Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

AIMS/HYPOTHESIS: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion. METHODS: Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging. RESULTS: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation. CONCLUSIONS/INTERPRETATION: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. DATA AVAILABILITY: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

Aims/hypothesis: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly-connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility which we explore here by focussing on the imprinted gene neuronatin (Nnat), which is required for normal insulin synthesis and secretion. Methods: Single cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing eGFP under the control of the Nnat enhancer/promoter regions were generated for fluorescence-activated cell (FAC) sorting of beta cells and downstream analysis of CpG methylation by bisulphite and RNA sequencing, respectively. Animals deleted for the de novo methyltransferase, DNMT3A from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 and Cal-590. Insulin secretion was measured using Homogeneous Time Resolved Fluorescence Imaging. Results: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic data sets demonstrated the early establishment of Nnat-positive and negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a sub-population specialised for insulin production, reminiscent of recently-described "ßHI" cells and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialization. Conclusions/interpretation: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may thus contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.

Habitat-based biodiversity responses to macroclimate and edaphic factors in European fen ecosystems.

Understanding large-scale drivers of biodiversity in palustrine wetlands is challenging due to the combined effects of macroclimate and local edaphic conditions. In boreal and temperate fen ecosystems, the influence of macroclimate on biodiversity is modulated by hydrological settings across habitats, making it difficult to assess their vulnerability to climate change. Here, we investigate the influence of macroclimate and edaphic factors on three Essential Biodiversity Variables across eight ecologically defined habitats that align with ecosystem classifications and red lists. We used 27,555 vegetation plot samples from European fens to assess the influence of macroclimate and groundwater pH predictors on the geographic distribution of each habitat type. Additionally, we modeled the relative influence of macroclimate, water pH, and water table depth on community species richness and composition, focusing on 309 plant specialists. Our models reveal strong effects of mean annual temperature, diurnal thermal range, and summer temperature on biodiversity variables, with contrasting differences among habitats. While macroclimatic factors primarily shape geographic distributions and species richness, edaphic factors emerge as the primary drivers of composition for vascular plants and bryophytes. Annual precipitation exhibits non-linear effects on fen biodiversity, with varying impact across habitats with different hydrological characteristics, suggesting a minimum requirement of 600 mm of annual precipitation for the occurrence of fen ecosystems. Our results anticipate potential impacts of climate warming on European fens, with predictable changes among habitat types and geographic regions. Moreover, we provide evidence that the drivers of biodiversity in boreal and temperate fens are closely tied to the ecological characteristics of each habitat type and the dispersal abilities of bryophytes and vascular plants. Given that the influence of macroclimate and edaphic factors on fen ecosystems is habitat specific, climate change research and conservation actions should consider ecological differentiation within functional IUCN ecosystems at continental and regional scales.

Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS.

DNA methylation is a critical epigenetic mark in mammalian cells. Many aspects of DNA methylation maintenance have been characterized; however, the exact kinetics of post-replicative methylation maintenance remain a subject of debate. Here we develop isolation of DNA by 5-ethynyl-deoxyuridine labelling for mass spectrometry (iDEMS), a highly sensitive, quantitative mass spectrometry-based method for measuring DNA modifications on metabolically labelled DNA. iDEMS reveals an unexpectedly hemi-methylated landscape on nascent DNA. Combining iDEMS with metabolic labelling reveals that methylation maintenance is outpaced by cell division in mouse embryonic stem cells. Our approach shows that hydroxymethylation is perpetually asymmetric between sister strands in favour of the parental, template strand. iDEMS can be coupled with immunoprecipitation of chromatin proteins, revealing features of DNA methylation-histone modification crosstalk and suggesting a model for interplay between methylation and nucleosome assembly. iDEMS therefore elucidates long-standing questions about DNA modification propagation and provides an important orthogonal technology to understanding this process in dynamic cellular contexts.

Conservation and restoration of Central European fens by mowing: A consensus from 20 years of experimental work.

