The filamentous fungi are an ecologically important group of organisms which also have important industrial applications but devastating effects as pathogens and agents of food spoilage. Protein ...kinases have been implicated in the regulation of virtually all biological processes but how they regulate filamentous fungal specific processes is not understood. The filamentous fungus Aspergillus nidulans has long been utilized as a powerful molecular genetic system and recent technical advances have made systematic approaches to study large gene sets possible. To enhance A. nidulans functional genomics we have created gene deletion constructs for 9851 genes representing 93.3% of the encoding genome. To illustrate the utility of these constructs, and advance the understanding of fungal kinases, we have systematically generated deletion strains for 128 A. nidulans kinases including expanded groups of 15 histidine kinases, 7 SRPK (serine-arginine protein kinases) kinases and an interesting group of 11 filamentous fungal specific kinases. We defined the terminal phenotype of 23 of the 25 essential kinases by heterokaryon rescue and identified phenotypes for 43 of the 103 non-essential kinases. Uncovered phenotypes ranged from almost no growth for a small number of essential kinases implicated in processes such as ribosomal biosynthesis, to conditional defects in response to cellular stresses. The data provide experimental evidence that previously uncharacterized kinases function in the septation initiation network, the cell wall integrity and the morphogenesis Orb6 kinase signaling pathways, as well as in pathways regulating vesicular trafficking, sexual development and secondary metabolism. Finally, we identify ChkC as a third effector kinase functioning in the cellular response to genotoxic stress. The identification of many previously unknown functions for kinases through the functional analysis of the A. nidulans kinome illustrates the utility of the A. nidulans gene deletion constructs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Obesity is a global epidemic and the underlying basis for numerous comorbidities. We report that the aryl hydrocarbon receptor (AHR) plays a key role in the metabolism of obesity. The AHR is a ...promiscuous, ligand-activated nuclear receptor primarily known for regulating genes involved in xenobiotic metabolism and T cell polarization. The aims of the work reported here were to understand the underlying mechanism of AHR-based obesity and to determine whether inhibition of AHR activity would reverse obesity.
Mice were fed control (low fat) and Western (high fat) diets with and without the AHR antagonist alpha-naphthoflavone (aNF). Gene expression of identified AHR-regulated genes from liver and adipose tissue was characterized. To determine the role of the AHR in obesity reversal, selected mice in control and Western diet regimens were switched at midpoint to the respective control and Western diets containing aNF, and the identified AHR-regulated genes characterized.
AHR inhibition prevented obesity in mice on a 40-week diet regimen. The likely AHR-regulated and cross-regulated downstream effectors of AHR-based obesity were shown to be CYP1B1, PPARα-target genes, SCD1, and SPP1 (osteopontin). Western diet caused an increase of mRNA and protein expression of the Cyp1b1, Scd1, and Spp1, and PPARα-target genes in the liver, and inhibition of the AHR maintained expression of these genes near control levels. The body weight of obese mice on Western diet switched to Western diet containing aNF decreased to that of mice on control diet concurrently with a reduction in the expression of liver CYP1B1, PPARα-target genes, SCD1, and SPP1. AHR inhibition prevented hypertrophy and hyperplasia in visceral adipose tissue and limited expression levels of CYP1B1 and SPP1 to that of mice on control diet.
AHR inhibition prevents and reverses obesity by likely reducing liver expression of the Cyp1b1, Scd1, Spp1, and PPARα-target genes; and the AHR is a potentially potent therapeutic target for the treatment and prevention of obesity and linked diseases.
