scSPLAT, a scalable plate-based protocol for single cell WGBS library preparation

DNA methylation is a central epigenetic mark that has diverse roles in gene regulation, development, and maintenance of genome integrity. 5 methyl cytosine (5mC) can be interrogated at base resolution in single cells by using bisulfite sequencing (scWGBS). Several different scWGBS strategies have been described in recent years to study DNA methylation in single cells. However, there remain limitations with respect to cost-efficiency and yield. Herein, we present a new development in the field of scWGBS library preparation; single cell Splinted Ligation Adapter Tagging (scSPLAT). scSPLAT employs a pooling strategy to facilitate sample preparation at a higher scale and throughput than previously possible. We demonstrate the accuracy and robustness of the method by generating data from 225 single K562 cells and from 309 single liver nuclei and compare scSPLAT against other scWGBS methods.

Interaction of Thymine DNA Glycosylase with Oxidised 5-Methyl-cytosines in Their Amino- and Imino-Forms

Thymine DNA Glycosylase (TDG) is an enzyme of the base excision repair mechanism and removes damaged or mispaired bases from DNA via hydrolysis of the glycosidic bond. Specificity is of high importance for such a glycosylase, so as to avoid the damage of intact DNA. Among the substrates reported for TDG are mispaired uracil and thymine but also formyl-cytosine and carboxyl-cytosine. Methyl-cytosine and hydroxylmethyl-cytosine are, in contrast, not processed by the TDG enzyme. We have in this work employed molecular dynamics simulations to explore the conformational dynamics of DNA carrying a formyl-cytosine or carboxyl-cytosine and compared those to DNA with the non-cognate bases methyl-cytosine and hydroxylmethyl-cytosine, as amino and imino tautomers. Whereas for the mispairs a wobble conformation is likely decisive for recognition, all amino tautomers of formyl-cytosine and carboxyl-cytosine exhibit the same Watson–Crick conformation, but all imino tautomers indeed form wobble pairs. The conformational dynamics of the amino tautomers in free DNA do not exhibit differences that could be exploited for recognition, and also complexation to the TDG enzyme does not induce any alteration that would indicate preferable binding to one or the other oxidised methyl-cytosine. The imino tautomers, in contrast, undergo a shift in the equilibrium between a closed and a more open, partially flipped state, towards the more open form upon complexation to the TDG enzyme. This stabilisation of the more open conformation is most pronounced for the non-cognate bases methyl-cytosine and hydroxyl-cytosine and is thus not a likely mode for recognition. Moreover, calculated binding affinities for the different forms indicate the imino forms to be less likely in the complexed DNA. These findings, together with the low probability of imino tautomers in free DNA and the indifference of the complexed amino tautomers, suggest that discrimination of the oxidised methyl-cytosines does not take place in the initial complex formation.

Methylation at CpG sites in genomes of aphid Acyrtosiphon pisum and its endosymbiont Buchnera

Pea aphid Acyrtosiphon pisum, a sap-feeding insect, has established a mutualistic relationship with an endosymbiotic bacteria (Buchnera aphidicola) that constitutes an evolutionary successful symbiosis to synthetize complex chemical compounds from a nutrient deprived diet. In this study, led by the presence of DNMT1 and a putative DNMT3 methylase in the aphid genome, we investigated the distribution of the methyl groups on 5'cytosine in CpG motifs on the whole genomes of host and endosymbiont, and looked into their correlation with gene expression. The DNA methylation turned to be present at low level in aphid (around 3% of total genomic cytosine) compared to mammals and plants, but increased to ~9% in genes. Interestingly, the reduced genome of the endosymbiont Buchnera also shows global low level of methyl cytosine despite the fact that its genome does not shelter any DNA methylase. This finding argues for the import of DNA methylase from the host to the endosymbiont. The observed differences in methylation patterns between two clonal variants (host plus endosymbiont) are reported along with the differences in their transcriptome profiles. Our data allowed to decipher a dynamic combinatorial DNA methylation and epigenetic cross talk between host and symbiont in a clonality context that might count for the aphid adaptation to environment.

Epigenetic MRI: Noninvasive Imaging of DNA Methylation in the Brain

It has been recognized that an integration of neuronal and genetic mechanisms supports brain function, regulates behaviour, and underpins response to environmental or disease stimuli. Several different technologies are available for imaging and studying neuronal activity in living brains, such as functional magnetic resonance imaging (fMRI), and have been translated to humans. However, the tools available to measure gene expression are destructive. Here we present a method, called epigenetic MRI (eMRI), that overcomes this limitation. eMRI achieves for the first time direct and noninvasive imaging of DNA methylation, a major gene expression regulator, in intact brains. eMRI exploits the methionine metabolic pathways that are responsible for DNA methylation to label the methyl-cytosine in brain genomic DNA through carbon-13 enriched diets. It then uses a novel carbon-13 magnetic resonance spectroscopic imaging (13C-MRSI) method to map the spatial distribution of labeled DNA. We demonstrated successful 13C labeling of brain DNA through diet using mass spectrometry, and robust and specific detection of labeled DNA using 13C-MRSI. We used eMRI and a biomedical piglet model to produce the first DNA methylation map of an intact brain hemisphere. With both noninvasive labeling and imaging, we expect eMRI to be readily translated to humans and thus enable many new investigations into the epigenetic basis of brain function, behavior, and disease.

