42 research outputs found
A molecular genetic toolbox for Yarrowia lipolytica
Background: Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain. Results: We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the "Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells. Conclusions: These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products
Transcription Factors in Light and Circadian Clock Signaling Networks Revealed by Genomewide Mapping of Direct Targets for Neurospora White Collar Complex
Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways
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The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth
Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the American Society for Cell Biology. The published article can be found at: http://www.molbiolcell.org/
Regulation of Yeast-to-Hyphae Transition in <i>Yarrowia lipolytica</i>
The yeast Yarrowia lipolytica undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the smooth mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5'-CCCCT-3') and upregulation of genes with cell cycle box (5'-ACGCG-3') motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p in Saccharomyces cerevisiae We confirmed that the Y. lipolyticamsn2 (Ylmsn2) ortholog is required for hyphal growth and found that overexpression of Ylmsn2 enables hyphal growth in smooth strains. The cell cycle box is bound by the Mbp1p/Swi6p complex in S. cerevisiae to regulate G1-to-S phase progression. We found that overexpression of either the Ylmbp1 or Ylswi6 homologs decreased hyphal growth and that deletion of either Ylmbp1 or Ylswi6 promotes hyphal growth in smooth strains. A second forward genetic screen for reversion to hyphal growth was performed with the smooth-33 mutant to identify additional genetic factors regulating hyphal growth in Y. lipolytica Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Ylchk1 and Ylnik1 and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Ylssk2, Ylpbs2, and Ylhog1 Together, these results demonstrate that Y. lipolytica transitions to hyphal growth in response to stress through multiple signaling pathways.IMPORTANCE Many yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition in Yarrowia lipolytica We confirmed that the transcription factor Ylmsn2 is required for the transition to hyphal growth and found that signaling by the histidine kinases Ylchk1 and Ylnik1 as well as the MAP kinases of the HOG pathway (Ylssk2, Ylpbs2, and Ylhog1) regulates the transition to hyphal growth. These results suggest that Y. lipolytica transitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response
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Genome-Wide Characterization of Light-Regulated Genes in Neurospora crassa
The filamentous fungus Neurospora crassa responds to light in complex ways. To thoroughly study the transcriptional response of this organism to light, RNA-seq was used to analyze capped and polyadenylated mRNA prepared from mycelium grown for 24 hr in the dark and then exposed to light for 0 (control) 15, 60, 120, and 240 min. More than three-quarters of all defined protein coding genes (79%) were expressed in these cells. The increased sensitivity of RNA-seq compared with previous microarray studies revealed that the RNA levels for 31% of expressed genes were affected two-fold or more by exposure to light. Additionally, a large class of mRNAs, enriched for transcripts specifying products involved in rRNA metabolism, showed decreased expression in response to light, indicating a heretofore undocumented effect of light on this pathway. Based on measured changes in mRNA levels, light generally increases cellular metabolism and at the same time causes significant oxidative stress to the organism. To deal with this stress, protective photopigments are made, antioxidants are produced, and genes involved in ribosome biogenesis are transiently repressed.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the Genetics Society of America. The published article can be found at: http://g3journal.org/.Keywords: Neurospora, Light, RNA-se
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Transcriptional networks controlled by the fatty acid regulators FAR-1 and FAR-2
Fungi are capable of growth on a wide variety of carbon sources, both living and dead. They can produce an arsenal of enzymes and transporters for harvesting sugars, polysaccharides, amino acids, lipids and micronutrients from their environments [1]. Within the nucleus of a cell, transcription factors (TF) control whether genes will be transcribed, after which the transcripts can be translated into functional protein. TF contact with DNA can be influenced by nucleosomal occupancy, DNA binding affinity, and competition with other DNA binding proteins [2], [3]. Sequence specific DNA binding transcription factors associate with promoter sequences in order to tune core metabolic pathways in response to nutrient availability, for example N. crassa’s major nitrogen regulator NIT-2, [4], or in response to oxidative stress, the alternative oxidase regulator, AOD-2 [5]. Change in the relative abundance of proteins within the cell or proteome can have broad effects from redirection of metabolic flux via carbon and nitrogen use, production of enzymes for detoxification, and altered growth and development from the examples above. A survey of transcription factor binding influenced by light is underway in Neurospora crassa as part of the Neurospora Functional Genomics and Systems Biology (NcFGSB) program project funded by the NIH (P01GM). This work began by testing the link of FAR-1, or Fatty Acid Regulator -1 (NCU08000) to light regulation, but has continued toward investigation of how both FAR-1 and a second TF, Fatty Acid Regulator-2, or FAR-2 (NCU03643) influence central metabolism, oxidative stress response, and development.