While the importance of conservation mowing for mesic grasslands is generally accepted, its use for fens and fen grasslands interspersed within agricultural land is still controversial. Although fens may persist naturally, ongoing environmental changes increase productivity and accelerate succession. These processes can be mitigated through conservation management with appropriate settings. However, long-term management experiments are challenging and provide only locally valid results. Here, we analysed vegetation data (bryophytes and vascular plants) from seven management experiments (spanning 3-20 years) conducted in Central European poor, moderately-rich, and calcareous spring fens (Czech Republic, Slovakia). Two of these experiments examined the effects of restoration of abandoned fens, while five experiments examined changes in mowing regimes in managed fens (cessation, intensification, delay to autumn, and litter removal). Data were analysed using unidimensional and multidimensional methods separately for the initial, extended, and entire period. Mowing had a statistically significant effect on species composition except for the shortest (3-year) experiment. Litter removal did not compensate for mowing. Mowing twice or delayed mowing significantly affected the species composition of calcareous fens. In all cases, cessation of mowing significantly reduced the richness of species, especially those of conservation importance. In contrast, any mowing of abandoned fens increased species richness. The effects of mowing intensification or cessation on species richness and composition of a restored calcareous fen were evident in the first 2-3 years. Other effects were initially weak or nonsignificant but later became stronger, such as mowing delay and restoration removal of litter, which became significant only after nearly 20 years. We found that cessation or restoration of mowing usually triggers a rapid vegetation change, whereas it can take decades to detect the response caused by changes in mowing timing. Importantly, mowing can stabilise or even restore vegetation of fen ecosystems that have been weakened by their fragmentation in the temperate agricultural landscapes.

Can Sphagnum removal reverse the undesired succession of rich fens under different alkalinity and fertility levels?

An undesired succession of rich fens leads to the formation of dense Sphagnum carpets that outcompete brown mosses and some vascular plants, resulting in biodiversity loss in fen habitats of high conservation importance. Small-scale Sphagnum removal is a rarely implemented conservational measure, whose success may depend on soil alkalinity and fertility (i.e., nutrient availability). Therefore, characterizing the effects of pH and fertility levels would potentially allow for the development of better Sphagnum removal strategies. Two experiments were conducted across 24 rich fens of different alkalinity and fertility located in an area of ~32,000 km2 spanning from the Bohemian Massif to the Western Carpathians (Europe). We hypothesized that high alkalinity and low fertility support the restoration of rich fen vegetation after Sphagnum removal. Our study focused on four different Sphagnum groups. In Experiment 1, the treatment plots remained unfenced. In Experiment 2, the treatment plots were fenced off and target brown mosses were transplanted from the surroundings to overcome dispersal limitations. A repeated-measures design was used, with vegetation composition recorded over a 5-year period. High alkalinity rather than fertility facilitated species richness and the appearance of target brown mosses. High alkalinity generally hindered Sphagnum recovery, whereas high fertility supported the recurrence of S. teres and S. recurvum agg. Under high pH conditions, enhanced fertility further correlated with the spread of nonsphagnaceous generalist bryophytes of low conservation value. Despite sustaining a significant overall reduction, all Sphagnum taxa began to recover throughout the experiment, albeit less obviously in fens with S. warnstorfii. Sphagnum removal may reverse biodiversity loss and allow for the restoration of brown mosses in rich fens where Sphagnum cover had increased due to slight eutrophication, acidification, or a decrease in the water table. In alkaline and nutrient-poor conditions (e.g., S. warnstorfii fens), the effect is evident and long lasting and the intervention may not be extensive. In fens dominated by S. teres or S. recurvum agg., repeated or large-scale removal may be needed if high nutrient availability (potassium, phosphorus) or low alkalinity supports Sphagnum recolonization. Treatment plots with S. subgenus Sphagnum exhibited the least promising brown-moss restoration prospects.

Rising temperature modulates pH niches of fen species.

