The effects of 5‐azacytidine and cadmium on global 5‐methylcytosine content and secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda

Epigenetic changes are important mechanisms in the regulation of chromatin structure and gene expression. Cytosine methylation is one of the major epigenetic modifications, mediated by DNA methyltransferases, which transfer methyl groups from S‐adenosyl‐L‐methionine (SAM) to the fifth carbon of cytosine. Various external environmental conditions can change the global hypo/hypermethylation pattern of DNA. These alterations may affect the organism's response to stress conditions. In this study, for the first time, we investigated the effects of 5‐azacytidine, a DNA methyltransferase inhibitor, and cadmium, a toxic metal and environmental pollutant, on the growth, biosynthesis of secondary metabolites (phenols, flavonoids, carotenoids), SAM, S‐adenosylhomocysteine, 5′‐methylthioadenosine and global 5‐methylcytosine (5‐mC) in the green microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda. The studied species showed major differences in 5‐mC content, secondary metabolite content, and antioxidant activity. Cadmium increased GSH (glutathione) content in C. reinhardtii by 60% whereas 5‐azacytidine did not affect GSH. The biosynthesis of GSH in S. quadricauda in response to the stressors was the opposite. Global 5‐mC content of C. reinhardtii was 1%–1.5%, and the content in S. quadricauda was 3.5%. Amount of some investigated methionine cycle metabolites (SAM, S‐adenosyl homocysteine [SAH], methionine) in S. quadricauda distinctly exceeded C. reinhardtii as well. However, chlorophylls a and b, carotenoids, total phenolic content, total flavonoid content and, antioxidant activity were significantly higher in C. reinhardtii than S. quadricauda. Therefore, in further studies it would be advisable to verify whether methylation of cytosine affects the expression of genes encoding certain secondary metabolites.     

TET1 suppresses colon cancer proliferation by impairing β-catenin signal pathway

The function of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in cancer is background dependent and may be involved in the initial step of active DNA demethylation, while there is little research to decipher the role of TET1 in DNA methylation-sensitive colon cancer. Downregulated TET1 expression assayed by quantitative real-time PCR (qRT-PCR) was observed in both colon cancer samples and cancer cell lines of HT29, HCT116, and SW48. Such downregulation could promote colon cancer cells proliferation as indicated by the fact that shTET1 could increase the viability of HT29 and HCT116 cells determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and cell count assay accompanied with upregulation of β-catenin (CTNNB1) and WNT luciferase activity, which was further confirmed as shTET1 could increase the tumor volume and tumor weight, and decrease the body weight in HT29 cells inoculated BALB/C nude mice. The CTNNB1 transfection could rescue the cell growth diminished by normal expression of TET1. shTET1 could promote axis inhibition protein1 (AXIN1) expression and the cell proliferation effect induced by TET1 short hairpin RNA was attenuated by co-inhibition of AXIN1. All of these indicate that TET1 can suppress colon cancer proliferation and the inhibition of the β-catenin pathway is AXIN1 dependent.     

Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure

The fertilized mouse egg actively demethylates the paternal genome within a few hours after fertilization, whereas the maternal genome is only passively demethylated by a replication-dependent mechanism after the two-cell stage. This evolutionarily conserved assymetry in the early diploid mammalian embryo may have a role in methylation reprogramming of the two very different sets of sperm and egg chromatin for somatic development and formation of totipotent cells. Immunofluorescence staining with an antibody against 5-methylcytosine (MeC) showed that the incidence of abnormal methylation patterns differs between mouse two-cell embryos from superovulated females, nonsuperovulated matings, and in vitro fertilization (IVF). It also depends on embryo culture conditions and genetic background. In general, there was a good correlation with the number of embryos (from the same experiment) which did not develop in vitro up to the blastocyst stage. Thus, aberrant genome-wide DNA methylation in early embryos may be an important mechanism contributing to the high incidence of developmental failure in mammals. Similar to the situation in abnormally methylated embryos from nuclear transfer, it may cause a high incidence of pregnancy loss and abnormal phenotypes.     

