Context-dependent DNA methylation signatures in animal livestock.

DNA methylation is an important epigenetic modification that is widely conserved across animal genomes. It is widely accepted that DNA methylation patterns can change in a context-dependent manner, including in response to changing environmental parameters. However, this phenomenon has not been analyzed in animal livestock yet, where it holds major potential for biomarker development. Building on the previous identification of population-specific DNA methylation in clonal marbled crayfish, we have now generated numerous base-resolution methylomes to analyze location-specific DNA methylation patterns. We also describe the time-dependent conversion of epigenetic signatures upon transfer from one environment to another. We further demonstrate production system-specific methylation signatures in shrimp, river-specific signatures in salmon and farm-specific signatures in chicken. Together, our findings provide a detailed resource for epigenetic variation in animal livestock and suggest the possibility for origin tracing of animal products by epigenetic fingerprinting.

Location-Dependent DNA Methylation Signatures in a Clonal Invasive Crayfish.

DNA methylation is an important epigenetic modification that has been repeatedly implied in organismal adaptation. However, many previous studies that have linked DNA methylation patterns to environmental parameters have been limited by confounding factors, such as cell-type heterogeneity and genetic variation. In this study, we analyzed DNA methylation variation in marbled crayfish, a clonal and invasive freshwater crayfish that is characterized by a largely tissue-invariant methylome and negligible genetic variation. Using a capture-based subgenome bisulfite sequencing approach that covers a small, variably methylated portion of the marbled crayfish genome, we identified specific and highly localized DNA methylation signatures for specimens from geographically and ecologically distinct wild populations. These results were replicated both biologically and technically by re-sampling at different time points and by using independent methodology. Finally, we show specific methylation signatures for laboratory animals and for laboratory animals that were reared at a lower temperature. Our results thus demonstrate the existence of context-dependent DNA methylation signatures in a clonal animal.

Phylogeographic reconstruction of the marbled crayfish origin.

The marbled crayfish (Procambarus virginalis) is a triploid and parthenogenetic freshwater crayfish species that has colonized diverse habitats around the world. Previous studies suggested that the clonal marbled crayfish population descended as recently as 25 years ago from a single specimen of P. fallax, the sexually reproducing parent species. However, the genetic, phylogeographic, and mechanistic origins of the species have remained enigmatic. We have now constructed a new genome assembly for P. virginalis to support a detailed phylogeographic analysis of the diploid parent species, Procambarus fallax. Our results strongly suggest that both parental haplotypes of P. virginalis were inherited from the Everglades subpopulation of P. fallax. Comprehensive whole-genome sequencing also detected triploid specimens in the same subpopulation, which either represent evolutionarily important intermediate genotypes or independent parthenogenetic lineages arising among the sexual parent population. Our findings thus clarify the geographic origin of the marbled crayfish and identify potential mechanisms of parthenogenetic speciation.

Genome analysis of the monoclonal marbled crayfish reveals genetic separation over a short evolutionary timescale.

The marbled crayfish (Procambarus virginalis) represents a very recently evolved parthenogenetic freshwater crayfish species that has invaded diverse habitats in Europe and in Madagascar. However, population genetic analyses have been hindered by the homogeneous genetic structure of the population and the lack of suitable tools for data analysis. We have used whole-genome sequencing to characterize reference specimens from various known wild populations. In parallel, we established a whole-genome sequencing data analysis pipeline for the population genetic analysis of nearly monoclonal genomes. Our results provide evidence for systematic genetic differences between geographically separated populations and illustrate the emerging differentiation of the marbled crayfish genome. We also used mark-recapture population size estimation in combination with genetic data to model the growth pattern of marbled crayfish populations. Our findings uncover evolutionary dynamics in the marbled crayfish genome over a very short evolutionary timescale and identify the rapid growth of marbled crayfish populations as an important factor for ecological monitoring.

