Transcriptional regulation of the ABCC6 gene and the background of impaired function of missense disease-causing mutations.

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein expressed primarily in the liver and to a lesser extent in the kidneys and the intestines. We review here the mechanisms of this restricted tissue-specific expression and the role of hepatocyte nuclear factor 4alpha which is responsible for the expression pattern. Detailed analyses uncovered further regulators of the expression of the gene pointing to an intronic primate-specific regulator region, an activator of the expression of the gene by binding CCAAT/enhancer-binding protein beta, which interacts with other proteins acting in the proximal promoter. This regulatory network is affected by various environmental stimuli including oxidative stress and the extracellular signal-regulated protein kinases 1 and 2 pathway. We also review here the structural and functional consequences of disease-causing missense mutations of ABCC6. A significant clustering of the missense disease-causing mutations was found at the domain-domain interfaces. This clustering means that the domain contacts are much less permissive to amino acid replacements than the rest of the protein. We summarize the experimental methods resulting in the identification of mutants with preserved transport activity but failure in intracellular targeting. These mutants are candidates for functional rescue by chemical chaperons. The results of such research can provide the basis of future allele-specific therapy of ABCC6-mediated disorders like pseudoxanthoma elasticum or the generalized arterial calcification in infancy

Inhibition of DNA methyltransferase leads to increased genomic 5-hydroxymethylcytosine levels in hematopoietic cells.

5-Hydroxymethylcytosine (5hmC) is produced from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC-MS/MS-based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5-aza-2\u27-deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5-aza-2\u27-deoxycytidine without significantly influencing 5mC levels

Altitude and life-history shape the evolution of Heliconius wings.

Phenotypic divergence between closely related species has long interested biologists. Taxa that inhabit a range of environments and have diverse natural histories can help understand how selection drives phenotypic divergence. In butterflies, wing color patterns have been extensively studied but diversity in wing shape and size is less well understood. Here, we assess the relative importance of phylogenetic relatedness, natural history, and habitat on shaping wing morphology in a large dataset of over 3500 individuals, representing 13 Heliconius species from across the Neotropics. We find that both larval and adult behavioral ecology correlate with patterns of wing sexual dimorphism and adult size. Species with solitary larvae have larger adult males, in contrast to gregarious Heliconius species, and indeed most Lepidoptera, where females are larger. Species in the pupal-mating clade are smaller than those in the adult-mating clade. Interestingly, we find that high-altitude species tend to have rounder wings and, in one of the two major Heliconius clades, are also bigger than their lowland relatives. Furthermore, within two widespread species, we find that high-altitude populations also have rounder wings. Thus, we reveal novel adaptive wing morphological divergence among Heliconius species beyond that imposed by natural selection on aposematic wing coloration

Evolutionary and ecological processes influencing chemical defense variation in an aposematic and mimetic Heliconius butterfly

Chemical defences against predators underlie the evolution of aposematic coloration and mimicry, which are classic examples of adaptive evolution. Surprisingly little is known about the roles of ecological and evolutionary processes maintaining defence variation, and how they may feedback to shape the evolutionary dynamics of species. Cyanogenic Heliconius butterflies exhibit diverse warning color patterns and mimicry, thus providing a useful framework for investigating these questions. We studied intraspecific variation in de novo biosynthesized cyanogenic toxicity and its potential ecological and evolutionary sources in wild populations of Heliconius erato along environmental gradients, in common-garden broods and with feeding treatments. Our results demonstrate substantial intraspecific variation, including detectable variation among broods reared in a common garden. The latter estimate suggests considerable evolutionary potential in this trait, although predicting the response to selection is likely complicated due to the observed skewed distribution of toxicity values and the signatures of maternal contributions to the inheritance of toxicity. Larval diet contributed little to toxicity variation. Furthermore, toxicity profiles were similar along steep rainfall and altitudinal gradients, providing little evidence for these factors explaining variation in biosynthesized toxicity in natural populations. In contrast, there were striking differences in the chemical profiles of H. erato from geographically distant populations, implying potential local adaptation in the acquisition mechanisms and levels of defensive compounds. The results highlight the extensive variation and potential for adaptive evolution in defense traits for aposematic and mimetic species, which may contribute to the high diversity often found in these systems.Peer reviewe

Common Practice Solvent Extraction Does not Reflect Actual Emission of a Sex Pheromone During Butterfly Courtship

Olfactory communication can be of critical importance for mate choice decisions. Lepidoptera are key model systems for understanding olfactory communication, particularly considering sex pheromone signaling in the context of sexual selection. Solvent extraction or rinsing of pheromone-producing structures is a widespread method for quantifying sex pheromones, but such measures reflect what is stored and may not represent what is actually emitted by an individual during courtship. Here, we address this point for the first time by quantifying the components of the male sex pheromone (MSP) of interacting Bicyclus anynana butterflies, a species for which much information is available onthe role played by MSPs in affecting mating success. Using headspace sampling during courtship and solvent extraction after completion of experiments using the same males, we were able to track individual traits. Our results show that solvent extracts do not reflect quantities of MSP components emitted by live butterflies. We further show that MSP amounts obtained using headspace sampling correlated with male mating success, but solvent extracts did not. Our results further strongly suggest that males actively control MSP emission when faced with increased male-male competition. Common practice solvent extracts may thus not serve as an adequate proxy for male sex pheromone signaling as they are perceived by choosy females. Our study serves as a proof of principle that quantification of male sex pheromone components depends on the method of collection, which could apply to many other insects using short-range chemical signals. This affects our understanding of how sexual selection shapes the evolution of sexually-selected chemical traits

Genomics of altitude-associated wing shape in two tropical butterflies.