Methylation of CpG dinucleotides in the lacI gene of the Big Blue® transgenic mouse

Cytosine residues at CpG dinucleotides can be methylated by endogenous methyltransferases in mammalian cells. The resulting 5-methylcytosine base may undergo spontaneous deamination to form thymine causing G/C to A/T transition mutations. Methylated CpGs also can form preferential targets for environmental mutagens and carcinogens. The Big Blue® transgenic mouse has been used to investigate tissue and organ specificity of mutations and to deduce mutational mechanisms in a mammal in vivo. The transgenic mouse contains approximately 40 concatenated lambda-like shuttle vectors, each of which contains one copy of an Escherichia coli lacI gene as a mutational target. lacI mutations in lambda transgenic mice are characterized by a high frequency of spontaneous mutations targeted to CpG dinucleotides suggesting an important contribution from methylation-mediated events. To study the methylation status of CpGs in the lacI gene, we have mapped the distribution of 5-methylcytosines along the DNA-binding domain and flanking sequences of the lacI gene of transgenic mice. We analyzed genomic DNA from various tissues including thymus, liver, testis, and DNA derived from two thymic lymphomas. The mouse genomic DNAs and methylated and unmethylated control DNAs were chemically cleaved, then the positions of 5-methylcytosines were mapped by ligation-mediated PCR which can be used to distinguish methylated from unmethylated cytosines. Our data show that most CpG dinucleotides in the DNA binding domain of the lacI gene are methylated to a high extent (>98%) in all tissues tested; only a few sites are partially (70–90%) methylated. We conclude that tissue-specific methylation is unlikely to contribute significantly to tissue-specific mutational patterns, and that the occurrence of common mutation sites at specific CpGs in the lacI gene is not related to selective methylation of only these sequences. The data confirm previous suggestions that the high frequency of CpG mutations in lacI transgenes is related to the presence of 5-methylcytosine bases.     

Origin and maintenance of sex: the evolutionary joys of self sex

Sex is generally thought of as meiosis, conjugation, and syngamy, with the primary function of sex believed to be genetic mixing. However, conjugation does not occur with complete automixis, whereas syngamy does not occur with restitutional automixis. Self sex in the forms of automixis and autogamy does not include genetic mixing. Yet sex, including self sex, is necessary for most eukaryotic lineages. What is the purpose of sex without genetic mixing? Obligate self sex is not an evolutionary dead end, but holds the key to understanding the evolutionary origin, function, maintenance, and ubiquity of sex. We extend the rejuvenescence hypothesis that sex provides a necessary developmental reset for multicellular eukaryotes and even many unicellular eukaryotes. Sex reduces additive genetic variance of epigenetic signals, especially cytosine methylation, and of ploidy levels. Furthermore, we argue that syngamy is a modified form of meiosis that maintains ploidy and resets epigenetic signals. Epigenetic resetting is consistent with sex being induced by starvation or desiccation. Diminution of additive genetic variance is consistent with the origin and maintenance of an adaptive trait, sex, that has been present for approximately two billion years. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 707–728.     

De novo genome assembly of the stress tolerant forest species Casuarina equisetifolia provides insight into secondary growth

