Longitudinal,genome-scale analysis of DNA methylation in twins from birth to 18 months of age reveals rapid epigenetic change in early life and pair-specific effects of discordance

Background

The extent to which development- and age-associated epigenetic changes are influenced by genetic, environmental and stochastic factors remains to be discovered. Twins provide an ideal model with which to investigate these influences but previous cross-sectional twin studies provide contradictory evidence of within-pair epigenetic drift over time. Longitudinal twin studies can potentially address this discrepancy.

Results

In a pilot, genome-scale study of DNA from buccal epithelium, a relatively homogeneous tissue, we show that one-third of the CpGs assayed show dynamic methylation between birth and 18 months. Although all classes of annotated genomic regions assessed show an increase in DNA methylation over time, probes located in intragenic regions, enhancers and low-density CpG promoters are significantly over-represented, while CpG islands and high-CpG density promoters are depleted among the most dynamic probes. Comparison of co-twins demonstrated that within-pair drift in DNA methylation in our cohort is specific to a subset of pairs, who show more differences at 18 months. The rest of the pairs show either minimal change in methylation discordance, or more similar, converging methylation profiles at 18 months. As with age-associated regions, sites that change in their level of within-pair discordance between birth and 18 months are enriched in genes involved in development, but the average magnitude of change is smaller than for longitudinal change.

Conclusions

Our findings suggest that DNA methylation in buccal epithelium is influenced by non-shared stochastic and environmental factors that could reflect a degree of epigenetic plasticity within an otherwise constrained developmental program.     

Contrasting evolutionary histories in Neotropical birds: Divergence across an environmental barrier in South America

Avian diversity in the Neotropics has been traditionally attributed to the effect of vicariant forces promoting speciation in allopatry. Recent studies have shown that phylogeographical patterns shared among codistributed species cannot be explained by a single vicariant event, as species responses to a common barrier depend on the biological attributes of each taxon. The open vegetation corridor (OVC) isolates Amazonia and the Andean forests from the Atlantic Forest, creating a notorious pattern of avian taxa that are disjunctly codistributed in these forests. Here, we studied and compared the evolutionary histories of Ramphotrigon megacephalum and Pipraeidea melanonota, two passerines with allopatric populations east and west of the OVC that represent different subspecies. These species differ in their biological attributes: R. megacephalum is a sedentary, forest specialist mostly confined to bamboo understorey, whereas P. melanonota is a seasonal migrant and generalist species that ranges in a variety of closed and semi‐open environments. We performed genetic and genomic analyses, complemented with the study of coloration and behavioural differentiation, to assess population divergence across the OVC. We found that the evolutionary histories of both R. megacephalum and P. melanonota have been shaped by this environmental barrier. However, these species responded in different and asynchronous manners to the establishment of the OVC and to past connections between the currently isolated South American forests, which can be mostly explained by their distinct ecologies and dispersal abilities. Our results support the fact that the biological attributes of species can make their evolutionary histories idiosyncratic.     

Autophagy in hematopoietic stem/progenitor cells exposed to heavy metals: Biological implications and toxicological relevance

The inherent toxicity of many metal compounds, together with their widespread environmental distribution, raises concerns of potential health hazards. Little is known about the impact of these important environmental toxicants on adult stem/progenitor cells, necessary for tissue homeostasis and repair. We recently reported that autophagy is implicated in the response of hematopoietic stem/progenitor cells to toxic concentrations of hexavalent chromium (Cr[VI]) and cadmium (Cd), two well known carcinogenic heavy metal cations. Autophagy may lead to cell death if carried out too extensively, but also acts as a survival pathway in cells under stress. In stem/progenitor cells, an autophagic phenotype could mitigate metal-induced toxicity, contributing to the conservation of tissue renewal capability. Given the key role of toxic damage to adult stem/progenitor cells in cancer, it is necessary to investigate whether autophagic responses modulate the carcinogenic potential of exposure to heavy metals during stem/progenitor cell differentiation.     

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation

1. Download : Download high-res image (198KB)
2. Download : Download full-size image

     

Evolution of a carbohydrate binding module into a protein-specific binder

A carbohydrate binding module, CBM4-2, derived from the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures.     