Evaluation of analytical and statistical approaches for predicting in vitro nitrogen solubility and in vivo pre-caecal crude protein digestibility of cereal grains in growing pigs.

Different analytical (enzyme system and near-infrared spectroscopy (NIRS)) and statistical (single and multiple regressions) approaches were used to predict in vivo standardized pre-caecal digestibility (PCD) of crude protein (CP) and amino acids (AA) in cereal grains for growing pigs as well as in vitro nitrogen (N) solubility. Furthermore, different chemical and physical characteristics were categorized (e.g. crude nutrients, AA, minerals, fibre components or combinations of these) and used for generating prediction equations. There were strong linear relationships (p < .05) between in vivo PCD of CP and essential AA and in vitro N solubility when grain species was considered as covariate in the model. Predicting in vivo PCD values using various chemical and physical characteristics produced inconsistent results among different grain species and AA and could therefore not be used for predicting PCD. It is possible to predict in vitro N solubility from chemical and physical characteristics for some grain species. However, the relationships between some of these categories and the in vitro N solubility were not consistent and not always causative or physiologically explainable. The R2 of NIRS for predicting in vitro N solubility was at a relatively high level (up to R2  = 0.80). This level of R2 indicates that a classification of the grain samples in, for example, high, medium and low in vitro N solubility levels is possible, but it does not allow for a quantitative prediction of the in vitro N solubility. In conclusion, the present database can be used for establishing a ranking of different cereal grain species for PCD of CP and essential AA values. However, it was not possible to create clear prediction equations for in vivo or in vitro digestibility values. Therefore, greater variation within grain species, for example due to different growing and harvesting conditions, is warranted for predicting PCD values of individual grain samples.

The methylome of the marbled crayfish links gene body methylation to stable expression of poorly accessible genes.

BACKGROUND: The parthenogenetic marbled crayfish (Procambarus virginalis) is a novel species that has rapidly invaded and colonized various different habitats. Adaptation to different environments appears to be independent of the selection of genetic variants, but epigenetic programming of the marbled crayfish genome remains to be understood. RESULTS: Here, we provide a comprehensive analysis of DNA methylation in marbled crayfish. Whole-genome bisulfite sequencing of multiple replicates and different tissues revealed a methylation pattern that is characterized by gene body methylation of housekeeping genes. Interestingly, this pattern was largely tissue invariant, suggesting a function that is unrelated to cell fate specification. Indeed, integrative analysis of DNA methylation, chromatin accessibility and mRNA expression patterns revealed that gene body methylation correlated with limited chromatin accessibility and stable gene expression, while low-methylated genes often resided in chromatin with higher accessibility and showed increased expression variation. Interestingly, marbled crayfish also showed reduced gene body methylation and higher gene expression variability when compared with their noninvasive mother species, Procambarus fallax. CONCLUSIONS: Our results provide novel insights into invertebrate gene body methylation and its potential role in adaptive gene regulation.

Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats.

The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as well as post-transcriptional mechanisms that control recombination of repeat regions and the expression and mobility of transposable elements. We report here that mutations in two (cytosine-5) RNA methyltransferases, Dnmt2 and NSun2, impact the accumulation of mobile element-derived sequences and DNA repeat integrity in Drosophila. Loss of Dnmt2 function caused moderate effects under standard conditions, while heat shock exacerbated these effects. In contrast, NSun2 function affected mobile element expression and genome integrity in a heat shock-independent fashion. Reduced tRNA stability in both RCMT mutants indicated that tRNA-dependent processes affected mobile element expression and DNA repeat stability. Importantly, further experiments indicated that complex formation with RNA could also contribute to the impact of RCMT function on gene expression control. These results thus uncover a link between tRNA modification enzymes, the expression of repeat DNA, and genomic integrity.

Clonal genome evolution and rapid invasive spread of the marbled crayfish.

