SAR11 marine bacteria require exogenous reduced sulphur for growth.

Sulphur is a universally required cell nutrient found in two amino acids and other small organic molecules. All aerobic marine bacteria are known to use assimilatory sulphate reduction to supply sulphur for biosynthesis, although many can assimilate sulphur from organic compounds that contain reduced sulphur atoms. An analysis of three complete 'Candidatus Pelagibacter ubique' genomes, and public ocean metagenomic data sets, suggested that members of the ubiquitous and abundant SAR11 alphaproteobacterial clade are deficient in assimilatory sulphate reduction genes. Here we show that SAR11 requires exogenous sources of reduced sulphur, such as methionine or 3-dimethylsulphoniopropionate (DMSP) for growth. Titrations of the algal osmolyte DMSP in seawater medium containing all other macronutrients in excess showed that 1.5 x 10(8) SAR11 cells are produced per nanomole of DMSP. Although it has been shown that other marine alphaproteobacteria use sulphur from DMSP in preference to sulphate, our results indicate that 'Cand. P. ubique' relies exclusively on reduced sulphur compounds that originate from other plankton.

Modulation of neural gene networks by estradiol in old rhesus macaque females.

The postmenopausal decrease in circulating estradiol (E2) levels has been shown to contribute to several adverse physiological and psychiatric effects. To elucidate the molecular effects of E2 on the brain, we examined differential gene expression and DNA methylation (DNAm) patterns in the nonhuman primate brain following ovariectomy (Ov) and subsequent subcutaneous bioidentical E2 chronic treatment. We identified several dysregulated molecular networks, including MAPK signaling and dopaminergic synapse response, that are associated with ovariectomy and shared across two different brain areas, the occipital cortex (OC) and prefrontal cortex (PFC). The finding that hypomethylation (p = 1.6 × 10-51) and upregulation (p = 3.8 × 10-3) of UBE2M across both brain regions provide strong evidence for molecular differences in the brain induced by E2 depletion. Additionally, differential expression (p = 1.9 × 10-4; interaction p = 3.5 × 10-2) of LTBR in the PFC provides further support for the role E2 plays in the brain, by demonstrating that the regulation of some genes that are altered by ovariectomy may also be modulated by Ov followed by hormone replacement therapy (HRT). These results present real opportunities to understand the specific biological mechanisms that are altered with depleted E2. Given E2's potential role in cognitive decline and neuroinflammation, our findings could lead to the discovery of novel therapeutics to slow cognitive decline. Together, this work represents a major step toward understanding molecular changes in the brain that are caused by ovariectomy and how E2 treatment may revert or protect against the negative neuro-related consequences caused by a depletion in estrogen as women approach menopause.

Selective sweeps and parallel mutation in the adaptive recovery from deleterious mutation in Caenorhabditis elegans.

Deleterious mutation poses a serious threat to human health and the persistence of small populations. Although adaptive recovery from deleterious mutation has been well-characterized in prokaryotes, the evolutionary mechanisms by which multicellular eukaryotes recover from deleterious mutation remain unknown. We applied high-throughput DNA sequencing to characterize genomic divergence patterns associated with the adaptive recovery from deleterious mutation using a Caenorhabditis elegans recovery-line system. The C. elegans recovery lines were initiated from a low-fitness mutation-accumulation (MA) line progenitor and allowed to independently evolve in large populations (N ∼ 1000) for 60 generations. All lines rapidly regained levels of fitness similar to the wild-type (N2) MA line progenitor. Although there was a near-zero probability of a single mutation fixing due to genetic drift during the recovery experiment, we observed 28 fixed mutations. Cross-generational analysis showed that all mutations went from undetectable population-level frequencies to a fixed state in 10-20 generations. Many recovery-line mutations fixed at identical timepoints, suggesting that the mutations, if not beneficial, hitchhiked to fixation during selective sweep events observed in the recovery lines. No MA line mutation reversions were detected. Parallel mutation fixation was observed for two sites in two independent recovery lines. Analysis using a C. elegans interactome map revealed many predicted interactions between genes with recovery line-specific mutations and genes with previously accumulated MA line mutations. Our study suggests that recovery-line mutations identified in both coding and noncoding genomic regions might have beneficial effects associated with compensatory epistatic interactions.

Methylome reorganization during in vitro dedifferentiation and regeneration of Populus trichocarpa.