Transcription factor networks consist of describing TFs that bind to multiple genes and individual genes controlled by multiple regulators. Further, the regulation of regulators describes how the transcription of transcription factor genes is controlled. Chromatin immunoprecipitation, or 'ChIP' experiments show that a variety of factors are enriched at the same promoter region as can be seen by comparing multiple datasets [6], [7]. Transcriptional regulators may promote or inhibit one another from binding DNA, leading to a total regulation as the sum of TF activity. This complexity cannot be explained by single genetic experiments and study of a limited number of loci. Even in the event of TF association with specific promoter DNA as analyzed by ChIP, proximal binding of a transcriptional activator does not directly mean that transcript levels will increase, as we have seen with the identification of WC-2 binding sites [7].
I used a reverse genetics approach, assaying deletion mutants of selected loci, to determine the cellular effects of FAR-1 and FAR-2 transcriptional activity in different carbon sources. The tools of high throughput sequencing, and bioinformatics data analyses were combined with experiments to characterize phenotypes observed. Here I report (1) a collection of binding sites found in the Neurospora genome for FAR-1 and FAR-2 in sucrose, butyrate, and oleate, (2) changes in transcription as a result of the loss of FAR-1 and/or FAR-2 function, and (3) how the combination of binding sites and transcriptional activities are reflected in phenotypes. This work has developed and utilized methods for combining genome-scale ChIP- and RNA-sequencing data to describe direct and indirect transcriptional regulation, and has added to the definition of transcription factor networks in N. crassa.KEYWORDS: peroxisome, glyoxylate cycle, heatmap, beta-oxidation, glyoxysome, gene ontology, transcription factor, perithecia, fatty acid, chromatin immunoprecipitation, reactive oxygen species, epitope tag, DNA bindin
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Characterization of Neurospora crassa “Fatty Acid Regulator” transcription factors, FAR-1 and FAR-2, by ChIP- and RNA-sequencing
Transcription factor (TF) genes were modified endogenously to include epitope tags for identification of TF protein binding sites by Chromatin Immunoprecipitation (ChIP), followed by high throughput sequencing. We used RNA-sequencing in carbon sources of sucrose, butyrate, and oleate in far-1, far-2, and a double far-1; far-2 mutant to find transcripts influenced by FAR regulation. This dataset includes (1) ChIP peaks indicative of genomic binding locations of FAR-1 and FAR-2 (4 files), (2) analysis of gene ontology (GO) by two methods (4 files), and (3) total RNA-sequencing for wild type, delta-far-1, delta-far-2, and delta-far-1; delta-far-2 mutants, with differential expression as evaluated by CuffDiff (2 files)
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TableS01_FAR1S_ChIP.xlsx
Transcription factor (TF) genes were modified endogenously to include epitope tags for identification of TF protein binding sites by Chromatin Immunoprecipitation (ChIP), followed by high throughput sequencing. We used RNA-sequencing in carbon sources of sucrose, butyrate, and oleate in far-1, far-2, and a double far-1; far-2 mutant to find transcripts influenced by FAR regulation. This dataset includes (1) ChIP peaks indicative of genomic binding locations of FAR-1 and FAR-2 (4 files), (2) analysis of gene ontology (GO) by two methods (4 files), and (3) total RNA-sequencing for wild type, delta-far-1, delta-far-2, and delta-far-1; delta-far-2 mutants, with differential expression as evaluated by CuffDiff (2 files)
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TableS10_MIPS-FAR2-RNAfiltered.xlsx
Transcription factor (TF) genes were modified endogenously to include epitope tags for identification of TF protein binding sites by Chromatin Immunoprecipitation (ChIP), followed by high throughput sequencing. We used RNA-sequencing in carbon sources of sucrose, butyrate, and oleate in far-1, far-2, and a double far-1; far-2 mutant to find transcripts influenced by FAR regulation. This dataset includes (1) ChIP peaks indicative of genomic binding locations of FAR-1 and FAR-2 (4 files), (2) analysis of gene ontology (GO) by two methods (4 files), and (3) total RNA-sequencing for wild type, delta-far-1, delta-far-2, and delta-far-1; delta-far-2 mutants, with differential expression as evaluated by CuffDiff (2 files)