Identification of a possible role of thymine DNA glycosylase (TDG) in epigenome maintenance

Thymine DNA glycosylase (TDG) was discovered as an enzyme capable of removing uracil (U) and thymine (T) from G/U and G/T mispairs, respectively. Owing to this ability, TDG was proposed to initiate restoration of C/G pairs at sites of cytosine or 5-methycytosine (5-meC) deamination. In addition to products of base deamination, the substrate spectrum of TDG covers a wide range of DNA base damages resulting from base oxidation and alkylation. TDG was also found to engage in physical and functional interactions with transcription factors, and more recent evidence supports additional interactions with the de novo DNA methyltransferases Dnmt3a and 3b in the context of gene transcription. Together with its biochemical properties, these observations suggest that TDG might be targeted to gene regulatory sequences as part of a macromolecular assembly to control their functional integrity. TDG may counteract the mutagenic effects of C and 5-meC deamination in CG-rich regions and/or be involved in the maintenance of CpG promoter methylation patterns. A tight regulation of CpG methylation at gene regulatory regions is critical for accurate gene expression, proper cellular differentiation and embryonic development. A somewhat surprising but in this context consistent finding was that, in contrast to other DNA glycosylases, TDG is essential for proper fetal development since a targeted knockout of the gene leads to embryonic lethality. To gain insights into the biological functions of TDG, we aimed to establish and apply biochemical fractionation procedures for high affinity purification and structural and functional characterization of TDG containing proteins complexes. The first part of the thesis was concerned with biochemical characterization of the protein interaction network of TDG in living mammalian cells. To this end, I applied different approaches allowing high affinity isolation of protein complexes from mammalian cells, such as the tandem affinity purification (TAP) method as well as immunoprecipitation of endogenous protein and of the TDGa isoform from TdgA overexpressing embryonic stem (ES) cells. These efforts, however, did not reveal any TDG interacting partners in subsequent mass spectrometry (MS) analyses. These results were surprising, as TDG was previously reported to interact with transcription factors and DNA methyltransferases. Remarkably, however, all previously identified protein interactors of TDG were discovered in screen with the respective partner proteins, and under conditions of simultaneous overexpression of both interacting proteins. The only proteins ever identified in screen with TDG were Sumo1 and Sumo3, which turned out to covalently modify the glycosylase. For this reason, we decided to pursue our search with classical cell fractionation experiments. We first did gel filtration experiments from total cell lysates and showed that TDG is indeed able to form distinct multiprotein complexes in undifferentiated mouse embryonic stem cells that may also contain the RNA helicase p68. Further subcellular fractionation experiments then revealed that TDG is present in all cell compartments, with a significant fraction of nuclear TDG being associated with chromatin, together with p68 and de novo DNA methyltransferases. Together with published findings, these results suggested that protein complexes containing TDG might act in a chromatin-associated context, at gene regulatory regions. The developmental phenotype of Tdg-/- knockout mice and the interactions of TDG with factors involved in developmental gene regulation (e.g. retinoic acid receptors RAR/RXR) implicate a function of TDG during early development and cell differentiation, at times governed by dynamic changes in gene expression, DNA methylation and histone modifications. Such changes have been studied using a well-established during in vitro differentiation of ES cells to lineage committed neuronal progenitors (NPs). We thus aimed to address the function of TDG as part of chromatin associated protein complexes during the process of retinoic acid induced differentiation of ES cells to NPs. In the second part of the thesis we made use of a this well-established in vitro differentiation system to examine the genome-wide localization of TDG to chromatin by TDG chromatin immunoprecipitation (ChIP) and to correlate TDG association to chromatin with gene expression and DNA methylation changes linked to cellular differentiation. TDG ChIP combined with high throughput sequencing showed that TDG associates with high preference to CpG islands in promoters of actively transcribed genes or genes poised for transcriptional activation. Such CpG rich sequences are normally unmethylated in mammalian genomes. Interestingly, we found TDG to localize to promoters of many genes controlling pluripotency (e.g. Oct4, Nanog) and developmental processes (e.g. Sfrp2, Tgfb2, Gata6), thus, supporting a function of TDG in cell differentiation and/or embryonic development. As different lines of circumstantial evidence have associated TDG with changes in CpG methylation following activation of hormone responsive gene promoters, we went on to further test genome-wide promoter methylation in Tdg+/- and Tdg-/- NPs making use of a combination of methylated DNA immunoprecipitation (MeDIP) and microarray technology. This showed that the loss of TDG does not affect global promoter DNA methylation. Nevertheless, there were a number of significant differences, suggesting that TDG might affect the CpG methylation pattern at some promoters. Also, owing to the limited resolution of the MeDIP method, however, we could not exclude an involvement of TDG in the control of DNA methylation of specific promoter CpGs. Additional bisulfite sequencing of promoters of TDG bound developmental genes (e.g. Sfrp2, Tgfb2) in NPs and differentiated mouse embryonic fibroblasts (MEFs) have indeed proved that loss of TDG affects local changes in DNA methylation at particular CpGs. Subsequent analysis of genome-wide gene expression profiles of ES cells and differentiated Tdg+/- and Tdg-/- NPs revealed that a limited number of genes (229) are differentially regulated in ES, whereas substantial differences in gene expression in were observed in NPs (1022 genes). This implicated a specific function of TDG in the regulation of cell differentiation triggered gene expression changes. Detailed analysis of the expression of the Pax6 gene, accurate regulation of which is essential for proper neuron development, showed that its promoter is bound by TDG and that its transcription is inappropriately regulated upon further differentiation of Tdg-/- NPs into the neuronal lineage. Whereas Tdg+/- NPs efficiently downregulated Pax6 (50x) and further differentiated into neuron-like cells, Tdg-/- NPs only partially downregulated Pax6 gene expression (6x) and underwent apoptosis at day 2 after plating in neuronal medium. This phenotype was complemented by expression of TDGa, clearly implicating TDG in the regulation of Pax6 expression during differentiation of ES cells to terminal neurons. We further observed misregulation of pluripotency genes (e.g. Oct4) regulated by TDG bound promoters during early differentiation of ES cells. In the absence of TDG, ES cells showed the tendency to enter spontaneous and/or RA induced differentiation, suggesting a role for TDG in the regulation of pluripotency. During RA induced differentiation we further observed the activation of the neuron specific gene Lrrtm2 exclusively in TDG proficient cells. In addition, ChIP experiments showed that transcription factors involved in the activation of the Lrrtm2 gene (e.g. COUP-TFI, RAR) are not recruited to the respective promoter in Tdg-/- cells, suggesting that TDG might act passively as a scaffold factor important for the recruitment of transcription factors to promoter regions. I set out to clarify the biological function of TDG by investigating its molecular interactions in mammalian cells. I found that TDG, as a DNA repair enzyme, associates tightly with chromatin, where it localizes with high preference to CpG island promoters of active genes and genes poised to be expressed. I also found that the loss of TDG causes misregulation of genes during cell differentiation and that this appears to be related to a function of TDG in establishing and/or maintaining CpG methylation pattern in gene regulatory sequences. These discoveries implicate a novel function of DNA repair, in the maintenance not only of the genome, but also the epigenome