Lung cancer accounts for the highest number of cancer-related deaths in the USA, highlighting the need for better prevention and therapy. Activation of the Nrf2 pathway detoxifies harmful insults and ...reduces oxidative stress, thus preventing carcinogenesis in various preclinical models. However, constitutive activation of the Nrf2 pathway has been detected in numerous cancers, which confers a survival advantage to tumor cells and a poor prognosis. In our study, we compared the effects of two clinically relevant classes of Nrf2 activators, dimethyl fumarate (DMF) and the synthetic oleanane triterpenoids, CDDO-imidazolide (CDDO-Im) and CDDO-methyl ester (CDDO-Me) in RAW 264.7 mouse macrophage-like cells, in VC1 lung cancer cells and in the A/J model of lung cancer. Although the triterpenoids and DMF both activated the Nrf2 pathway, CDDO-Im and CDDO-Me were markedly more potent than DMF. All of these drugs reduced the production of reactive oxygen species and inhibited nitric oxide production in RAW264.7 cells, but the triterpenoids were 100 times more potent than DMF in these assays. Microarray analysis revealed that only 52 of 99 Nrf2-target genes were induced by all three compounds, and each drug regulated a unique subset of Nrf2 genes. These drugs also altered the expression of other genes important in lung cancer independent of Nrf2. Although all three compounds enhanced the phosphorylation of CREB, only DMF increased the phosphorylation of Akt. CDDO-Me, at either 12.5 or 50mg/kg of diet, was the most effective drug in our lung cancer mouse model. Specifically, CDDO-Me significantly reduced the average tumor number, size and burden compared with the control group (P < 0.05). Additionally, 52% of the tumors in the control group were high-grade tumors compared with only 14% in the CDDO-Me group. Though less potent, CDDO-Im had similar activity as CDDO-Me. In contrast, 61-63% of the tumors in the DMF groups (400-1200mg/kg diet) were high-grade tumors compared with 52% for the controls (P < 0.05). Additionally, DMF significantly increased the average number of tumors compared with the controls (P < 0.05). Thus, in contrast to the triterpenoids, which effectively reduced pathogenesis in A/J mice, DMF enhanced the severity of lung carcinogenesis in these mice. Collectively, these results suggest that although CDDO-Im, CDDO-Me and DMF all activate the Nrf2 pathway, they target distinct genes and signaling pathways, resulting in opposite effects for the prevention of experimental lung cancer.
Significance Circadian clocks regulate gene expression levels to allow an organism to anticipate environmental conditions. These clocks reside in all the major branches of life and confer a ...competitive advantage to the organisms that maintain them. The clock in the fungus Neurospora crassa is an excellent model for basic understanding of core circadian architecture as well as for filamentous fungi. Here, we identify genes whose expression is clock regulated; indeed, as much as 40% of the transcriptome may be clock regulated, broadly directing daytime catabolism and nighttime growth. Both transcriptional control and posttranscriptional regulation play major roles in control of cycling transcripts such that DNA binding of transcription factors alone appears insufficient to set the phase of circadian transcription.
Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation–based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes ( ccg s) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora , revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccg s. Correlations between ccg abundance from RNA-Seq, the degree of ccg -promoter activation as reported by ccg- promoter–luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.
Light is a pervasive environmental factor that regulates development, stress resistance, and even virulence in numerous fungal species. Though much research has focused on signaling pathways in ...Aspergillus fumigatus, an understanding of how this pathogen responds to light is lacking. In this report, we demonstrate that the fungus does indeed respond to both blue and red portions of the visible spectrum. Included in the A. fumigatus light response is a reduction in conidial germination rates, increased hyphal pigmentation, enhanced resistance to acute ultraviolet and oxidative stresses, and an increased susceptibility to cell wall perturbation. By performing gene deletion analyses, we have found that the predicted blue light receptor LreA and red light receptor FphA play unique and overlapping roles in regulating the described photoresponsive behaviors of A. fumigatus. However, our data also indicate that the photobiology of this fungus is complex and likely involves input from additional photosensory pathways beyond those analyzed here. Finally, whole-genome microarray analysis has revealed that A. fumigatus broadly regulates a variety of metabolic genes in response to light, including those involved in respiration, amino acid metabolism, and metal homeostasis. Together, these data demonstrate the importance of the photic environment on the physiology of A. fumigatus and provide a basis for future studies into this unexplored area of its biology.
White collar‐1 (WC‐1) and white collar‐2 (WC‐2) are essential for light‐mediated responses in Neurospora crassa, but the molecular mechanisms underlying gene induction and the roles of other real and ...putative photoreceptors remain poorly characterized. Unsupervised hierarchical clustering of genome‐wide microarrays reveals 5.6% of detectable transcripts, including several novel mediators, that are either early or late light responsive. Evidence is shown for photoreception in the absence of the dominant, and here confirmed, white collar complex (WCC) that regulates both types of light responses. VVD primarily modulates late responses, whereas light‐responsive submerged protoperithecia‐1 (SUB‐1), a GATA family transcription factor, is essential for most late light gene expression. After a 15‐min light stimulus, the WCC directly binds the sub‐1 promoter. Bioinformatics analysis detects many early light response elements (ELREs), as well as identifying a late light response element (LLRE) required for wild‐type activity of late light response promoters. The data provide a global picture of transcriptional response to light, as well as illuminating the cis‐ and trans‐acting elements comprising the regulatory signalling cascade that governs the photobiological response.
Objective
The aryl hydrocarbon receptor (AHR) plays a key role in obesity. In vitro studies revealed that the tryptophan metabolite kynurenine (Kyn) activates AHR signaling in cultured hepatocytes. ...The objective of this study was to determine whether Kyn activated the AHR in mice to induce obesity.