The marbled crayfish Procambarus virginalis is a unique freshwater crayfish characterized by very recent speciation and parthenogenetic reproduction. Marbled crayfish also represent an emerging invasive species and have formed wild populations in diverse freshwater habitats. However, our understanding of marbled crayfish biology, evolution and invasive spread has been hampered by the lack of freshwater crayfish genome sequences. We have now established a de novo draft assembly of the marbled crayfish genome. We determined the genome size at approximately 3.5 gigabase pairs and identified >21,000 genes. Further analysis confirmed the close relationship to the genome of the slough crayfish, Procambarus fallax, and also established a triploid AA'B genotype with a high level of heterozygosity. Systematic fieldwork and genotyping demonstrated the rapid expansion of marbled crayfish on Madagascar and established the marbled crayfish as a potent invader of freshwater ecosystems. Furthermore, comparative whole-genome sequencing demonstrated the clonality of the population and their genetic identity with the oldest known stock from the German aquarium trade. Our study closes an important gap in the phylogenetic analysis of animal genomes and uncovers the unique evolutionary history of an emerging invasive species.

Comprehensive DNA methylation analysis of the Aedes aegypti genome.

Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play key roles in virus control and it has been suggested that dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the Ae. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the Ae. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided first evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus regulation.

Determination of basal ileal endogenous losses and standardized ileal digestibility of amino acids in barley fed to growing pigs.

BACKGROUND: Basal ileal endogenous amino acid (AA) losses (IAAend) and standardized ileal digestibility (SID) values of cereal grains, such as barley, are apparently underestimated when determined according to the nitrogen (N)-free method. Regression analysis between the dietary apparent ileal digestible content (cAID) and total crude protein (CP) and AA can be considered as alternative approach to obtain more accurate values for IAAend and SID of AA in cereal grains. METHODS: Eight hulled barley genotypes were used, with barley being the only source of CP and AA in the assay diets. The diets contained 95 % as-fed of these eight barley genotypes each, ranging in CP content between 109.1 and 123.8 g/kg dry matter (DM). Nine ileally T-cannulated barrows, average body weight (BW) 30 ± 2 kg, were allotted to a row-column design comprising eight periods with 6 d each and nine pigs. On d 5 and the night of d 6 of every period, ileal digesta were collected for a total of 12 h. The IAAend and the SID were determined by linear regression analysis between cAID and total dietary CP and AA. RESULTS: There exist linear relationships between cAID and total CP and AA (P < 0.001). The IAAend of CP, Lys, Met, Thr and Trp amounted to 35.34, 1.08, 0.25, 1.02 and 0.38 g/kg DM intake (DMI), respectively, which are greater compared to average IAAend determined previously under N-free feeding conditions. The SID of CP, Lys, Met, Thr and Trp was 90, 79, 85, 79 and 86 %, respectively, and was greater when compared to tabulated values. Moreover, these SID values were greater than those reported in literature, based on correction of apparent ileal digestibility (AID) of CP and AA for their IAAend values. Summarized, the results of the present regression analysis indicate greater IAAend in barley-based diets compared to those obtained by N-free feeding. CONCLUSIONS: For low-protein feed ingredients like barley the regression method may be preferred over correction of AID values for their IAAend determined under N-free feeding conditions, as intercepts and slopes of the linear regression equations between cAID and total dietary CP and AA provide direct estimates of IAAend and SID of CP and AA in the presence of the assay feed ingredient.

The marbled crayfish as a paradigm for saltational speciation by autopolyploidy and parthenogenesis in animals.

