Description: Epithelial Na + channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na + influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ~20 Å above the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed—in contrast to overexpression of wild type—a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.—Roy, S., Boiteux, C., Alijevic, O., Liang, C., Bernèche, S., Kellenberger, S. Molecular determinants of desensitization in an ENaC/degenerin channel.

Description: The vitamin E family includes both tocopherols and tocotrienols, where α -tocopherol ( α TOC) is the most bioavailable form. Clinical trials testing the therapeutic efficacy of high-dose α TOC against stroke have largely failed or reported negative outcomes when a "more is better" approach to supplementation (〉400 IU/d) was used. This work addresses mechanisms by which supraphysiologic α TOC may contribute to stroke-induced brain injury. Ischemic stroke injury and the neuroinflammatory response were studied in tocopherol transfer protein-deficient mice maintained on a diet containing α TOC vitamin E at the equivalent human dose of 1680 IU/d. Ischemic stroke-induced brain injury was exacerbated in the presence of supraphysiologic brain α TOC levels. At 48 h after stroke, S100B and RAGE expression was increased in stroke-affected cortex of mice with elevated brain α TOC levels. Such increases were concomitant with aggravated microglial activation and neuroinflammatory signaling. A poststroke increase in markers of oxidative injury and neurodegeneration in the presence of elevated brain α TOC establish that at supraphysiologic levels, α TOC potentiates neuroinflammatory responses to acute ischemic stroke. Exacerbation of microglial activation by excessive α TOC likely depends on its unique cell signaling regulatory properties independent of antioxidant function. Against the background of clinical failure for high-dose α TOC, outcomes of this work identify risk for exacerbating stroke-induced brain injury as a result of supplementing diet with excessive levels of α TOC.—Khanna, S., Heigel,M., Weist, J., Gnyawali, S., Teplitsky, S., Roy, S., Sen, C. K., Rink, C. Excessive α -tocopherol exacerbates microglial activation and brain injury caused by acute ischemic stroke.

Description: Changes in chromatin state play important roles in cell fate transitions. Current computational approaches to analyze chromatin modifications across multiple cell types do not model how the cell types are related on a lineage or over time . To overcome this limitation, we developed a method called Chromatin Module INference on Trees (CMINT), a probabilistic clustering approach to systematically capture chromatin state dynamics across multiple cell types. Compared to existing approaches, CMINT can handle complex lineage topologies, capture higher quality clusters, and reliably detect chromatin transitions between cell types. We applied CMINT to gain novel insights in two complex processes: reprogramming to induced pluripotent stem cells (iPSCs) and hematopoiesis. In reprogramming, chromatin changes could occur without large gene expression changes, different combinations of activating marks were associated with specific reprogramming factors, there was an order of acquisition of chromatin marks at pluripotency loci, and multivalent states (comprising previously undetermined combinations of activating and repressive histone modifications) were enriched for CTCF. In the hematopoietic system, we defined critical decision points in the lineage tree, identified regulatory elements that were enriched in cell-type–specific regions, and found that the underlying chromatin state was achieved by specific erasure of preexisting chromatin marks in the precursor cell or by de novo assembly. Our method provides a systematic approach to model the dynamics of chromatin state to provide novel insights into the relationships among cell types in diverse cell-fate specification processes.

Description: Comparative functional genomics studies the evolution of biological processes by analyzing functional data, such as gene expression profiles, across species. A major challenge is to compare profiles collected in a complex phylogeny. Here, we present Arboretum, a novel scalable computational algorithm that integrates expression data from multiple species with species and gene phylogenies to infer modules of coexpressed genes in extant species and their evolutionary histories. We also develop new, generally applicable measures of conservation and divergence in gene regulatory modules to assess the impact of changes in gene content and expression on module evolution. We used Arboretum to study the evolution of the transcriptional response to heat shock in eight species of Ascomycota fungi and to reconstruct modules of the ancestral environmental stress response (ESR). We found substantial conservation in the stress response across species and in the reconstructed components of the ancestral ESR modules. The greatest divergence was in the most induced stress, primarily through module expansion. The divergence of the heat stress response exceeds that observed in the response to glucose depletion in the same species. Arboretum and its associated analyses provide a comprehensive framework to systematically study regulatory evolution of condition-specific responses.

