while the differences between percentage of unchanged, methylated and demethylated sites were significant in stem and root tissues. The total numbers of methylated polymorphic bands ranged from 137 to 154 bands in Sel95th1716 and Arman, accounting for 36.81%–44.64% of all bands, respectively. Ten fragments that were differentially amplified between infected and control plants were isolated and sequenced in three tissues separately. Most of sequenced fragments showed homology with disease related genes in GenBank. The results suggest that significant differences in cytosine methylation exist between resistant and sensitive chickpea genotypes, and that hypermethylation or hypomethylation of specific genes may be involved in the chickpea resistance to Fusarium wilt.

Salt tolerance and alterations in cytosine methylation in the interspecific hybrids of Fraxinus velutina and Fraxinus mandshurica

For cross-pollination trees, the optimal breeding method is hybridization. Tree heterosis is commonly present and is the main research focus in tree crossbreeding. Salt stress and interspecific hybridization may lead to DNA methylation changes. The study crossed Fraxinus mandshurica (female parent) with Fraxinus velutina (male parent) to obtain interspecific F1 hybrid progenies that could obtain the good characters of parents. The results showed that growth and survival rate of the interspecific hybrid progenies (F1 hybrids of F. mandshurica × F. velutina) were significantly higher than those of intraspecific open pollinated plants from parental F. mandshurica and F. velutina. Salt tolerance and cytosine methylation in interspecific F1 hybrids and the intraspecific open pollinated plants from parents were examined. Membrane permeability, ROS and antioxidant activity, malondialdehyde, and photosynthesis were measured after salt treatment and genomic methylation was analyzed using a methylation-sensitive amplified polymorphism protocol. F1 hybrids exhibited heterosis for growth in normal as well as high salt conditions. DNA methylation in the F1 hybrids was lower than the intraspecific open pollinated plants from parents. Salt treatments changed DNA methylation patterns in F1 hybrids. Genomic DNA of the intraspecific open pollinated plants from parents had internal cytosine methylation (average of 13.22 %), whereas F1 hybrid seedlings had external cytosine methylation (average of 7.34 %). Such changes in DNA methylation patterns in F1 hybrids suggest a connection between salt tolerance and epigenetic mechanisms in plants. We observed that alteration of DNA methylation was closely correlated with the adaptation to the salt stress and provided epigenetic mechanisms of salt tolerance in the interspecific hybridization of trees.

Short Communication An efficient method for simultaneous extraction of high-quality RNA and DNA from various plant tissues.

Determination of gene expression is an important tool to study biological processes and relies on the quality of the extracted RNA. Changes in gene expression profiles may be directly related to mutations in regulatory DNA sequences or alterations in DNA cytosine methylation, which is an epigenetic mark. Correlation of gene expression with DNA sequence or epigenetic mark polymorphism is often desirable; for this, a robust protocol to isolate high-quality RNA and DNA simultaneously from the same sample is required. Although commercial kits and protocols are available, they are mainly optimized for animal tissues and, in general, restricted to RNA or DNA extraction, not both. In the present study, we describe an efficient and accessible method to extract both RNA and DNA simultaneously from the same sample of various plant tissues, using small amounts of starting material. The protocol was efficient in the extraction of high-quality nucleic acids from several Arabidopsis thaliana tissues (e.g., leaf, inflorescence stem, flower, fruit, cotyledon, seedlings, root, and embryo) and from other tissues of non-model plants, such as Avicennia schaueriana (Acanthaceae), Theobroma cacao (Malvaceae), Paspalum notatum (Poaceae), and Sorghum bicolor (Poaceae). The obtained nucleic acids were used as templates for downstream analyses, such as mRNA sequencing, quantitative real time-polymerase chain reaction, bisulfite treatment, and others; the results were comparable to those obtained with commercial kits. We believe that this protocol could be applied to a broad range of plant species, help avoid technical and sampling biases, and facilitate several RNA- and DNA-dependent analyses.

Characterization of in vitro haploid and doubled haploid Chrysanthemum morifolium plants via unfertilized ovule culture for phenotypical traits and DNA methylation pattern.