The parthenogenetic all-female marbled crayfish is a novel research model and potent invader of freshwater ecosystems. It is a triploid descendant of the sexually reproducing slough crayfish, Procambarus fallax, but its taxonomic status has remained unsettled. By cross-breeding experiments and parentage analysis we show here that marbled crayfish and P. fallax are reproductively separated. Both crayfish copulate readily, suggesting that the reproductive barrier is set at the cytogenetic rather than the behavioural level. Analysis of complete mitochondrial genomes of marbled crayfish from laboratory lineages and wild populations demonstrates genetic identity and indicates a single origin. Flow cytometric comparison of DNA contents of haemocytes and analysis of nuclear microsatellite loci confirm triploidy and suggest autopolyploidisation as its cause. Global DNA methylation is significantly reduced in marbled crayfish implying the involvement of molecular epigenetic mechanisms in its origination. Morphologically, both crayfish are very similar but growth and fecundity are considerably larger in marbled crayfish, making it a different animal with superior fitness. These data and the high probability of a divergent future evolution of the marbled crayfish and P. fallax clusters suggest that marbled crayfish should be considered as an independent asexual species. Our findings also establish the P. fallax-marbled crayfish pair as a novel paradigm for rare chromosomal speciation by autopolyploidy and parthenogenesis in animals and for saltational evolution in general.

The RNA methyltransferase Dnmt2 is required for efficient Dicer-2-dependent siRNA pathway activity in Drosophila.

Transfer RNA (tRNA) fragmentation in response to stress conditions has been described in many organisms. tRNA fragments have been found in association with small interfering RNA (siRNA) components, but the biological role of these interactions remains unclear. We report here that the tRNA methyltransferase Dnmt2 is essential for efficient Dicer-2 (Dcr-2) function in Drosophila. Using small RNA (sRNA) sequencing, we confirmed that Dnmt2 limits the extent of tRNA fragmentation during the heat-shock response. tRNAs as well as tRNA fragments serve as Dcr-2 substrates, and Dcr-2 degrades tRNA-derived sequences, especially under heat-shock conditions. tRNA-derived RNAs are able to inhibit Dcr-2 activity on long double-stranded RNAs (dsRNAs). Consequently, heat-shocked Dnmt2 mutant animals accumulate dsRNAs, produce fewer siRNAs, and show misregulation of siRNA pathway-dependent genes. These results reveal the impact of tRNA fragmentation on siRNA pathways and implicate tRNA modifications in the regulation of sRNA homeostasis during the heat-shock response.

Efficient RNA virus control in Drosophila requires the RNA methyltransferase Dnmt2.

Drosophila use small-interfering RNA mechanisms to limit the amplification of viral genomes. However, it is unclear how small RNA interference components recognize and separate viral from cellular RNA. Dnmt2 enzymes are highly conserved RNA methyltransferases with substrate specificity towards cellular tRNAs. We report here that Dnmt2 is required for efficient innate immune responses in Drosophila. Dnmt2 mutant flies accumulate increasing levels of Drosophila C virus and show activated innate immune responses. Binding of Dnmt2 to DCV RNA suggests that Dnmt2 contributes to virus control directly, possibly by RNA methylation. These observations demonstrate a role for Dnmt2 in antiviral defence.

Detection of cytosine methylation in RNA using bisulfite sequencing.

Post-transcriptional RNA modifications are a characteristic feature of noncoding RNAs and have been described for ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and various other small RNAs. However, the biological function of most of these modifications remains uncharacterized. Cytosine-5 methylation (5mC) has been detected in abundant and long-lived RNA molecules such as rRNAs and tRNAs, but, because of technical limitations, the occurrence of base-methylated cytosines in other RNAs is not known. To facilitate the detection of RNA methylation, we have established a method for analyzing base-methylated cytosines in RNA using bisulfite sequencing. Treatment of RNA with bisulfite causes the chemical deamination of nonmethylated cytosines to uracil, while methylated cytosines remain unaffected. cDNA synthesis followed by polymerase chain reaction (PCR) amplification and DNA sequencing allows investigators to reproducibly and quantitatively distinguish unmethylated cytosines (as thymines) from methylated cytosines in tRNAs and rRNAs. Using high-throughput sequencing approaches, this protocol should enable the characterization of 5mC methylation patterns in any RNA molecule, including low abundance RNAs.

RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage.