Description: Black-pearl (Blp) is a highly conserved, essential inner-mitochondrial membrane protein. The yeast Blp homologue, Magmas/Pam16, is required for mitochondrial protein transport, growth, and survival. Our purpose was to determine the role of Drosophila Blp in mitochondrial function, cell survival, and proliferation. To this end, we performed mitotic recombination in Drosophila melanogaster , RNAi-mediated knockdown, MitoTracker staining, measurement of reactive oxygen species (ROS), flow cytometry, electron transport chain complex assays, and hemocyte isolation from Drosophila larvae. Proliferation-defective, Blp-deficient Drosophila Schneider cells exhibited mitochondrial membrane depolarization, a 60% decrease in ATP levels, increased amounts of ROS (3.5-fold), cell cycle arrest, and activation of autophagy that were associated with a selective 65% reduction of cytochrome c oxidase activity. N -acetyl cysteine (NAC) rescued Blp-RNAi-treated cells from cell cycle arrest, indicating that increased production of ROS is the primary cause of the proliferation and survival defects in Blp-depleted cells. blp hypomorph larvae had a 35% decreased number of plasmatocytes with a 45% reduced active mitochondrial staining and their viability was increased 2-fold by administration of NAC, which blocked melanotic lesions. Loss of Blp decreases cytochrome c oxidase activity and uncouples oxidative phosphorylation, causing ROS production, which selectively affects mitochondria-rich plasmatocyte survival and function, leading to melanotic lesions in Blp-deficient flies.—Roy, S., Short, M. K., Stanley, E. R., Jubinsky, P. T. Essential role of Drosophila black-pearl is mediated by its effects on mitochondrial respiration.

Description: Gaining insights on gene regulation from large-scale functional data sets is a grand challenge in systems biology. In this article, we develop and apply methods for transcriptional regulatory network inference from diverse functional genomics data sets and demonstrate their value for gene function and gene expression prediction. We formulate the network inference problem in a machine-learning framework and use both supervised and unsupervised methods to predict regulatory edges by integrating transcription factor (TF) binding, evolutionarily conserved sequence motifs, gene expression, and chromatin modification data sets as input features. Applying these methods to Drosophila melanogaster, we predict ~300,000 regulatory edges in a network of ~600 TFs and 12,000 target genes. We validate our predictions using known regulatory interactions, gene functional annotations, tissue-specific expression, protein–protein interactions, and three-dimensional maps of chromosome conformation. We use the inferred network to identify putative functions for hundreds of previously uncharacterized genes, including many in nervous system development, which are independently confirmed based on their tissue-specific expression patterns. Last, we use the regulatory network to predict target gene expression levels as a function of TF expression, and find significantly higher predictive power for integrative networks than for motif or ChIP-based networks. Our work reveals the complementarity between physical evidence of regulatory interactions (TF binding, motif conservation) and functional evidence (coordinated expression or chromatin patterns) and demonstrates the power of data integration for network inference and studies of gene regulation at the systems level.

Description: The order of genes in eukaryotic genomes has generally been assumed to be neutral, since gene order is largely scrambled over evolutionary time. Only a handful of exceptional examples are known, typically involving deeply conserved clusters of tandemly duplicated genes (e.g., Hox genes and histones). Here we report the first systematic survey of microsynteny conservation across metazoans, utilizing 17 genome sequences. We identified nearly 600 pairs of unrelated genes that have remained tightly physically linked in diverse lineages across over 600 million years of evolution. Integrating sequence conservation, gene expression data, gene function, epigenetic marks, and other genomic features, we provide extensive evidence that many conserved ancient linkages involve (1) the coordinated transcription of neighboring genes, or (2) genomic regulatory blocks (GRBs) in which transcriptional enhancers controlling developmental genes are contained within nearby bystander genes. In addition, we generated ChIP-seq data for key histone modifications in zebrafish embryos, which provided further evidence of putative GRBs in embryonic development. Finally, using chromosome conformation capture (3C) assays and stable transgenic experiments, we demonstrate that enhancers within bystander genes drive the expression of genes such as Otx and Islet , critical regulators of central nervous system development across bilaterians. These results suggest that ancient genomic functional associations are far more common than previously thought—involving ~12% of the ancestral bilaterian genome—and that cis -regulatory constraints are crucial in determining metazoan genome architecture.