Methods
Mice were fed a low‐fat diet and the same diet supplemented with Kyn. Body mass, liver status, and the expression of identified relevant genes were determined.
Results
Kyn caused mice to gain significant body mass, develop fatty liver and hyperglycemia, and increase expression levels of cytochrome P450 1B1 and stearoyl‐CoA desaturase 1. The hyperglycemia was accompanied with decreased insulin levels, which may have been due to the repression of genes involved in insulin secretion. Kyn plasma concentrations and BMI were measured in female patients, and a significant association was observed between Kyn and age in patients with obesity but not in patients who were lean.
Conclusions
Results show that (1) Kyn or a metabolite thereof is a ligand responsible for inducing AHR‐based obesity, fatty liver, and hyperglycemia in mice; (2) plasma Kyn levels increase with age in women with obesity but not in lean women; and (3) an activated AHR is necessary but not sufficient to attain obesity, a status that also requires fat in the diet.
The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this ...study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.
Obesity is an increasingly urgent global problem, yet, little is known about its causes and less is known how obesity can be effectively treated. We showed previously that the aryl hydrocarbon ...receptor (AHR) plays a role in the regulation of body mass in mice fed Western diet. The AHR is a ligand-activated nuclear receptor that regulates genes involved in a number of biological pathways, including xenobiotic metabolism and T cell polarization. This study was an investigation into whether inhibition of the AHR prevents Western diet-based obesity. Male C57Bl/6J mice were fed control and Western diets with and without the AHR antagonist α-naphthoflavone or CH-223191, and a mouse hepatocyte cell line was used to delineate relevant cellular pathways. Studies are presented showing that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduce obesity and adiposity and ameliorates liver steatosis in male C57Bl/6J mice fed a Western diet. Mice deficient in the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) were also resistant to obesity. Using an AHR-directed, luciferase-expressing mouse hepatocyte cell line, we show that the transforming growth factor β1 (TGFβ1) signaling pathway via PI3K and NF-κB and the toll-like receptor 2/4 (TLR2/4) signaling pathway stimulated by oxidized low-density lipoproteins via NF-κB, each induce luciferase expression; however, TLR2/4 signaling was significantly reduced by inhibition of IDO1. At physiological levels, kynurenine but not kynurenic acid (both tryptophan metabolites and known AHR agonists) activated AHR-directed luciferase expression. We propose a hepatocyte-based model, in which kynurenine production is increased by enhanced IDO1 activity stimulated by TGFβ1 and TLR2/4 signaling, via PI3K and NF-κB, to perpetuate a cycle of AHR activation to cause obesity; and inhibition of the AHR, in turn, blocks the cycle's output to prevent obesity. The AHR with its broad ligand binding specificity is a promising candidate for a potentially simple therapeutic approach for the prevention and treatment of obesity and associated complications.
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•The AHR acts as a hub in Western diet-based obesity.•Inhibition of AHR signaling by antagonists prevents obesity and liver steatosis.•ox-LDL stimulates AHR activity via a TLR2/4, NF-kB, IDO1, kynurenine axis.•TGFβ stimulates AHR activity in Hepa-1c1c7 cells via PI3K and NF-kB.•The AHR offers a simple and promising approach for treating obesity.
Mutants in the period-1 (prd-1) gene, characterized by a recessive allele, display a reduced growth rate and period lengthening of the developmental cycle controlled by the circadian clock. We ...refined the genetic location of prd-1 and used whole genome sequencing to find the mutation defining it, confirming the identity of prd-1 by rescuing the mutant circadian phenotype via transformation. PRD-1 is an RNA helicase whose orthologs, DDX5 DEAD (Asp-Glu-Ala-Asp) Box Helicase 5 and DDX17 in humans and DBP2 (Dead Box Protein 2) in yeast, are implicated in various processes, including transcriptional regulation, elongation, and termination, ribosome biogenesis, and mRNA decay. Although prd-1 mutants display a long period (∼25 h) circadian developmental cycle, they interestingly display a WT period when the core circadian oscillator is tracked using a frq-luciferase transcriptional fusion under conditions of limiting nutritional carbon; the core oscillator in the prd-1 mutant strain runs with a long period under glucose-sufficient conditions. Thus, PRD-1 clearly impacts the circadian oscillator and is not only part of a metabolic oscillator ancillary to the core clock. PRD-1 is an essential protein, and its expression is neither light-regulated nor clock-regulated. However, it is transiently induced by glucose; in the presence of sufficient glucose, PRD-1 is in the nucleus until glucose runs out, which elicits its disappearance from the nucleus. Because circadian period length is carbon concentration-dependent, prd-1 may be formally viewed as a clock mutant with defective nutritional compensation of circadian period length.
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