BACKGROUND: Cytosine DNA methylation (5mC) is an epigenetic modification that is important to genome stability and regulation of gene expression. Perturbations of 5mC have been implicated as a cause of phenotypic variation among plants regenerated through in vitro culture systems. However, the pattern of change in 5mC and its functional role with respect to gene expression, are poorly understood at the genome scale. A fuller understanding of how 5mC changes during in vitro manipulation may aid the development of methods for reducing or amplifying the mutagenic and epigenetic effects of in vitro culture and plant transformation. RESULTS: We investigated the in vitro methylome of the model tree species Populus trichocarpa in a system that mimics routine methods for regeneration and plant transformation in the genus Populus (poplar). Using methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq), we compared the methylomes of internode stem segments from micropropagated explants, dedifferentiated calli, and internodes from regenerated plants. We found that more than half (56%) of the methylated portion of the genome appeared to be differentially methylated among the three tissue types. Surprisingly, gene promoter methylation varied little among tissues, however, the percentage of body-methylated genes increased from 9% to 14% between explants and callus tissue, then decreased to 8% in regenerated internodes. Forty-five percent of differentially-methylated genes underwent transient methylation, becoming methylated in calli, and demethylated in regenerants. These genes were more frequent in chromosomal regions with higher gene density. Comparisons with an expression microarray dataset showed that genes methylated at both promoters and gene bodies had lower expression than genes that were unmethylated or only promoter-methylated in all three tissues. Four types of abundant transposable elements showed their highest levels of 5mC in regenerated internodes. CONCLUSIONS: DNA methylation varies in a highly gene- and chromosome-differential manner during in vitro differentiation and regeneration. 5mC in redifferentiated tissues was not reset to that in original explants during the study period. Hypermethylation of gene bodies in dedifferentiated cells did not interfere with transcription, and may serve a protective role against activation of abundant transposable elements.

Cross-species epigenetic regulation of nucleus accumbens KCNN3 transcripts by excessive ethanol drinking.

The underlying genetic and epigenetic mechanisms driving functional adaptations in neuronal excitability and excessive alcohol intake are poorly understood. Small-conductance Ca2+-activated K+ (KCa2 or SK) channels encoded by the KCNN family of genes have emerged from preclinical studies as a key contributor to alcohol-induced functional neuroadaptations in alcohol-drinking monkeys and alcohol dependent mice. Here, this cross-species analysis focused on KCNN3 DNA methylation, gene expression, and single nucleotide polymorphisms including alternative promoters in KCNN3 that could influence surface trafficking and function of KCa2 channels. Bisulfite sequencing analysis of the nucleus accumbens tissue from alcohol-drinking monkeys and alcohol dependent mice revealed a differentially methylated region in exon 1A of KCNN3 that overlaps with a predicted promoter sequence. The hypermethylation of KCNN3 in the accumbens paralleled an increase in expression of alternative transcripts that encode apamin-insensitive and dominant-negative KCa2 channel isoforms. A polymorphic repeat in macaque KCNN3 encoded by exon 1 did not correlate with alcohol drinking. At the protein level, KCa2.3 channel expression in the accumbens was significantly reduced in very heavy drinking monkeys. Together, our cross-species findings on epigenetic dysregulation of KCNN3 represent a complex mechanism that utilizes alternative promoters to impact firing of accumbens neurons. Thus, these results provide support for hypermethylation of KCNN3 as a possible key molecular mechanism underlying harmful alcohol intake and alcohol use disorder.

Cross-species epigenetic regulation of nucleus accumbens KCNN3 transcripts by excessive ethanol drinking.

The underlying genetic and epigenetic mechanisms driving functional adaptations in neuronal excitability and excessive alcohol intake are poorly understood. Small-conductance Ca2+-activated K+ (KCa2 or SK) channels encoded by the KCNN family of genes have emerged from preclinical studies as a key contributor to alcohol-induced functional neuroadaptations in alcohol-drinking monkeys and alcohol-dependent mice. Here, this cross-species analysis focused on KCNN3 DNA methylation, gene expression, and single nucleotide polymorphisms, including alternative promoters in KCNN3, that could influence surface trafficking and function of KCa2 channels. Bisulfite sequencing analysis of the nucleus accumbens tissue from alcohol-drinking monkeys and alcohol-dependent mice revealed a differentially methylated region in exon 1A of KCNN3 that overlaps with a predicted promoter sequence. The hypermethylation of KCNN3 in the accumbens paralleled an increase in the expression of alternative transcripts that encode apamin-insensitive and dominant-negative KCa2 channel isoforms. A polymorphic repeat in macaque KCNN3 encoded by exon 1 did not correlate with alcohol drinking. At the protein level, KCa2.3 channel expression in the accumbens was significantly reduced in very heavy-drinking monkeys. Together, our cross-species findings on epigenetic dysregulation of KCNN3 represent a complex mechanism that utilizes alternative promoters to potentially impact the firing of accumbens neurons. Thus, these results provide support for hypermethylation of KCNN3 as a possible key molecular mechanism underlying harmful alcohol intake and alcohol use disorder.