Description: Understanding the mechanism that allows the intracellular protozoan parasite Leishmania donovani ( Ld ) to respond to reactive oxygen species (ROS) is of increasing therapeutic importance because of the continuing resistance toward antileishmanial drugs and for determining the illusive survival strategy of these parasites. A shift in primary carbon metabolism is the fastest response to oxidative stress. A 14 CO 2 evolution study, expression of glucose transporters together with consumption assays, indicated a shift in metabolic flux of the parasites from glycolysis toward pentose phosphate pathway (PPP) when exposed to different oxidants in vitro/ex vivo . Changes in gene expression, protein levels, and enzyme activities all pointed to a metabolic reconfiguration of the central glucose metabolism in response to oxidants. Generation of glucose-6-phosphate dehydrogenase (G6PDH) (~5-fold) and transaldolase (TAL) (~4.2-fold) overexpressing Ld cells reaffirmed that lethal doses of ROS were counterbalanced by effective manipulation of NADPH:NADP + ratio and stringent maintenance of reduced thiol content. The extent of protein carbonylation and accumulation of lipid peroxidized products were also found to be less in overexpressed cell lines. Interestingly, the LD 50 of sodium antimony gluconate (SAG), amphotericin-B (AmB), and miltefosine were significantly high toward overexpressing parasites. Consequently, this study illustrates that Ld strategizes a metabolic reconfiguration for replenishment of NADPH pool to encounter oxidative challenges.—Ghosh, A. K., Sardar, A. H., Mandal, A., Saini, S., Abhishek, K., Kumar, A., Purkait, B., Singh, R., Das, S., Mukhopadhyay, R., Roy, S., Das, P. Metabolic reconfiguration of the central glucose metabolism: a crucial strategy of Leishmania donovani for its survival during oxidative stress.

Description: Ischemic stroke results in excessive release of glutamate, which contributes to neuronal cell death. Here, we test the hypothesis that otherwise neurotoxic glutamate can be productively metabolized by glutamate oxaloacetate transaminase (GOT) to maintain cellular energetics and protect the brain from ischemic stroke injury. The GOT-dependent metabolism of glutamate was studied in primary neural cells and in stroke-affected C57-BL6 mice using magnetic resonance spectroscopy and GC-MS. Extracellular Glu sustained cell viability under hypoglycemic conditions and increased GOT-mediated metabolism in vitro . Correction of stroke-induced hypoxia using supplemental oxygen in vivo lowered Glu levels as measured by 1 H magnetic resonance spectroscopy. GOT knockdown abrogated this effect and caused ATP loss in the stroke-affected brain. GOT overexpression increased anaplerotic refilling of tricarboxylic acid cycle intermediates in mouse brain during ischemic stroke. Furthermore, GOT overexpression not only reduced ischemic stroke lesion volume but also attenuated neurodegeneration and improved poststroke sensorimotor function. Taken together, our results support a new paradigm that GOT enables metabolism of otherwise neurotoxic extracellular Glu through a truncated tricarboxylic acid cycle under hypoglycemic conditions.—Rink, C., Gnyawali, S., Stewart, R., Teplitsky, S., Harris, H., Roy, S., Sen, C. K., Khanna, S. Glutamate oxaloacetate transaminase enables anaplerotic refilling of TCA cycle intermediates in stroke-affected brain.