Optimization of prednisolone-loaded long-circulating liposomes via application of Quality by Design (QbD) approach

Quality by design principles (QbD) were used to assist the formulation of prednisolone-loaded long-circulating liposomes (LCL-PLP) in order to gain a more comprehensive understanding of the preparation process. This approach enables us to improve the final product quality in terms of liposomal drug concentration, encapsulation efficiency and size, and to minimize preparation variability. A 19-run D-optimal experimental design was used to study the impact of the highest risk factors on PLP liposomal concentration (Y₁- μg/ml), encapsulation efficiency (Y₂-%) and size (Y₃-nm). Out of six investigated factors, four of them were identified as critical parameters affecting the studied responses. PLP molar concentration and the molar ratio of DPPC to MPEG-2000-DSPE had a positive impact on both Y₁ and Y₂, while the rotation speed at the formation of the lipid film had a negative impact. Y₃ was highly influenced by prednisolone molar concentration and extrusion temperature. The accuracy and robustness of the model was further on confirmed. The developed model was used to optimize the formulation of LCL-PLP for efficient accumulation of the drug to tumor tissue. The cytotoxicity of the optimized LCL-PLP on C26 murine colon carcinoma cells was assessed. LCL-PLP exerted significant anti-angiogenic and anti-inflammatory effects on M2 macrophages, affecting indirectly the C26 colon carcinoma cell proliferation and development.     

Calibrating the molecular clock beyond cytochrome b: assessing the evolutionary rate of COI in birds

Estimating the age of species or their component lineages based on sequence data is crucial for many studies in avian evolutionary biology. Although calibrations of the molecular clock in birds have been performed almost exclusively using cytochrome b (cyt b), they are commonly extrapolated to other mitochondrial genes. The existence of a large, standardized cytochrome c oxidase subunit I (COI) library generated as a result of the DNA barcoding initiative provides the opportunity to obtain a calibration for this mitochondrial gene in birds. In this study we compare the evolutionary rate of COI relative to cyt b across ten different avian orders. We obtained divergence estimates for both genes from nearly 300 phylogenetically independent pairs of species through the analysis of almost 5000 public sequences. For each pair of species we calculated the difference in divergence between COI and cyt b. Our results indicate that COI evolves on average 14% slower than cyt b, but also reveal considerable variation both among and within avian orders, precluding the use of this value as a standard adjustment for the COI molecular clock for birds. Our findings suggest that this variation is partially explained by a clear negative relationship between the difference in divergence in these genes and the age of species. Distances for cyt b are higher than those for COI for closely related species, but the values become similar as the divergence between the species increases. This appears to be the result of a stronger pattern of negative time‐dependency in the rate of cyt b than in that of COI, a difference that could be related to lower functional constraints on a small number of sites in cyt b that allow it to initially accumulate mutations more rapidly than COI.     

The nuclear protein HMGB1 is secreted by monocytes via a non-classical,vesicle-mediated secretory pathway

HMGB1, a non-histone nuclear factor, acts extracellularly as a mediator of delayed endotoxin lethality, which raises the question of how a nuclear protein can reach the extracellular space. We show that activation of monocytes results in the redistribution of HMGB1 from the nucleus to cytoplasmic organelles, which display ultrastructural features of endolysosomes. HMGB1 secretion is induced by stimuli triggering lysosome exocytosis. The early mediator of inflammation interleukin (IL)-1beta is also secreted by monocytes through a non-classical pathway involving exocytosis of secretory lysosomes. However, in keeping with their respective role of early and late inflammatory factors, IL-1beta and HMGB1 respond at different times to different stimuli: IL-1beta secretion is induced earlier by ATP, autocrinally released by monocytes soon after activation; HMGB1 secretion is triggered by lysophosphatidylcholine, generated later in the inflammation site. Thus, in monocytes, non-classical secretion can occur through vescicle compartments that are at least partially distinct.     

Synthesis and properties of triplex-forming oligonucleotides containing 2′-O-(2-methoxyethyl)-5-(3-aminoprop-1-ynyl)-uridine

2′-O-(2-Methoxyethyl)-5-(3-aminoprop-1-ynyl)-uridine phosphoramidite (MEPU) has been synthesized from d-ribose and 5-iodouracil and incorporated into triplex-forming oligonucleotides (TFOs) by automated solid-phase oligonucleotide synthesis. The TFOs gave very high triplex stability with their target duplexes as measured by ultraviolet/fluorescence melting and DNase I footprinting. The incorporation of MEPU into TFOs renders them resistant to degradation by serum nucleases.