Casuarina equisetifolia (C. equisetifolia), a conifer‐like angiosperm with resistance to typhoon and stress tolerance, is mainly cultivated in the coastal areas of Australasia. C. equisetifolia, making it a valuable model to study secondary growth associated genes and stress‐tolerance traits. However, the genome sequence is unavailable and therefore wood‐associated growth rate and stress resistance at the molecular level is largely unexplored. We therefore constructed a high‐quality draft genome sequence of C. equisetifolia by a combination of Illumina second‐generation sequencing reads and Pacific Biosciences single‐molecule real‐time (SMRT) long reads to advance the investigation of this species. Here, we report the genome assembly, which contains approximately 300 megabases (Mb) and scaffold size of N50 is 1.06 Mb. Additionally, gene annotation, assisted by a combination of prediction and RNA‐seq data, generated 29 827 annotated protein‐coding genes and 1983 non‐coding genes, respectively. Furthermore, we found that the total number of repetitive sequences account for one‐third of the genome assembly. Here we also construct the genome‐wide map of DNA modification, such as two novel forms N⁶‐adenine (6mA) and N4‐methylcytosine (4mC) at the level of single‐nucleotide resolution using single‐molecule real‐time (SMRT) sequencing. Interestingly, we found that 17% of 6mA modification genes and 15% of 4mC modification genes also included alternative splicing events. Finally, we investigated cellulose, hemicellulose, and lignin‐related genes, which were associated with secondary growth and contained different DNA modifications. The high‐quality genome sequence and annotation of C. equisetifolia in this study provide a valuable resource to strengthen our understanding of the diverse traits of trees.     

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease ^1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest ^5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5''mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) ⁸. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA ^{2, 4, 9-11}.     

Ascorbate-induced generation of 5-hydroxymethylcytosine is unaffected by varying levels of iron and 2-oxoglutarate

Tet (ten–eleven translocation) methylcytosine dioxygenases, which belong to the iron and 2-oxoglutarate (2OG)-dependent dioxygenase superfamily, convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. We recently reported that ascorbate (vitamin C) induces Tet-mediated generation of 5hmC. To initially delineate the role of ascorbate on 5hmC generation, we analyzed whether the effect of ascorbate is dependent upon the conditions of other components involved in the hydroxylation of 5mC catalyzed by Tet. We found that removing iron from the culture medium did not affect the induction of 5hmC by ascorbate (10 μM) in mouse embryonic fibroblasts (MEFs). The effect of ascorbate did not involve an increased expression of Tet1–3 or isocitrate dehydrogenases (IDH1–2), the enzymes responsible for producing 2OG. Interestingly, MEFs cultured with different concentrations of glucose, a major precursor of 2OG, exhibited nearly identical responses to ascorbate treatment. Further, blocking the uptake of the reduced form of vitamin C, ascorbic acid, through the sodium-dependent vitamin C transporters (SVCTs) inhibited the effect of ascorbate on 5hmC. However, inhibition of the facilitative glucose transporters (GLUTs), which mediate the incorporation of the oxidized form of vitamin C, dehydroascorbic acid (DHA), did not modify the ability of ascorbate to induce 5hmC generation. These results indicate that the effect of ascorbate on 5hmC is not dependent upon iron uptake, the expression of Tet and IDH, or the production of 2OG, suggesting that ascorbate may directly participate in the generation of 5hmC, most likely as a cofactor of Tet.     

Epigenetic patterns within the haplotype phased fig (Ficus carica L.) genome

Due to DNA heterozygosity and repeat content, assembly of non‐model plant genomes is challenging. Herein, we report a high‐quality genome reference of one of the oldest known domesticated species, fig (Ficus carica L.), using Pacific Biosciences single‐molecule, real‐time sequencing. The fig genome is ~333 Mbp in size, of which 80% has been anchored to 13 chromosomes. Genome‐wide analysis of N⁶‐methyladenine and N⁴‐methylcytosine revealed high methylation levels in both genes and transposable elements, and a prevalence of methylated over non‐methylated genes. Furthermore, the characterization of N⁶‐methyladenine sites led to the identification of ANHGA, a species‐specific motif, which is prevalent for both genes and transposable elements. Finally, exploiting the contiguity of the 13 pseudomolecules, we identified 13 putative centromeric regions. The high‐quality reference genome and the characterization of methylation profiles, provides an important resource for both fig breeding and for fundamental research into the relationship between epigenetic changes and phenotype, using fig as a model species.