Rising temperatures may endanger fragile ecosystems because their character and key species show different habitat affinities under different climates. This assumption has only been tested in limited geographical scales. In fens, one of the most endangered ecosystems in Europe, broader pH niches have been reported from cold areas and are expected for colder past periods. We used the largest European-scale vegetation database from fens to test the hypothesis that pH interacts with macroclimate temperature in forming realized niches of fen moss and vascular plant species. We calibrated the data set (29,885 plots after heterogeneity-constrained resampling) with temperature, using two macroclimate variables, and with the adjusted pH, a variable combining pH and calcium richness. We modelled temperature, pH and water level niches for one hundred species best characterizing European fens using generalized additive models and tested the interaction between pH and temperature. Fifty-five fen species showed a statistically significant interaction between pH and temperature (adj p Ë‚ .01). Forty-six of them (84%) showed a positive interaction manifested by a shift or restriction of their niche to higher pH in warmer locations. Nine vascular plants and no moss showed the opposite interaction. Mosses showed significantly greater interaction. We conclude that climate significantly modulates edaphic niches of fen plants, especially bryophytes. This result explains previously reported regional changes in realized pH niches, a current habitat-dependent decline of endangered taxa, and distribution changes in the past. A warmer climate makes growing seasons longer and warmer, increases productivity, and may lower the water level. These effects prolong the duration and intensity of interspecific competition, support highly competitive Sphagnum mosses, and, as such, force niches of specialized fen species towards narrower high-pH ranges. Recent anthropogenic landscape changes pose a severe threat to many fen species and call for mitigation measures to lower competition pressure in their refugia.

Sex-specific chromatin remodelling safeguards transcription in germ cells.

Stability of the epigenetic landscape underpins maintenance of the cell-type-specific transcriptional profile. As one of the main repressive epigenetic systems, DNA methylation has been shown to be important for long-term gene silencing; its loss leads to ectopic and aberrant transcription in differentiated cells and cancer1. The developing mouse germ line endures global changes in DNA methylation in the absence of widespread transcriptional activation. Here, using an ultra-low-input native chromatin immunoprecipitation approach, we show that following DNA demethylation the gonadal primordial germ cells undergo remodelling of repressive histone modifications, resulting in a sex-specific signature in mice. We further demonstrate that Polycomb has a central role in transcriptional control in the newly hypomethylated germline genome as the genetic loss of Ezh2 leads to aberrant transcriptional activation, retrotransposon derepression and dramatic loss of developing female germ cells. This sex-specific effect of Ezh2 deletion is explained by the distinct landscape of repressive modifications observed in male and female germ cells. Overall, our study provides insight into the dynamic interplay between repressive chromatin modifications in the context of a developmental reprogramming system.

Land snail community patterns related to regional habitat conservation status of European spring fens.

Human activities have enormous impact on current biodiversity distribution across all spatial scales. Despite the numerous studies showing the difference between preserved and impaired sites, only little is known about the regional scale. Therefore, we selected four European regions differing in habitat conservation status (HCS) to explore if the variation in land snail communities reflects regional differences. We collected quantitative land snail samples at 169 isolated spring fen sites and measured environmental parameters. The species richness of habitat specialists expressed low variation and weak associations with local conditions in the two regions of adequate HCS, presumably because of their common occurrence throughout most sites. In contrast, the richness of matrix-derived species, i.e. predominantly habitat generalists, was highly variable in these two regions and also tightly associated with local conditions, especially moisture. In both the intermediate and the inadequate HCS region, these associations were much weaker as the fens are less extreme and allow for penetration of matrix-derived species. Population densities of Vertigo geyeri, an umbrella species internationally protected by the EU Habitats Directive, were highest in the two adequate HCS regions. Species composition was primarily controlled by moisture in the regions of adequate HCS, while in the remaining regions, those predictors that are less easily jeopardized by human impact, such as climate, water chemistry and terrain topography, gained importance. In the inadequate HCS region, none of the analysed predictors was associated with the main compositional gradient, suggesting a complete disruption of community-environment relationships. Our results suggest that the species richness and community responses to natural gradients might be substantially modified by human impact, although the effect of some other region-specific factors cannot be easily disentangled because of inevitably low number of studied regions.

Specification and epigenomic resetting of the pig germline exhibit conservation with the human lineage.

