A major challenge in biology is to understand how complex gene expression patterns are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional ...silencers have been identified, and they remain poorly understood. Here, we used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Almost all of the transcriptional silencers that we identified were also active enhancers in other cellular contexts. These elements are bound by more transcription factors than non-silencers. A subset of these silencers forms long-range contacts with promoters. Deletion of a silencer caused derepression of its target gene. Our results challenge the common practice of treating enhancers and silencers as separate classes of regulatory elements and suggest the possibility that thousands or more bifunctional CRMs remain to be discovered in Drosophila and 104–105 in humans.
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•Silencers are bifunctional and can act as enhancers in other cellular contexts•A subset of silencers forms long-range contacts to promoters•Deletion of a silencer by genome editing caused derepression of its target gene•Results suggest that thousands of bifunctional elements in flies remain to be discovered
Gisselbrecht et al. performed a screen in developing Drosophila embryos for genomic sequences that can act as transcriptional silencers. They report that nearly all silencers are enhancers in other tissues or at other developmental stages. Their silencers fall into two classes, one of which forms physical chromosomal contacts with promoters.
How disease-associated mutations impair protein activities in the context of biological networks remains mostly undetermined. Although a few renowned alleles are well characterized, functional ...information is missing for over 100,000 disease-associated variants. Here we functionally profile several thousand missense mutations across a spectrum of Mendelian disorders using various interaction assays. The majority of disease-associated alleles exhibit wild-type chaperone binding profiles, suggesting they preserve protein folding or stability. While common variants from healthy individuals rarely affect interactions, two-thirds of disease-associated alleles perturb protein-protein interactions, with half corresponding to "edgetic" alleles affecting only a subset of interactions while leaving most other interactions unperturbed. With transcription factors, many alleles that leave protein-protein interactions intact affect DNA binding. Different mutations in the same gene leading to different interaction profiles often result in distinct disease phenotypes. Thus disease-associated alleles that perturb distinct protein activities rather than grossly affecting folding and stability are relatively widespread.
Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these regulatory elements in development, responses to environmental ...stresses and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here we describe 'enhancer-FACS-seq' (eFS) for highly parallel identification of active, tissue-specific enhancers in Drosophila melanogaster embryos. Analysis of enhancers identified by eFS as being active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, previously uncharacterized mesodermal transcription factors. Naive Bayes classifiers using transcription factor binding site motifs accurately predicted mesodermal enhancer activity. Application of eFS to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded in them.
Spider venoms represent vast sources of bioactive molecules whose diversity remains largely unknown. Indeed, only a small subset of species have been studied out of the ~43,000 extant spider species. ...The present study investigated inter- and intra-species venom complexity in 18 samples collected from a variety of lethal Australian funnel-web spiders (Mygalomorphae: Hexathelidae: Atracinae) using C4 reversed-phase separation coupled to offline MALDI-TOF mass spectrometry (LC-MALDI-TOF MS). An in-depth investigation focusing on four atracine venoms (male Illawarra wisharti, male and female Hadronyche cerberea, and female Hadronyche infensa Toowoomba) revealed, on average, ~800 peptides in female venoms while male venoms contained ~400 peptides, distributed across most HPLC fractions. This is significantly higher than previous estimates of peptide expression in mygalomorph venoms. These venoms also showed distinct intersexual as well as intra- and inter-species variation in peptide masses. Construction of both 3D and 2D contour plots revealed that peptide mass distributions in all 18 venoms were centered around the 3200–5400m/z range and to a lesser extent the 6600–8200m/z range, consistent with previously described hexatoxins. These findings highlight the extensive diversity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules.
In the present study we describe the complexity of 18 venoms from lethal Australian funnel-web spiders using LC-MALDI-TOF MS. The study includes an in-depth investigation, focusing on four venoms, that revealed the presence of ~800 peptides in female venoms and ~400 peptides in male venoms. This is significantly higher than previous estimates of peptide expression in spider venoms. By constructing both 3D and 2D contour plots we were also able to reveal the distinct intersexual as well as intra- and inter-species variation in venom peptide masses. We show that peptide mass distributions in all 18 venoms were centered around the 3200–5400 m/z range and to a lesser extent the 6600–8200 m/z range, consistent with the small number of previously described hexatoxins from these spiders. These findings highlight the extensive diversity of peptide toxins in Australian funnel-web spider venoms that that can be exploited as novel therapeutic and biopesticide lead molecules. The present study has greatly expanded our understanding of peptide variety and complexity in these lethal mygalomorph spiders. Specifically it highlights both the utility of LC-MALDI-TOF in spider taxonomy and the massive combinatorial peptide libraries that spider venoms offer the pharmaceutical and agrochemical industry.
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► LC-MALDI-TOF MS was used to determine venom complexity in funnel-web spiders. ► On average there were 800 and 400 peptides in female and male venoms, respectively. ► Venoms showed intersexual, intra- and inter-species variation in peptide masses. ► All peptides were centered around the 3200–5400 and 6600–8200m/z ranges. ► Vast peptide diversity can be exploited for novel therapeutics and biopesticides.