NSUN6, an RNA methyltransferase of 5-mC controls glioblastoma response to Temozolomide (TMZ) via NELFB and RPS6KB2 interaction.

Nop2/Sun RNA methyltransferase (NSUN6) is an RNA 5 - methyl cytosine (5mC) transferase with little information known of its function in cancer and response to cancer therapy. Here, we show that NSUN6 methylates both large and small RNA in glioblastoma and controls glioblastoma response to temozolomide with or without influence of the MGMT promoter status, with high NSUN6 expression conferring survival benefit to glioblastoma patients and in other cancers. Mechanistically, our results show that NSUN6 controls response to TMZ therapy via 5mC mediated regulation of NELFB and RPS6BK2. Taken together, we present evidence that show that NSUN6 mediated 5mC deposition regulates transcriptional pause (by accumulation of NELFB and the general transcription factor complexes (POLR2A, TBP, TFIIA, TFIIE) on the preinitiation complex at TATA binding site to control translation machinery in glioblastoma response to alkylating agents. Our findings open a new frontier into controlling of transcriptional regulation by RNA methyltransferase and 5mC.

Prenatal Trimethyltin Exposure Induces Long-Term DNA Methylation Changes in the Male Mouse Hippocampus

Trimethyltin (TMT) is an irreversible neurotoxicant. Because prenatal TMT exposure has been reported to induce behavioral changes, this study was conducted to observe gender differences and epigenetic changes using a mouse model. In behavioral testing of offspring at 5 weeks of age, the total times spent in the center, corner, or border zones in the male prenatal TMT-exposed mice were less than those of control unexposed mice in the open-field test. Female TMT-exposed mice scored lower on total numbers of arm entries and percentages of alternations than controls in the Y-maze test with lower body weight. We found that only TMT-exposed males had fewer copies of mtDNA in the hippocampus and prefrontal cortex region than controls. Additional epigenetic changes, including increased 5-methyl cytosine/5-hydroxymethyl cytosine levels in the male TMT hippocampus, were observed. After methylation binding domain (MBD) sequencing, multiple signaling pathways related to metabolism and neurodevelopment, including FoxO signaling, were identified by pathway analysis for differentially methylated regions (DMRs). Increased FOXO3 and decreased ASCL1 expression were also observed in male TMT hippocampi. This study suggests that sex differences and epigenetics should be more carefully considered in prenatal toxicology studies.

The Shu Complex Prevents Mutagenesis and Cytotoxicity of Single-Strand Specific Alkylation Lesions

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans, express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells, exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Finally, the Shu complex exhibits increased affinity for 3meC-containing DNA. Thus, our work identifies a novel mechanism for coping with alkylation adducts.

Ultrastructural immunocytochemistry shows impairment of RNA pathways in skeletal muscle nuclei of old mice: A link to sarcopenia?

During aging, skeletal muscle is affected by sarcopenia, a progressive decline in muscle mass, strength and endurance that leads to loss of function and disability. Cell nucleus dysfunction is a possible factor contributing to sarcopenia because aging-associated alterations in mRNA and rRNA transcription/maturation machinery have been shown in several cell types including muscle cells. In this study, the distribution and density of key molecular factors involved in RNA pathways namely, nuclear actin (a motor protein and regulator of RNA transcription), 5-methyl cytosine (an epigenetic regulator of gene transcription), and ribonuclease A (an RNA degrading enzyme) were compared in different nuclear compartments of late adult and old mice myonuclei by means of ultrastructural immunocytochemistry. In all nuclear compartments, an age-related decrease of nuclear actin suggested altered chromatin structuring and impaired nucleus-to-cytoplasm transport of both mRNA and ribosomal subunits, while a decrease of 5-methyl cytosine and ribonuclease A in the nucleoli of old mice indicated an age-dependent loss of rRNA genes. These findings provide novel experimental evidence that, in the aging skeletal muscle, nuclear RNA pathways undergo impairment, likely hindering protein synthesis and contributing to the onset and progression of sarcopenia.

Current Advances in DNA Methylation Analysis Methods

DNA methylation is one of the epigenetic changes, which plays a major role in regulating gene expression and, thus, many biological processes and diseases. There are several methods for determining the methylation of DNA samples. However, selecting the most appropriate method for answering biological questions appears to be a challenging task. The primary methods in DNA methylation focused on identifying the state of methylation of the examined genes and determining the total amount of 5-methyl cytosine. The study of DNA methylation at a large scale of genomic levels became possible following the use of microarray hybridization technology. The new generation of sequencing platforms now allows the preparation of genomic maps of DNA methylation at the single-open level. This review includes the majority of methods available to date, introducing the most widely used methods, the bisulfite treatment, biological identification, and chemical cutting along with their advantages and disadvantages. The techniques are then scrutinized according to their robustness, high throughput capabilities, and cost.