Investigations of the human germline and programming are challenging because of limited access to embryonic material. However, the pig as a model may provide insights into transcriptional network and epigenetic reprogramming applicable to both species. Here we show that, during the pre- and early migratory stages, pig primordial germ cells (PGCs) initiate large-scale epigenomic reprogramming, including DNA demethylation involving TET-mediated hydroxylation and, potentially, base excision repair (BER). There is also macroH2A1 depletion and increased H3K27me3 as well as X chromosome reactivation (XCR) in females. Concomitantly, there is dampening of glycolytic metabolism genes and re-expression of some pluripotency genes like those in preimplantation embryos. We identified evolutionarily young transposable elements and gene coding regions resistant to DNA demethylation in acutely hypomethylated gonadal PGCs, with potential for transgenerational epigenetic inheritance. Detailed insights into the pig germline will likely contribute significantly to advances in human germline biology, including in vitro gametogenesis.

TBPL2/TFIIA complex establishes the maternal transcriptome through oocyte-specific promoter usage.

During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.

Oral and vaginal microbiota in selected field mice of the genus Apodemus: a wild population study.

Animal-associated microbiota is expected to impose crucial effects on the host's fitness-related performance, including reproduction. Most research to date has focused on interactions between the host with its gut microbiota; however, there remain considerable gaps in knowledge regarding microbial consortia in other organs, including interspecific divergence, temporal stability, variation drivers, and their effects on the host. To fill these gaps, we examined oral and vaginal microbiota composition in four free-living mouse species of the genus Apodemus, each varying in the degree of female promiscuity. To assess temporal stability and microbiota resistance to environmental change, we exposed one of the species, Apodemus uralensis, to standardized captive conditions and analyzed longitudinal changes in its microbiota structure. Our results revealed the existence of a "core" oral microbiota that was not only shared among all four species but also persisted almost unchanged in captivity. On the other hand, vaginal microbiota appears to be more plastic in captive conditions and less species-specific in comparison with oral microbiota. This study is amongst the first to describe oral microbiota dynamics. Furthermore, the vaginal microbiota results are especially surprising in light of the well-known role of stable vaginal microbiota as a defense against pathogens. The results indicate the existence of diverse mechanisms that shape each microbiota. On the other hand, our data provides somewhat ambiguous support for the systematic effect of phylogeny and social system on both oral and vaginal microbiota structures.

Evolutionary Persistence of DNA Methylation for Millions of Years after Ancient Loss of a De Novo Methyltransferase.

Cytosine methylation of DNA is a widespread modification of DNA that plays numerous critical roles. In the yeast Cryptococcus neoformans, CG methylation occurs in transposon-rich repeats and requires the DNA methyltransferase Dnmt5. We show that Dnmt5 displays exquisite maintenance-type specificity in vitro and in vivo and utilizes similar in vivo cofactors as the metazoan maintenance methylase Dnmt1. Remarkably, phylogenetic and functional analysis revealed that the ancestral species lost the gene for a de novo methylase, DnmtX, between 50-150 mya. We examined how methylation has persisted since the ancient loss of DnmtX. Experimental and comparative studies reveal efficient replication of methylation patterns in C. neoformans, rare stochastic methylation loss and gain events, and the action of natural selection. We propose that an epigenome has been propagated for >50 million years through a process analogous to Darwinian evolution of the genome.

Habitat extremity and conservation management stabilise endangered calcareous fens in a changing world.

