Genetic and Physiological Activation of Osmosensitive Gene Expression Mimics Transcriptional Signatures of Pathogen Infection in C. elegans

The soil-dwelling nematode C. elegans is a powerful system for comparative molecular analyses of environmental stress response mechanisms. Infection of worms with bacterial and fungal pathogens causes the activation of well-characterized innate immune transcriptional programs in pathogen-exposed hypodermal and intestinal tissues. However, the pathophysiological events that drive such transcriptional responses are not understood. Here, we show that infection-activated transcriptional responses are, in large part, recapitulated by either physiological or genetic activation of the osmotic stress response. Microarray profiling of wild type worms exposed to non-lethal hypertonicity identified a suite of genes that were also regulated by infection. Expression profiles of five different osmotic stress resistant (osr) mutants under isotonic conditions reiterated the wild type transcriptional response to osmotic stress and also showed substantial similarity to infection-induced gene expression under isotonic conditions. Computational, transgenic, and functional approaches revealed that two GATA transcription factors previously implicated in infection-induced transcriptional responses, elt-2 and elt-3, are also essential for coordinated tissue-specific activation of osmosensitive gene expression and promote survival under osmotically stressful conditions. Together, our data suggest infection and osmotic adaptation share previously unappreciated transcriptional similarities which might be controlled via regulation of tissue-specific GATA transcription factors

An unexpectedly high degree of specialization and a widespread involvement in sterol metabolism among the C. elegans putative aminophospholipid translocases

<p>Abstract</p> <p>Background</p> <p>P-type ATPases in subfamily IV are exclusively eukaryotic transmembrane proteins that have been proposed to directly translocate the aminophospholipids phosphatidylserine and phosphatidylethanolamine from the exofacial to the cytofacial monolayer of the plasma membrane. Eukaryotic genomes contain many genes encoding members of this subfamily. At present it is unclear why there are so many genes of this kind per organism or what individual roles these genes perform in organism development.</p> <p>Results</p> <p>We have systematically investigated expression and developmental function of the six, <it>tat-1 </it>through <it>6</it>, subfamily IV P-type ATPase genes encoded in the <it>Caenorhabditis elegans </it>genome. <it>tat-5 </it>is the only ubiquitously-expressed essential gene in the group. <it>tat-6 </it>is a poorly-transcribed recent duplicate of <it>tat-5</it>. <it>tat-2 </it>through <it>4 </it>exhibit tissue-specific developmentally-regulated expression patterns. Strong expression of both <it>tat-2 </it>and <it>tat-4 </it>occurs in the intestine and certain other cells of the alimentary system. The two are also expressed in the uterus, during spermatogenesis and in the fully-formed spermatheca. <it>tat-2 </it>alone is expressed in the pharyngeal gland cells, the excretory system and a few cells of the developing vulva. The expression pattern of <it>tat-3 </it>is almost completely different from those of <it>tat-2 </it>and <it>tat-4</it>. <it>tat-3 </it>expression is detectable in the steroidogenic tissues: the hypodermis and the XXX cells, as well as in most cells of the pharynx (except gland), various tissues of the reproductive system (except uterus and spermatheca) and seam cells. Deletion of <it>tat-1 </it>through <it>4 </it>individually interferes little or not at all with the regular progression of organism growth and development under normal conditions. However, <it>tat-2 </it>through <it>4 </it>become essential for reproductive growth during sterol starvation.</p> <p>Conclusion</p> <p><it>tat-5 </it>likely encodes a housekeeping protein that performs the proposed aminophospholipid translocase function routinely. Although individually dispensable, <it>tat-1 </it>through <it>4 </it>seem to be at most only partly redundant. Expression patterns and the sterol deprivation hypersensitivity deletion phenotype of <it>tat-2 </it>through <it>4 </it>suggest that these genes carry out subtle metabolic functions, such as fine-tuning sterol metabolism in digestive or steroidogenic tissues. These findings uncover an unexpectedly high degree of specialization and a widespread involvement in sterol metabolism among the genes encoding the putative aminophospholipid translocases.</p

Tcf21 regulates the specification and maturation of proepicardial cells

The epicardium is a mesothelial cell layer essential for vertebrate heart development and pertinent for cardiac repair post-injury in the adult. The epicardium initially forms from a dynamic precursor structure, the proepicardial organ, from which cells migrate onto the heart surface. During the initial stage of epicardial development crucial epicardial-derived cell lineages are thought to be determined. Here, we define an essential requirement for transcription factor Tcf21 during early stages of epicardial development in Xenopus, and show that depletion of Tcf21 results in a disruption in proepicardial cell specification and failure to form a mature epithelial epicardium. Using a mass spectrometry-based approach we defined Tcf21 interactions and established its association with proteins that function as transcriptional co-repressors. Furthermore, using an in vivo systems-based approach, we identified a panel of previously unreported proepicardial precursor genes that are persistently expressed in the epicardial layer upon Tcf21 depletion, thereby confirming a primary role for Tcf21 in the correct determination of the proepicardial lineage. Collectively, these studies lead us to propose that Tcf21 functions as a transcriptional repressor to regulate proepicardial cell specification and the correct formation of a mature epithelial epicardium

