an-alternative-culture-method-to-maintain-genomic-hypomethylation

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

Chinese Academy of Sciences

In order to shorten the start-up time of anaerobic ammonium oxidation (ANAMMOX) reactor, biological activated cabon reactor was applied. Three lab scale UASB reactors were seeded with anaerobic sludge, fed with synthetic wastewater containing ammonia and nitrite, and supplemented with granular activated carbon on day 0, 33 and 56, respectively. The nitrogen removal performance of the first reactor, into which GAC was added on day 0, showed no significant improvement in 90 days. After being suspended for about one month, the secondary start-up of this reactor succeeded in another 33 days (totally 123 days). 49 d and 85 d were taken for the other two reactors started up by the addition of GAC on day 33 and 56, respectively. After the reactors were started up, the average removal rates of total nitrogen were 89.8%, 86.7% and 86.7%, respectively. The start-up process could be divided into four stages, namely, the bacterial autolysis phase, the lag phase, the improve phase and the stationary phase, and the best time for adding GAC carrier was right after the start of the lag phase.

[Study on the start-up of anaerobic ammonium oxidation process in biological activated carbon reactor].

DNA N-methyl-2'-deoxyadenosine (6mdA) is an epigenetic modification in both eukaryotes and bacteria. Here we exploited stable isotope-labeled deoxynucleoside [N]-2'-deoxyadenosine ([N]-dA) as an initiation tracer and for the first time developed a metabolically differential tracing code for monitoring DNA 6mdA in human cells. We demonstrate that the initiation tracer [N]-dA undergoes a specific and efficient adenine deamination reaction leading to the loss the exocyclic amine N, and further utilizes the purine salvage pathway to generate mainly both [N]-dA and [N]-2'-deoxyguanosine ([N]-dG) in mammalian genomes. However, [N]-dA is largely retained in the genomes of mycoplasmas, which are often found in cultured cells and experimental animals. Consequently, the methylation of dA generates 6mdA with a consistent coding pattern, with a predominance of [N]-6mdA. Therefore, mammalian DNA 6mdA can be potentially discriminated from that generated by infecting mycoplasmas. Collectively, we show a promising approach for identification of authentic DNA 6mdA in human cells and determine if the human cells are contaminated with mycoplasmas.

Metabolically Generated Stable Isotope-Labeled Deoxynucleoside Code for Tracing DNA N-Methyladenine in Human Cells.

Androgenetic haploid embryonic stem cells (AG-haESCs) hold great promise for exploring gene functions and generating gene-edited semi-cloned (SC) mice. However, the high incidence of self-diploidization and low efficiency of SC mouse production are major obstacles preventing widespread use of these cells. Moreover, although SC mice generation could be greatly improved by knocking out the differentially methylated regions of two imprinted genes, 50% of the SC mice did not survive into adulthood. Here, we found that the genome-wide DNA methylation level in AG-haESCs is extremely low. Subsequently, downregulation of both de novo methyltransferase Dnmt3b and other methylation-related genes was determined to be responsible for DNA hypomethylation. We further demonstrated that ectopic expression of Dnmt3b in AG-haESCs could effectively improve DNA methylation level, and the high incidence of self-diploidization could be markedly rescued. More importantly, the developmental potential of SC embryos was improved, and most SC mice could survive into adulthood.

Reduced Self-Diploidization and Improved Survival of Semi-cloned Mice Produced from Androgenetic Haploid Embryonic Stem Cells through Overexpression of Dnmt3b.

N-methyladenine (6mA) is a rediscovered DNA modification in eukaryotic genomes. To explore the distribution and functions of 6mA, it is of paramount option to use immunoprecipitation to select 6mA-containing DNA fragments for genome-wide sequencing. Presumably, most of the 6mA-free fragments are removed, and the copulling down of the residual is stochastic and sequence-independent and thus they should not be called as peaks by computation. Surprisingly, here we show the predominance of 6mA-free fragments in the pulled-down fractions. By taking advantage of the submicromolar affinity of the antibodies, we further develop an elegant, multiple-round immunoprecipitation (MrIP) approach and show that 6mA-containing fragments can be enriched over 9100-fold and dominate in the final pulled-down fractions. This biochemical approach would greatly reduce the peak calling bias, which is caused by handling of dominated 6mA-free DNA fragments with an assumption-based algorithm computation and facilitates 6mA-pertinent data mining. The MrIP concept is extendable for the genome-wide sequencing of diverse DNA modifications.

Predominance of N-Methyladenine-Specific DNA Fragments Enriched by Multiple Immunoprecipitation.

LC-MS/MS technologies provide important and powerful analytical tools for chemical structure-dependent identification and quantification of epigenetically crucial DNA modifications. To perform LC-MS/MS analysis, it is better to convert DNA to 2'-deoxynucleosides through enzymatic digestion. Here, we observed that inorganic cations Na and K and phosphate buffers, which were often found in various DNA solutions, significantly inhibited DNA digestion as catalyzed by typical set of DNase I, snake venom phosphodiesterase, and calf intestine alkaline phosphatase, leading to poor or varying performance on UHPLC-MS/MS analysis. We then developed an efficient and unique vertical-ultrafiltration approach, enabling us to remove these inorganic salts without DNA loss. Consequently, the removal of inorganic salts by ultrafiltration facilitated the followed DNA digestion and thus enhanced the final UHPLC-MS/MS detection. Benefiting from the developed vertical-ultrafiltration approach, it is also feasible to integrate the desalting step with the other two steps of DNA digestion and protein removal. By investigating the time course of DNA digestion, we observed a differential release rate of 2'-deoxycytidine and 5-methyl-2'-deoxycytidine causing a measurement bias on the methylation frequency. We further exploited Mg to eliminate this bias by stimulating DNase set-based DNA digestion. These innovative approaches enable us to perform rapid, sensitive, and robust UHPLC-MS/MS analysis of methylated DNA 2'-deoxycytidine, demethylation intermediates, and probably other DNA modifications.

Weiyi Lai

State Key Laboratory of Environmental Chemistry and Ecotoxicology,
Research Center for Eco-Environmental Sciences,
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences

Chinese Academy of Sciences

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C