Pig farmers’ willingness to pay for management strategies to reduce aggression between pigs

When deciding whether to invest in an improvement to animal welfare, farmers must trade-off the relative costs and benefits. Despite the existence of effective solutions to many animal welfare issues, farmers’ willingness to pay for them is largely unknown. This study modelled pig farmers’ decisions to improve animal welfare using a discrete choice experiment focused on alleviating aggression between growing/finishing pigs at regrouping. Eighty-two UK and Irish pig farm owners and managers were asked to choose between hypothetical aggression control strategies described in terms of four attributes; installation cost, on-going cost, impact on skin lesions from aggression and impact on growth rate. If they did not like any of the strategies they could opt to keep their current farm practice. Systematic variations in product attributes allowed farmers’ preferences and willingness to pay to be estimated and latent class modelling accounted for heterogeneity in responses. The overall willingness to pay to reduce lesions was low at £0.06 per pig place (installation cost) and £0.01 per pig produced (running cost) for each 1% reduction in lesions. Results revealed three independent classes of farmers. Farmers in Class 1 were unlikely to regroup unfamiliar growing/finishing pigs, and thus were unwilling to adopt measures to reduce aggression at regrouping. Farmers in Classes 2 and 3 were willing to adopt measures providing certain pre-conditions were met. Farmers in Class 2 were motivated mainly by business goals, whilst farmers in Class 3 were motivated by both business and animal welfare goals, and were willing to pay the most to reduce aggression; £0.11 per pig place and £0.03 per pig produced for each 1% reduction in lesions. Farmers should not be considered a homogeneous group regarding the adoption of animal welfare innovations. Instead, campaigns should be targeted at subgroups according to their independent preferences and willingness to pay

Genome-Wide Functional Divergence after the Symbiosis of Proteobacteria with Insects Unraveled through a Novel Computational Approach