Modulation of neural gene networks by estradiol in old rhesus macaque females.

The postmenopausal decrease in circulating estradiol (E2) levels has been shown to contribute to several adverse physiological and psychiatric effects. To elucidate the molecular effects of E2 on the brain, we examined differential gene expression and DNA methylation (DNAm) patterns in the nonhuman primate brain following ovariectomy (Ov) and subsequent E2 treatment. We identified several dysregulated molecular networks, including MAPK signaling and dopaminergic synapse response, that are associated with ovariectomy and shared across two different brain areas, the occipital cortex (OC) and prefrontal cortex (PFC). The finding that hypomethylation (p=1.6×10-51) and upregulation (p=3.8×10-3) of UBE2M across both brain regions, provide strong evidence for molecular differences in the brain induced by E2 depletion. Additionally, differential expression (p=1.9×10-4; interaction p=3.5×10-2) of LTBR in the PFC, provides further support for the role E2 plays in the brain, by demonstrating that the regulation of some genes that are altered by ovariectomy may also be modulated by Ov followed by hormone replacement therapy (HRT). These results present real opportunities to understand the specific biological mechanisms that are altered with depleted E2. Given E2's potential role in cognitive decline and neuroinflammation, our findings could lead to the discovery of novel therapeutics to slow cognitive decline. Together, this work represents a major step towards understanding molecular changes in the brain that are caused by ovariectomy and how E2 treatment may revert or protect against the negative neuro-related consequences caused by a depletion in estrogen as women approach menopause.

Methylation analysis by targeted bisulfite sequencing in large for gestational age (LGA) newborns: the LARGAN cohort.

BACKGROUND: In 1990, David Barker proposed that prenatal nutrition is directly linked to adult cardiovascular disease. Since then, the relationship between adult cardiovascular risk, metabolic syndrome and birth weight has been widely documented. Here, we used the TruSeq Methyl Capture EPIC platform to compare the methylation patterns in cord blood from large for gestational age (LGA) vs adequate for gestational age (AGA) newborns from the LARGAN cohort. RESULTS: We found 1672 differentially methylated CpGs (DMCs) with a nominal p < 0.05 and 48 differentially methylated regions (DMRs) with a corrected p < 0.05 between the LGA and AGA groups. A systems biology approach identified several biological processes significantly enriched with genes in association with DMCs with FDR < 0.05, including regulation of transcription, regulation of epinephrine secretion, norepinephrine biosynthesis, receptor transactivation, forebrain regionalization and several terms related to kidney and cardiovascular development. Gene ontology analysis of the genes in association with the 48 DMRs identified several significantly enriched biological processes related to kidney development, including mesonephric duct development and nephron tubule development. Furthermore, our dataset identified several DNA methylation markers enriched in gene networks involved in biological pathways and rare diseases of the cardiovascular system, kidneys, and metabolism. CONCLUSIONS: Our study identified several DMCs/DMRs in association with fetal overgrowth. The use of cord blood as a material for the identification of DNA methylation biomarkers gives us the possibility to perform follow-up studies on the same patients as they grow. These studies will not only help us understand how the methylome responds to continuum postnatal growth but also link early alterations of the DNA methylome with later clinical markers of growth and metabolic fitness.

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression.

BACKGROUND: DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. RESULTS: We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. CONCLUSIONS: We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

Genome resequencing reveals multiscale geographic structure and extensive linkage disequilibrium in the forest tree Populus trichocarpa.

• Plant population genomics informs evolutionary biology, breeding, conservation and bioenergy feedstock development. For example, the detection of reliable phenotype-genotype associations and molecular signatures of selection requires a detailed knowledge about genome-wide patterns of allele frequency variation, linkage disequilibrium and recombination. • We resequenced 16 genomes of the model tree Populus trichocarpa and genotyped 120 trees from 10 subpopulations using 29,213 single-nucleotide polymorphisms. • Significant geographic differentiation was present at multiple spatial scales, and range-wide latitudinal allele frequency gradients were strikingly common across the genome. The decay of linkage disequilibrium with physical distance was slower than expected from previous studies in Populus, with r(2) dropping below 0.2 within 3-6 kb. Consistent with this, estimates of recent effective population size from linkage disequilibrium (N(e) ≈ 4000-6000) were remarkably low relative to the large census sizes of P. trichocarpa stands. Fine-scale rates of recombination varied widely across the genome, but were largely predictable on the basis of DNA sequence and methylation features. • Our results suggest that genetic drift has played a significant role in the recent evolutionary history of P. trichocarpa. Most importantly, the extensive linkage disequilibrium detected suggests that genome-wide association studies and genomic selection in undomesticated populations may be more feasible in Populus than previously assumed.