Genetic interaction analysis of transcription factors and in the regulation of respiration and fluconazole susceptibility.

is the second most common cause of invasive candidiasis and is widely known to have reduced susceptibility to fluconazole relative to many other spp. Upc2A is a transcription factor that regulates ergosterol biosynthesis gene expression under conditions of sterol stress such as azole drug treatment or hypoxia. Through an microevolution experiment, we found that loss-of-function mutants of the ATF/CREB transcription factor suppresses the fluconazole hyper-susceptibility of the ∆ mutant.

Gene-specific transcriptional activation by the AtrR factor requires a conserved C-terminal domain.

Unlabelled: Treatment of fungal infections associated with the filamentous fungus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in the treatment of aspergillosis with this drug class acting by inhibiting a key step in the biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the gene.

Loss of a conserved C-terminal region of the AtrR transcriptional regulator leads to a gene-specific defect in target gene expression.

Treatment of fungal infections associated with the filamentous fungus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in treatment of aspergillosis with this drug class acting by inhibiting a key step in biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the gene.

Cryptococcus neoformans adapts to the host environment through TOR-mediated remodeling of phospholipid asymmetry.

Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients. Host CO concentrations are 100-fold higher than the external environment and strains unable to grow at host CO concentrations are not pathogenic.

Interference with the HNF4-dependent gene regulatory network diminishes endoplasmic reticulum stress in hepatocytes.

Background: In all eukaryotic cell types, the unfolded protein response (UPR) upregulates factors that promote protein folding and misfolded protein clearance to help alleviate endoplasmic reticulum (ER) stress. Yet, ER stress in the liver is uniquely accompanied by the suppression of metabolic genes, the coordination and purpose of which are largely unknown.

Methods: Here, we combined in silico machine learning, in vivo liver-specific deletion of the master regulator of hepatocyte differentiation HNF4α, and in vitro manipulation of hepatocyte differentiation state to determine how the UPR regulates hepatocyte identity and toward what end.

The reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates filamentation, biofilm formation, and oral commensalism.

is a commensal fungus that colonizes the human oral cavity and gastrointestinal tract but also causes mucosal as well as invasive disease. The expression of virulence traits in clinical isolates is heterogeneous and the genetic basis of this heterogeneity is of high interest. The reference strain SC5314 is highly invasive and expresses robust filamentation and biofilm formation relative to many other clinical isolates.

Interactions between the transcription factors FfmA and AtrR are required to properly regulate gene expression in the fungus Aspergillus fumigatus.

Transcriptional regulation of azole resistance in the filamentous fungus Aspergillus fumigatus is a key step in development of this problematic clinical phenotype. We and others have previously described a C2H2-containing transcription factor called FfmA that is required for normal levels of voriconazole susceptibility. Null alleles of ffmA exhibit a strongly compromised growth rate even in the absence of any external stress.

The reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates biofilm formation and oral commensalism.

Unlabelled: is a diploid human fungal pathogen that displays significant genomic and phenotypic heterogeneity over a range of virulence traits and in the context of a variety of environmental niches. Here, we show that the effects of Rob1 on biofilm and filamentation virulence traits is dependent on both the specific environmental condition and the clinical strain of . The reference strain SC5314 is a heterozygote with two alleles that differ by a single nucleotide polymorphism at position 946 resulting in a serine or proline containing isoform.

Transcription factor FfmA interacts both physically and genetically with AtrR to properly regulate gene expression in the fungus .

Transcriptional regulation of azole resistance in the filamentous fungus is a key step in development of this problematic clinical phenotype. We and others have previously described a C2H2-containing transcription factor called FfmA that is required for normal levels of voriconazole susceptibility and expression of an ATP-binding cassette transporter gene called . Null alleles of exhibit a strongly compromised growth rate even in the absence of any external stress.

Interference with the HNF4-dependent gene regulatory network diminishes ER stress in hepatocytes.