Calcareous fens represent an endangered type of peatlands, acting as refugia for stress-tolerant species in the currently changing landscapes. The resurveys across many regions have reported their recent disappearance or deterioration despite both the extreme habitat conditions (carbonate richness, presence of calcareous tufa, nutrient limitation, high water level) and conservation management. To test the stability of their biotic communities in different environmental and management configurations, we repeatedly sampled molluscs (terrestrial and aquatic), vascular plants, and bryophytes at 30 calcareous fens in the Inner Western Carpathians (Slovakia, Poland) after 13-17 years of warm summers and land-use changes. We found a small yet statistically significant effect of sampling period (old versus new survey) on the species composition of all three groups of organisms when the effect of various positions of sites along ecological gradients was controlled for. The compositional changes, interpreted with the help of Ellenberg Indicator Values, suggest an incipient succession towards grasslands and shrublands, driven by decreasing soil moisture and increasing nutrient availability. Although the number of habitat specialists did not change, the number of matrix-derived vascular plant and bryophyte species significantly increased, with six ubiquitous species of productive habitats being significantly more represented currently, while the richness of aquatic molluscs significantly decreased. Fens in which potentially strongly competitive plant species were less stressed because of less intense management and lower habitat extremity were more prone to such succession. There was no single factor that could predict the magnitude of composition changes; instead, tested factors were found to act synergistically. Conservation management was predominantly important for bryophytes, while extreme habitat conditions were predominantly important for terrestrial snails. We suggested a way how nature conservancy authorities can prioritise the management needs by applying an abiotic indicator system, with less environmentally extreme fens requiring more intense conservation management.

TET2 Regulates the Neuroinflammatory Response in Microglia.

Epigenomic mechanisms regulate distinct aspects of the inflammatory response in immune cells. Despite the central role for microglia in neuroinflammation and neurodegeneration, little is known about their epigenomic regulation of the inflammatory response. Here, we show that Ten-eleven translocation 2 (TET2) methylcytosine dioxygenase expression is increased in microglia upon stimulation with various inflammogens through a NF-κB-dependent pathway. We found that TET2 regulates early gene transcriptional changes, leading to early metabolic alterations, as well as a later inflammatory response independently of its enzymatic activity. We further show that TET2 regulates the proinflammatory response in microglia of mice intraperitoneally injected with LPS. We observed that microglia associated with amyloid ß plaques expressed TET2 in brain tissue from individuals with Alzheimer's disease (AD) and in 5xFAD mice. Collectively, our findings show that TET2 plays an important role in the microglial inflammatory response and suggest TET2 as a potential target to combat neurodegenerative brain disorders.

Oral vitamin C supplementation to patients with myeloid cancer on azacitidine treatment: Normalization of plasma vitamin C induces epigenetic changes.

BACKGROUND: Patients with haematological malignancies are often vitamin C deficient, and vitamin C is essential for the TET-induced conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), the first step in active DNA demethylation. Here, we investigate whether oral vitamin C supplementation can correct vitamin C deficiency and affect the 5hmC/5mC ratio in patients with myeloid cancers treated with DNA methyltransferase inhibitors (DNMTis). RESULTS: We conducted a randomized, double-blinded, placebo-controlled pilot trial (NCT02877277) in Danish patients with myeloid cancers performed during 3 cycles of DNMTi-treatment (5-azacytidine, 100 mg/m2/d for 5 days in 28-day cycles) supplemented by oral dose of 500 mg vitamin C (n = 10) or placebo (n = 10) daily during the last 2 cycles. Fourteen patients (70%) were deficient in plasma vitamin C (< 23 µM) and four of the remaining six patients were taking vitamin supplements at inclusion. Global DNA methylation was significantly higher in patients with severe vitamin C deficiency (< 11.4 µM; 4.997 vs 4.656% 5mC relative to deoxyguanosine, 95% CI [0.126, 0.556], P = 0.004). Oral supplementation restored plasma vitamin C levels to the normal range in all patients in the vitamin C arm (mean increase 34.85 ± 7.94 µM, P = 0.0004). We show for the first time that global 5hmC/5mC levels were significantly increased in mononuclear myeloid cells from patients receiving oral vitamin C compared to placebo (0.037% vs - 0.029%, 95% CI [- 0.129, - 0.003], P = 0.041). CONCLUSIONS: Normalization of plasma vitamin C by oral supplementation leads to an increase in the 5hmC/5mC ratio compared to placebo-treated patients and may enhance the biological effects of DNMTis. The clinical efficacy of oral vitamin C supplementation to DNMTis should be investigated in a large randomized, placebo-controlled clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02877277 . Registered on 9 August 2016, retrospectively registered.

Mechanistic Insights into Cytosine-N3 Methylation by DNA Methyltransferase DNMT3A.