Symbiosis has been among the most important evolutionary steps to generate biological complexity. The establishment of symbiosis required an intimate metabolic link between biological systems with different complexity levels. The strict endo-cellular symbiotic bacteria of insects are beautiful examples of the metabolic coupling between organisms belonging to different kingdoms, a eukaryote and a prokaryote. The host (eukaryote) provides the endosymbiont (prokaryote) with a stable cellular environment while the endosymbiont supplements the host's diet with essential metabolites. For such communication to take place, endosymbionts' genomes have suffered dramatic modifications and reconfigurations of proteins' functions. Two of the main modifications, loss of genes redundant for endosymbiotic bacteria or the host and bacterial genome streamlining, have been extensively studied. However, no studies have accounted for possible functional shifts in the endosymbiotic proteomes. Here, we develop a simple method to screen genomes for evidence of functional divergence between two species clusters, and we apply it to identify functional shifts in the endosymbiotic proteomes. Despite the strong effects of genetic drift in the endosymbiotic systems, we unexpectedly identified genes to be under stronger selective constraints in endosymbionts of aphids and ants than in their free-living bacterial relatives. These genes are directly involved in supplementing the host's diet with essential metabolites. A test of functional divergence supports a strong relationship between the endosymbiosis and the functional shifts of proteins involved in the metabolic communication with the insect host. The correlation between functional divergence in the endosymbiotic bacterium and the ecological requirements of the host uncovers their intimate biochemical and metabolic communication and provides insights on the role of symbiosis in generating species diversity

Model Cortical Association Fields Account for the Time Course and Dependence on Target Complexity of Human Contour Perception

Can lateral connectivity in the primary visual cortex account for the time dependence and intrinsic task difficulty of human contour detection? To answer this question, we created a synthetic image set that prevents sole reliance on either low-level visual features or high-level context for the detection of target objects. Rendered images consist of smoothly varying, globally aligned contour fragments (amoebas) distributed among groups of randomly rotated fragments (clutter). The time course and accuracy of amoeba detection by humans was measured using a two-alternative forced choice protocol with self-reported confidence and variable image presentation time (20-200 ms), followed by an image mask optimized so as to interrupt visual processing. Measured psychometric functions were well fit by sigmoidal functions with exponential time constants of 30-91 ms, depending on amoeba complexity. Key aspects of the psychophysical experiments were accounted for by a computational network model, in which simulated responses across retinotopic arrays of orientation-selective elements were modulated by cortical association fields, represented as multiplicative kernels computed from the differences in pairwise edge statistics between target and distractor images. Comparing the experimental and the computational results suggests that each iteration of the lateral interactions takes at least ms of cortical processing time. Our results provide evidence that cortical association fields between orientation selective elements in early visual areas can account for important temporal and task-dependent aspects of the psychometric curves characterizing human contour perception, with the remaining discrepancies postulated to arise from the influence of higher cortical areas

Nitazoxanide Stimulates Autophagy and Inhibits mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis

Tuberculosis, caused by Mycobacterium tuberculosis infection, is a major cause of morbidity and mortality in the world today. M. tuberculosis hijacks the phagosome-lysosome trafficking pathway to escape clearance from infected macrophages. There is increasing evidence that manipulation of autophagy, a regulated catabolic trafficking pathway, can enhance killing of M. tuberculosis. Therefore, pharmacological agents that induce autophagy could be important in combating tuberculosis. We report that the antiprotozoal drug nitazoxanide and its active metabolite tizoxanide strongly stimulate autophagy and inhibit signaling by mTORC1, a major negative regulator of autophagy. Analysis of 16 nitazoxanide analogues reveals similar strict structural requirements for activity in autophagosome induction, EGFP-LC3 processing and mTORC1 inhibition. Nitazoxanide can inhibit M. tuberculosis proliferation in vitro. Here we show that it inhibits M. tuberculosis proliferation more potently in infected human THP-1 cells and peripheral monocytes. We identify the human quinone oxidoreductase NQO1 as a nitazoxanide target and propose, based on experiments with cells expressing NQO1 or not, that NQO1 inhibition is partly responsible for mTORC1 inhibition and enhanced autophagy. The dual action of nitazoxanide on both the bacterium and the host cell response to infection may lead to improved tuberculosis treatment