Dnmt2 proteins are the most conserved members of the DNA methyltransferase enzyme family, but their substrate specificity and biological functions have been a subject of controversy. We show here that, in addition to tRNA(Asp-GTC), tRNA(Val-AAC) and tRNA(Gly-GCC) are also methylated by Dnmt2. Drosophila Dnmt2 mutants showed reduced viability under stress conditions, and Dnmt2 relocalized to stress granules following heat shock. Strikingly, stress-induced cleavage of tRNAs was Dnmt2-dependent, and Dnmt2-mediated methylation protected tRNAs against ribonuclease cleavage. These results uncover a novel biological function of Dnmt2-mediated tRNA methylation, and suggest a role for Dnmt2 enzymes during the biogenesis of tRNA-derived small RNAs.

Azacytidine inhibits RNA methylation at DNMT2 target sites in human cancer cell lines.

The cytosine analogues azacytidine and decitabine are currently being developed as drugs for epigenetic cancer therapy. Although various studies have shown that both drugs are effective in inhibiting DNA methylation, it has also become clear that their mode of action is not limited to DNA demethylation. Because azacytidine is a ribonucleoside, the primary target of this drug may be cellular RNA rather than DNA. We have now analyzed the possibility that azacytidine inhibits the RNA methyltransferase DNMT2. We found that DNMT2 is variably expressed in human cancer cell lines. RNA bisulfite sequencing showed that azacytidine, but not decitabine, inhibits cytosine 38 methylation of tRNA(Asp), a major substrate of DNMT2. Azacytidine caused a substantially stronger effect than decitabine on the metabolic rate of all the cancer cell lines tested, consistent with an effect of this drug on RNA metabolism. Of note, drug-induced loss of RNA methylation seemed specific for DNMT2 target sites because we did not observe any significant demethylation at sites known to be methylated by other RNA methyltransferases. Our results uncover a novel and quantifiable drug activity of azacytidine and raise the possibility that tRNA hypomethylation might contribute to patient responses.

RNA cytosine methylation analysis by bisulfite sequencing.

Covalent modifications of nucleic acids play an important role in regulating their functions. Among these modifications, (cytosine-5) DNA methylation is best known for its role in the epigenetic regulation of gene expression. Post-transcriptional RNA modification is a characteristic feature of noncoding RNAs, and has been described for rRNAs, tRNAs and miRNAs. (Cytosine-5) RNA methylation has been detected in stable and long-lived RNA molecules, but its function is still unclear, mainly due to technical limitations. In order to facilitate the analysis of RNA methylation patterns we have established a protocol for the chemical deamination of cytosines in RNA, followed by PCR-based amplification of cDNA and DNA sequencing. Using tRNAs and rRNAs as examples we show that cytosine methylation can be reproducibly and quantitatively detected by bisulfite sequencing. The combination of this method with deep sequencing allowed the analysis of a large number of RNA molecules. These results establish a versatile method for the identification and characterization of RNA methylation patterns, which will be useful for defining the biological function of RNA methylation.

MCP-1 induces activation of MAP-kinases ERK, JNK and p38 MAPK in human endothelial cells.

Activation of vascular endothelial cells (ECs) plays an important pathogenic role in the development of atherosclerosis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant of monocytes. Besides induction of monocyte recruitment, it has been suggested that MCP-1 can also affect the cellular responses of ECs. We investigated whether MCP-1 activated the three major mitogen activated protein (MAP)-kinases extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38 MAPK. Stimulation of ECs with MCP-1 induced a time- and concentration-dependent activation of all three MAP-kinases, concentrations as low as 0.1 ng/ml were sufficient for this mechanism. MCP-1 also induced secretion of matrix metalloproteinase (MMP)-2 which along with ERK activation was inhibited by PD098059. The results demonstrate that MCP-1 can lead to direct activation of MAP kinases together with induction of MMP2 in ECs. Our data thus propose a new mechanism for the proatherogenic effect of MCP-1.