Understanding how organisms adapt to their local environment is central to evolution. With new whole-genome sequencing technologies and the explosion of data, deciphering the genomic basis of complex traits that are ecologically relevant is becoming increasingly feasible. Here, we studied the genomic basis of wing shape in two Neotropical butterflies that inhabit large geographical ranges. Heliconius butterflies at high elevations have been shown to generally have rounder wings than those in the lowlands. We reared over 1,100 butterflies from 71 broods of H. erato and H. melpomene in common-garden conditions and showed that wing aspect ratio, that is, elongatedness, is highly heritable in both species and that elevation-associated wing aspect ratio differences are maintained. Genome-wide associations with a published data set of 666 whole genomes from across a hybrid zone, uncovered a highly polygenic basis to wing aspect ratio variation in the wild. We identified several genes that have roles in wing morphogenesis or wing aspect ratio variation in Drosophila flies, making them promising candidates for future studies. There was little evidence for molecular parallelism in the two species, with only one shared candidate gene, nor for a role of the four known colour pattern loci, except for optix in H. erato. Thus, we present the first insights into the heritability and genomic basis of within-species wing aspect ratio in two Heliconius species, adding to a growing body of evidence that polygenic adaptation may underlie many ecologically relevant traits

The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2

Hepatocyte nuclear factor 4 alpha (HNF4alpha) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4alpha is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4alpha. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4alpha. However, based on our previous results we hypothesized that HNF4alpha is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4alpha in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4alpha leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4alpha. Accordingly, we have observed decreasing but not disappearing binding of HNF4alpha to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4alpha chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4alpha-dependent hepatic gene expression

Impronta genómica del gen Peg1 en el roedor tetraploide Tympanoctpmys barrerae (Rodentia, Octodontidae)

La impronta génica es un fenómeno que se caracteriza por la expresión monoalélica de un gen en forma dependiente del origen parental del alelo. Los genes que poseen impronta están involucrados en procesos como crecimiento prenatal, formación de la placenta, diferenciación de linajes celulares, y establecimiento de la conducta postnatal. En ratones, el gen Peg1 (o Mest) es de copia única; está ubicado en el cromosoma 6 y posee impronta genómica, expresándose desde el alelo paterno. Mediante análisis de las secuencias nucleotídicas de Peg1 se indagó su expresión en Tympanoctomys barrerae, un roedor con duplicación genómica total. Para ello, se extrajo RNA total de cuatro embriones y se amplificó un fragmento del gen, que fue posteriormente clonado y secuenciado. Al clonar esta región a partir de amplificación de DNA genómico, usando dos de los embriones y su madre, se pudo caracterizar hasta tres alelos por individuo. Sólo uno de estos alelos se expresa en los embriones, lo que demuestra impronta genómica en T. barrerae. Además, se caracterizaron cuatro loci para retropseudogenes en esta especie. Dos de ellos se expresan en los embriones, a pesar de poseer corrimiento en el marco de lectura y codones de término que imposibilitan a estos transcritos para codificar una proteína funcional. Al estudiar la estructura del fragmento genómico obtenido se encontró también un sitio de splicing alternativo, que da origen a una secuencia no funcional de cDNA.Genomic imprinting is the monoallelic expression of genes in a parent-dependent manner. Imprinted genes are implicated in fetal growth, function of the placenta, development of particular cell lineages and postnatal behavior. In mice, Peg1 (also called Mest) is an imprinted, single copy gene, located on chromosome 6, and it is expressed from the paternal allele. Using nucleotide sequence analysis Peg1 gene expression was studied in the rodent Tympanoctomys barrerae, a mammal with whole genome duplication. We purified total RNA from four embryos, and performed RT-PCR in order to amplify a Peg1 fragment, which was cloned and sequenced. When the same fragment was cloned from genomic DNA amplification of two embryos and their mother, three different alleles were characterized. Expression was confirmed for only one of them, indicating genomic imprinting for Peg1. Four retropseudogenes loci were also characterized in this species, two of which are expressed in the embryos, despite they presented frameshifts and stop codons, which make impossible for those transcripts to produce a functional protein. Finally, by analyzing the structure of this genomic region, an alternative splicing site was found, which gave origin to one of the non functional cDNA sequences

A universal dynamical metabolic model representing mixotrophic growth of Chlorella sp. on wastes

International audienceAn emerging idea is to couple wastewater treatment and biofuel production using microalgae to achieve higher productivities and lower costs. This paper proposes a metabolic modeling of Chlorella sp. growing on fermentation wastes (blend of acetate, butyrate and other acids) in mixotrophic conditions, accounting also for the possible inhibitory substrates. This model extends previous works by modifying the metabolic network to include the consumption of glycerol and glucose by Chlorella sp., with the goal to test the addition of these substrates in order to overcome butyrate inhibition. The metabolic model was built using the DRUM framework and consists of 188 reactions and 173 metabolites. After a calibration phase, the model was successfully challenged with data from 122 experiments collected from scientific literature in autotrophic, heterotrophic and mixotrophic conditions. The optimal feeding strategy estimated with the model reduces the time to consume the volatile fatty acids from 16 days to 2 days. The high prediction capability of this model opens new routes for enhancing design and operation in waste valorization using microalgae