Recently, it has been discovered that different DNA-(cytosine C5)-methyltransferases including DNMT3A generate low levels of 3mC [Rosic et al. (2018), Nat. Genet., 50, 452-459]. This reaction resulted in the co-evolution of DNMTs and ALKB2 DNA repair enzymes, but its mechanism remained elusive. Here, we investigated the catalytic mechanism of DNMT3A for cytosine N3 methylation. We generated several DNMT3A variants with mutated catalytic residues and measured their activities in 5mC and 3mC generation by liquid chromatography linked to tandem mass spectrometry. Our data suggest that the methylation of N3 instead of C5 is caused by an inverted binding of the flipped cytosine target base into the active-site pocket of the DNA methyltransferase, which is partially compatible with the arrangement of catalytic amino acid residues. Given that all DNA-(cytosine C5)-methyltransferases have a common catalytic mechanism, it is likely that other enzymes of this class generate 3mC following the same mechanism.

SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA.

Cap analysis of gene expression (CAGE) is a methodology for genome-wide quantitative mapping of mRNA 5' ends to precisely capture transcription start sites at a single nucleotide resolution. In combination with high-throughput sequencing, CAGE has revolutionized our understanding of the rules of transcription initiation, led to discovery of new core promoter sequence features, and discovered transcription initiation at enhancers genome-wide. The biggest limitation of CAGE is that even the most recently improved version (nAnT-iCAGE) still requires large amounts of total cellular RNA (5 µg), preventing its application to scarce biological samples such as those from early embryonic development or rare cell types. Here, we present SLIC-CAGE, a Super-Low Input Carrier-CAGE approach to capture 5' ends of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. This dramatic increase in sensitivity is achieved by specially designed, selectively degradable carrier RNA. We demonstrate the ability of SLIC-CAGE to generate data for genome-wide promoterome with 1000-fold less material than required by existing CAGE methods, by generating a complex, high-quality library from mouse embryonic day 11.5 primordial germ cells.

Oxidative stress in sperm affects the epigenetic reprogramming in early embryonic development.

BACKGROUND: Reactive oxygen species (ROS)-induced oxidative stress is well known to play a major role in male infertility. Sperm are sensitive to ROS damaging effects because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. However, how oxidative DNA lesions in sperm affect early embryonic development remains elusive. RESULTS: Using cattle as model, we show that fertilization using sperm exposed to oxidative stress caused a major developmental arrest at the time of embryonic genome activation. The levels of DNA damage response did not directly correlate with the degree of developmental defects. The early cellular response for DNA damage, γH2AX, is already present at high levels in zygotes that progress normally in development and did not significantly increase at the paternal genome containing oxidative DNA lesions. Moreover, XRCC1, a factor implicated in the last step of base excision repair (BER) pathway, was recruited to the damaged paternal genome, indicating that the maternal BER machinery can repair these DNA lesions induced in sperm. Remarkably, the paternal genome with oxidative DNA lesions showed an impairment of zygotic active DNA demethylation, a process that previous studies linked to BER. Quantitative immunofluorescence analysis and ultrasensitive LC-MS-based measurements revealed that oxidative DNA lesions in sperm impair active DNA demethylation at paternal pronuclei, without affecting 5-hydroxymethylcytosine (5hmC), a 5-methylcytosine modification that has been implicated in paternal active DNA demethylation in mouse zygotes. Thus, other 5hmC-independent processes are implicated in active DNA demethylation in bovine embryos. The recruitment of XRCC1 to damaged paternal pronuclei indicates that oxidative DNA lesions drive BER to repair DNA at the expense of DNA demethylation. Finally, this study highlighted striking differences in DNA methylation dynamics between bovine and mouse zygotes that will facilitate the understanding of the dynamics of DNA methylation in early development. CONCLUSIONS: The data demonstrate that oxidative stress in sperm has an impact not only on DNA integrity but also on the dynamics of epigenetic reprogramming, which may harm the paternal genetic and epigenetic contribution to the developing embryo and affect embryo development and embryo quality.