Famine food of vegetal origin consumed in the Netherlands during World War II

Background: Periods of extreme food shortages during war force people to eat food that they normally do not consider edible. The last time that countries in Western Europe experienced severe scarcities was during World War II. The so-called Dutch famine or Hunger Winter (1944-1945) made at least 25,000 victims. The Dutch government took action by opening soup kitchens and providing information on wild plants and other famine food sources in "wartime cookbooks." The Dutch wartime diet has never been examined from an ethnobotanical perspective. Methods: We interviewed 78 elderly Dutch citizens to verify what they remembered of the consumption of vegetal and fungal famine food during World War II by them and their close surroundings. We asked whether they experienced any adverse effects from consuming famine food plants and how they knew they were edible. We identified plant species mentioned during interviews by their local Dutch names and illustrated field guides and floras. We hypothesized that people living in rural areas consumed more wild species than urban people. A Welch t test was performed to verify whether the number of wild and cultivated species differed between urban and rural citizens. Results: A total number of 38 emergency food species (14 cultivated and 21 wild plants, three wild fungi) were mentioned during interviews. Sugar beets, tulip bulbs, and potato peels were most frequently consumed. Regularly eaten wild species were common nettle, blackberry, and beechnuts. Almost one third of our interviewees explicitly described to have experienced extreme hunger during the war. People from rural areas listed significantly more wild species than urban people. The number of cultivated species consumed by both groups was similar. Negative effects were limited to sore throats and stomachache from the consumption of sugar beets and tulip bulbs. Knowledge on the edibility of famine food was obtained largely by oral transmission; few people remembered the written recipes in wartime cookbooks. Conclusion: This research shows that 71years after the Second World War, knowledge on famine food species, once crucial for people's survival, is still present in the Dutch society. The information on famine food sources supplied by several institutions was not distributed widely. For the necessary revival of famine food knowledge during the 1940s, people needed to consult a small group of elders. Presumed toxicity was a major reason given by our participants to explain why they did not collect wild plants or mushrooms during the war

Principles of genetic circuit design

Cells navigate environments, communicate and build complex patterns by initiating gene expression in response to specific signals. Engineers seek to harness this capability to program cells to perform tasks or create chemicals and materials that match the complexity seen in nature. This Review describes new tools that aid the construction of genetic circuits. Circuit dynamics can be influenced by the choice of regulators and changed with expression 'tuning knobs'. We collate the failure modes encountered when assembling circuits, quantify their impact on performance and review mitigation efforts. Finally, we discuss the constraints that arise from circuits having to operate within a living cell. Collectively, better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792)National Institute of General Medical Sciences (U.S.) (Grant R01 GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)Life Technologies, Inc. (A114510)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant 4500000552

Sex differences in cardiovascular complications and mortality in hospital patients with covid-19: registry based observational study

Objective To assess whether the risk of cardiovascular complications of covid-19 differ between the sexes and to determine whether any sex differences in risk are reduced in individuals with pre-existing cardiovascular disease. Design Registry based observational study. Setting 74 hospitals across 13 countries (eight European) participating in CAPACITY-COVID (Cardiac complicAtions in Patients With SARS Corona vIrus 2 regisTrY), from March 2020 to May 2021 Participants All adults (aged ≥18 years), predominantly European, admitted to hospital with highly suspected covid-19 disease or covid-19 disease confirmed by positive laboratory test results (n=11 167 patients). Main outcome measures Any cardiovascular complication during admission to hospital. Secondary outcomes were in-hospital mortality and individual cardiovascular complications with ≥20 events for each sex. Logistic regression was used to examine sex differences in the risk of cardiovascular outcomes, overall and grouped by pre-existing cardiovascular disease. Results Of 11 167 adults (median age 68 years, 40% female participants) included, 3423 (36% of whom were female participants) had pre-existing cardiovascular disease. In both sexes, the most common cardiovascular complications were supraventricular tachycardias (4% of female participants, 6% of male participants), pulmonary embolism (3% and 5%), and heart failure (decompensated or de novo) (2% in both sexes). After adjusting for age, ethnic group, pre-existing cardiovascular disease, and risk factors for cardiovascular disease, female individuals were less likely than male individuals to have a cardiovascular complication (odds ratio 0.72, 95% confidence interval 0.64 to 0.80) or die (0.65, 0.59 to 0.72). Differences between the sexes were not modified by pre-existing cardiovascular disease; for the primary outcome, the female-to-male ratio of the odds ratio in those without, compared with those with, pre-existing cardiovascular disease was 0.84 (0.67 to 1.07). Conclusions In patients admitted to hospital for covid-19, female participants were less likely than male participants to have a cardiovascular complication. The differences between the sexes could not be attributed to the lower prevalence of pre-existing cardiovascular disease in female individuals. The reasons for this advantage in female individuals requires further research

Emerging roles of ATF2 and the dynamic AP1 network in cancer

Cooperation among transcription factors is central for their ability to execute specific transcriptional programmes. The AP1 complex exemplifies a network of transcription factors that function in unison under normal circumstances and during the course of tumour development and progression. This Perspective summarizes our current understanding of the changes in members of the AP1 complex and the role of ATF2 as part of this complex in tumorigenesis.Fil: Lopez Bergami, Pablo Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; ArgentinaFil: Lau, Eric . Burnham Institute for Medical Research; Estados UnidosFil: Ronai, Zeev . Burnham Institute for Medical Research; Estados Unido