The Caenorhabditis elegans Mucin-Like Protein OSM-8 Negatively Regulates Osmosensitive Physiology Via the Transmembrane Protein PTR-23

The molecular mechanisms of animal cell osmoregulation are poorly understood. Genetic studies of osmoregulation in yeast have identified mucin-like proteins as critical regulators of osmosensitive signaling and gene expression. Whether mucins play similar roles in higher organisms is not known. Here, we show that mutations in the Caenorhabditis elegans mucin-like gene osm-8 specifically disrupt osmoregulatory physiological processes. In osm-8 mutants, normal physiological responses to hypertonic stress, such as the accumulation of organic osmolytes and activation of osmoresponsive gene expression, are constitutively activated. As a result, osm-8 mutants exhibit resistance to normally lethal levels of hypertonic stress and have an osmotic stress resistance (Osr) phenotype. To identify genes required for Osm-8 phenotypes, we performed a genome-wide RNAi osm-8 suppressor screen. After screening ∼18,000 gene knockdowns, we identified 27 suppressors that specifically affect the constitutive osmosensitive gene expression and Osr phenotypes of osm-8 mutants. We found that one suppressor, the transmembrane protein PTR-23, is co-expressed with osm-8 in the hypodermis and strongly suppresses several Osm-8 phenotypes, including the transcriptional activation of many osmosensitive mRNAs, constitutive glycerol accumulation, and osmotic stress resistance. Our studies are the first to show that an extracellular mucin-like protein plays an important role in animal osmoregulation in a manner that requires the activity of a novel transmembrane protein. Given that mucins and transmembrane proteins play similar roles in yeast osmoregulation, our findings suggest a possible evolutionarily conserved role for the mucin-plasma membrane interface in eukaryotic osmoregulation

A proteomics view of SIRT6 functions and the anti-viral roles of sirtuin proteins

Viruses have evolved effective means for hijacking host molecular mechanisms for their replication and spread. Recent work from our laboratory and other groups has established that protein deacetylases are targeted by viral proteins and play critical roles during infection. Importantly, we have discovered that human sirtuins are a new family of anti-viral proteins with broad-range properties against both DNA and RNA viruses. One of the sirtuins with prominent anti-viral functions is SIRT6, a nuclear enzyme just recently established as a critical factor in human disease. Multiple biological processes, including DNA repair, gene expression, telomere maintenance, and metabolism, have been linked to SIRT6. Still, the mechanisms involved in regulating its functions and protein interactions have remained largely undefined. Here, we utilize a multidisciplinary approach integrating proteomics, virology, genetics, microscopy, and bioinformatics to characterize SIRT6 protein interaction networks, and assess their dynamic regulation during human cytomegalovirus (HCMV) infection. First, we provide an overview of advancements in proteomic approaches for studying protein interactions, several of which have been pioneered by our laboratory. These methods can provide direct insight into associations required for host defense against infections or virus spread. Second, we present the first network of SIRT6 interactions and identify prominent phosphorylations within a C-terminus naturally disordered region. Importantly, we report a previously unrecognized interplay between SIRT6 enzymatic activity and its associations. Next, we identify infection-triggered changes in SIRT6 interactions and sub-cellular localization. We demonstrate SIRT6 redistribution from the nucleus to the cytoplasm during late stages of HCMV infection, suggesting a repurposing of its functions and associations with different substrates and protein complexes. Notably, we report that SIRT6 is specifically targeted by viral proteins, including the viral kinase pUL97, at different time points during infection. Further strengthening the SIRT6 contribution to immune responses following infection, we identify and validate its interactions with the viral tegument protein pUL83--involved in viral immune evasion, and the host DNA sensor IFI16--necessary for inducing antiviral cytokines after infection. Finally, we demonstrate that the antiviral properties of sirtuins are evolutionarily conserved, showing that the bacterial homolog CobB in Escherichia coli has protective functions during bacteriophage infection

Temperature downshift induces antioxidantresponse in fungi isolated from Antarctica

Although investigators have been studying the cold-shock response in a variety of organisms for the last two decades or more, comparatively little is known about the difference between antioxidant cell response to cold stress in Antarctic and temperate microorganisms. The change of environmental temperature, which is one of the most common stresses, could be crucial for their use in the biotechnological industry and in ecological research. We compared the effect of short-term temperature downshift on antioxidant cell response in Antarctic and temperate fungi belonging to the genus Penicillium. Our study showed that downshift from an optimal temperature to 15° or 6°C led to a cell response typical of oxidative stress: significant reduction of biomass production; increase in the levels of oxidative damaged proteins and accumulation of storage carbohydrates (glycogen and trehalose) in comparison to growth at optimal temperature. Cell response against cold stress includes also increase in the activities of SOD and CAT, which are key enzymes for directly scavenging reactive oxygen species. This response is more species-dependent than dependent on the degree of cold-shock. Antarctic psychrotolerant strain Penicillium olsonii p14 that is adapted to life in extremely cold conditions demonstrated enhanced tolerance to temperature downshift in comparison with both mesophilic strains (Antarctic Penicillium waksmanii m12 and temperate Penicillium sp. t35)