Chrysanthemum is one of important ornamental species in the world. Its highly heterozygous state complicates molecular analysis, so it is of interest to derive haploid forms. A total of 2579 non-fertilized chrysanthemum ovules pollinated by Argyranthemum frutescens were cultured in vitro to isolate haploid progeny. One single regenerant emerged from each of three of the 105 calli produced. Chromosome counts and microsatellite fingerprinting showed that only one of the regenerants was a true haploid. Nine doubled haploid derivatives were subsequently generated by colchicine treatment of 80 in vitro cultured haploid nodal segments. Morphological screening showed that the haploid plant was shorter than the doubled haploids, and developed smaller leaves, flowers, and stomata. An in vitro pollen germination test showed that few of the haploid's pollen were able to germinate and those which did so were abnormal. Both the haploid and the doubled haploids produced yellow flowers, whereas those of the maternal parental cultivar were mauve. Methylation-sensitive amplification polymorphism (MSAP) profiling was further used to detect alterations in cytosine methylation caused by the haploidization and/or the chromosome doubling processes. While 52.2% of the resulting amplified fragments were cytosine methylated in the maternal parent's genome, the corresponding proportions for the haploid's and doubled haploids' genomes were, respectively, 47.0 and 51.7%, demonstrating a reduction in global cytosine methylation caused by haploidization and a partial recovery following chromosome doubling.

Comparison of Salinity-Induced Changes in Two Cultivars of Barley

In vitro effects of salinity were compared in two cultivars of barley (Hordeum vulgare cv. Bornova-92 and Hilal). Mature embryos were cultured on Murashige and Skoog media supplemented with 0, 50 and 100 mM NaCl for 20 days. NaCl-treatment decreased maximum shoot length, total soluble protein and DNA contents in both cultivars but decreased maximum root length in Hilal. Changes in fresh and dry weight and water content were not statistically-significant. Inhibitory effects were more dramatic in Hilal. Salinity did not cause genotoxic effects in both cultivars yet slightly affected protein patterns in Bornova-92. However, salinity altered cytosine methylation patterns from CCG to CG in Bornova-92, from CG to CCG in Hilal. Bornova-92 and Hilal may be regarded as salt-tolerant and salt-susceptible, respectively and the relative salt-tolerance of Bornova-92 may be due to cytosine methylation patterns and/or regulation of protein synthesis.

DNA methylation changes in photoperiod-thermo-sensitive male sterile rice PA64S under two different conditions

Epigenetic modification can occur at a high frequency in crop plants and might generate phenotypic variation without changes in DNA sequences. DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by regulating gene expression. Rice Photoperiod-Thermo-Sensitive Genic Male Sterile (PTGMS) lines can transform from sterility to fertility under lower temperatures and short-day (SD) conditions during anther development. So far, little is known about the DNA methylation variation of PTGMS throughout the genome in rice. In this study, we investigated DNA cytosine methylation alterations in the young panicles of PTGMS line PA64S under two different conditions using methylation sensitive amplified polymorphism (MSAP) method. Compared with the DNA methylation level of PA64S under lower temperatures and SD conditions (fertility), higher methylation was observed in PA64S (sterility). The sequences of 25 differentially amplified fragments were successfully obtained and annotated. Three methylated fragments, which are homologous to D2, NAD7 and psaA, were confirmed by bisulfite sequencing and their expression levels were also evaluated by qPCR. Real time quantitative PCR analysis revealed that five of the six selected methylated genes were downregulated in PA64S (sterility). These results suggested that DNA methylation may be involved in the sterility–fertility transition of PA64S under two different environmental conditions.

DNA Methylation Alterations at 5′-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus

DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5′-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5′-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.

Integrated genetic and epigenetic analysis of childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy and is characterized by recurring structural genetic alterations. Previous studies of DNA methylation suggest epigenetic alterations may also be important, but an integrated genome-wide analysis of genetic and epigenetic alterations in ALL has not been performed. We analyzed 137 B-lineage and 30 T-lineage childhood ALL cases using microarray analysis of DNA copy number alterations and gene expression, and genome-wide cytosine methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. We found that the different genetic subtypes of ALL are characterized by distinct DNA methylation signatures that exhibit significant correlation with gene expression profiles. We also identified an epigenetic signature common to all cases, with correlation to gene expression in 65% of these genes, suggesting that a core set of epigenetically deregulated genes is central to the initiation or maintenance of lymphoid transformation. Finally, we identified aberrant methylation in multiple genes also targeted by recurring DNA copy number alterations in ALL, suggesting that these genes are inactivated far more frequently than suggested by structural genomic analyses alone. Together, these results demonstrate subtype- and disease-specific alterations in cytosine methylation in ALL that influence transcriptional activity, and are likely to exert a key role in leukemogenesis.