Un des enjeux majeurs de la biologie moderne est de comprendre les mécanismes complexes régissant l’expression de gènes d’un organisme en développement. Alors que les activateurs (enhancers) ont été ...abondamment étudiés et analysés, seul un relatif petit nombre de répresseurs (silencers) a été identifié à ce jour et restent jusqu’à présent assez mal compris. Un nombre non négligeable de CRMs jouent par ailleurs un double rôle à la fois d’amplificateurs et d’inactivateurs de transcription en fonction de l’état ou du type cellulaire dans lequel ils se trouvent, rajoutant un niveau supplémentaire de à la régulation génique dans différents types cellulaires et tissus. De façon surprenante, nous avons découvert que tous les éléments ayant une activité de répression transcriptionnelle que nous avons identifiés, s’avèrent aussi avoir une activité d’activation transcriptionnelle dans d’autres contextes cellulaires. Nos résultats remettent donc en question le paradigme de deux catégories distinctes de CRMs et suggèrent que des milliers, ou plus, d’éléments bifonctionnels restent à être découverts chez la Drosophile et potentiellement 104-105 chez l’humain. Le référencement et la caractérisation de ces éléments devraient s’avérer utiles, si ce n’est cruciaux, afin de comprendre la façon par laquelle ces motifs d’expression sont encodés au sein des génomes d'organismes métazoaires et donc éventuellement chez l’Homme.
A major challenge in biology is to understand how complex gene expression patterns in organismal development are encoded in the genome. While transcriptional enhancers have been studied extensively, few transcriptional silencers have been identified and they remain poorly understood. Here we used a novel strategy to screen hundreds of sequences for tissue-specific silencer activity in whole Drosophila embryos. Strikingly, 100% of the tested elements that we found to act as transcriptional silencers were also active enhancers in other cellular contexts. These elements were enriched in highly occupied target (HOT) region overlap (Roy et al., 2010) and specific transcription factor (TF) motif combinations. CRM bifunctionality complicates the understanding of how gene regulation is specified in the genome and how it is read out differently in different cell types. Our results challenge the common practice of treating elements with enhancer activity identified in one cell type as serving exclusively activating roles in the organism and suggest that thousands or more bifunctional CRMs remain to be discovered in Drosophila and perhaps 104-105 in human (Heintzman et al., 2009). Characterization of bifunctional elements should aid in investigations of how precise gene expression patterns are encoded in the genome.
The aim of this paper was to evaluate the effects of the inclusion of essential oils and live yeasts (Saccharomyces cerevisiae; SC) compound on fecal parameters, apparent digestibility of nutrients ...and blood parameters of horses. Eight geldings Mini-Horse breed (age 48 ± 6 months and body weight 147 ± 15kg) were used, randomly distributed in two Latin squares (2 × 2). The individual dry matter intake adopted was 1.75% of body weight. The experimental diet (concentrate: hay as 60:40) was divided into Control group (CO – without additives), live yeast S. cerevisiae group (SC – 2g/day addition), Essential Oils group (EO – 150mg/day addition) and Live Yeast + Essential Oils group (LE – both additives). Four periods of 23 days each, being 15 days of adaptation to the diet, 5 days of total collection of feces, and 3 days of washout were performed. Variance and orthogonal contrasts (CO vs others; SC and EO vs LE; EO vs SC) analysis using p < 0.05 as a reference for significant values. The apparent digestibility of nutrients was evaluated through the total collection of feces of each animal by calculating the relationship between the ingested and the excreted nutrients. We were calculated the contents of dry matter (DM), organic matter (OM), crude protein (CP), ethereal extract (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), and starch. Lipids (triglycerides (TG), total cholesterol (TC) and fractions of high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL)) were evaluated into serum, which was obtained during the total collection of feces period. The glycemic and insulinemic responses were evaluated based on the area under the curve of glucose (AUC-G) and insulin (AUC-I). The microbial population (Fibrobacter succinogenes, Ruminococcus flavefaciens and Lactobacillus genus) and pH were evaluated in feces. The treatments did not influence (p > 0.05) the apparent digestibility coefficients of DM, OM, PB, EE, NDF, ADF and starch. No differences (p > 0.05) in blood parameters CT, TG, HDL, LDL, VLDL, AUC-G and AUC-I were observed. There was no effect (p > 0.05) of treatments, time and treatment*time interaction in fecal pH of horses. No differences in the relative population of microorganisms (p > 0.05) between treatments were observed. The use of essential oils and live yeasts (CNCM I-1077), allied or not, in equine diets does not alter blood, fecal and digestible parameters in horses.
•The contrasts did not indicate a synergistic effect between treatments.•Additives do not interfere in fecal, blood and digestible parameters of horses.•The addition of essential oils and yeast does not alter the microbial population.•The additives did not change the apparent digestibility coefficients of the nutrients.
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