In all eukaryotic cell types, the unfolded protein response (UPR) upregulates factors that promote protein folding and misfolded protein clearance to help alleviate endoplasmic reticulum (ER) stress. Yet ER stress in the liver is uniquely accompanied by the suppression of metabolic genes, the coordination and purpose of which is largely unknown. Here, we used unsupervised machine learning to identify a cluster of correlated genes that were profoundly suppressed by persistent ER stress in the liver.

Loss-of-Function Mutations Suppress the Fluconazole Susceptibility of Δ Mutation in Candida glabrata, Implicating Additional Positive Regulators of Ergosterol Biosynthesis.

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress-induced transcription factors such as Upc2 and Upc2A as well as homologs of sterol response element binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other spp.

The Candida glabrata Upc2A transcription factor is a global regulator of antifungal drug resistance pathways.

The most commonly used antifungal drugs are the azole compounds, which interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1.

The Ndr/LATS Kinase Cbk1 Regulates a Specific Subset of Ace2 Functions and Suppresses the Hypha-to-Yeast Transition in Candida albicans.

The egulation of ce2 and orphogenesis (RAM) pathway is an important regulatory network in the human fungal pathogen The RAM pathway's two most well-studied components, the NDR/Lats kinase Cbk1 and its putative substrate, the transcription factor Ace2, have a wide range of phenotypes and functions. It is not clear, however, which of these functions are specifically due to the phosphorylation of Ace2 by Cbk1. To address this question, we first compared the transcriptional profiles of and deletion mutants.

Elimination of SOX2/OCT4-Associated Prostate Cancer Stem Cells Blocks Tumor Development and Enhances Therapeutic Response.

SOX2 and OCT4 are key regulators of embryonic stem cell pluripotency. They are overexpressed in prostate cancers and have been associated with cancer stem cell (CSC) properties. However, reliable tools for detecting and targeting SOX2/OCT4-overexpressing cells are lacking, limiting our understanding of their roles in prostate cancer initiation, progression, and therapeutic resistance.

MALTA: a calculator for estimating the coverage with shRNA, CRISPR, and cDNA libraries.

Genetic screens using shRNA, CRISPR, or cDNA libraries rely on adequately transferring the library into cells for further assay. These libraries can have many different elements and each element can be present at different copy numbers within a given pooled library. Calculating how many recipient cells are needed to adequately sample all or most of the different elements within a library is important, especially if one wants to compare the outcomes of different genetic screens that rely on accurately reproducing the starting population of library-containing cells.

AtrR Is an Essential Determinant of Azole Resistance in Aspergillus fumigatus.

Aspergillosis associated with azole-resistant has a mortality rate that can approach 90% in certain patient populations. The best-understood avenue for azole resistance involves changes in the gene that encodes the target of azole drugs, lanosterol α-14 demethylase. The most common azole resistance allele currently described is a linked change corresponding to a change in the coding sequence of and a duplication of a 34-bp region in the promoter leading to a tandem repeat (TR).

High content screening identifies monensin as an EMT-selective cytotoxic compound.

Epithelial-to-mesenchymal transition (EMT) is implicated in cancer metastasis and drug resistance. Specifically targeting cancer cells in an EMT-like state may have therapeutic value. In this study, we developed a cell imaging-based high-content screening protocol to identify EMT-selective cytotoxic compounds.

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens.

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay.

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved.

DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions.

We adapted the yeast 2-hybrid assay to simultaneously uncover multiple transient protein interactions within a single screen by using a strategy termed DEEPN (dynamic enrichment for evaluation of protein networks). This approach incorporates high-throughput DNA sequencing and computation to follow competition among a plasmid population encoding interacting partners. To demonstrate the capacity of DEEPN, we identify a wide range of ubiquitin-binding proteins, including interactors that we verify biochemically.

Tetraspan cargo adaptors usher GPI-anchored proteins into multivesicular bodies.

Ubiquitinated membrane proteins are sorted into intralumenal endosomal vesicles on their way for degradation in lysosomes. Here we summarize the discovery of the Cos proteins, which work to organize and segregate ubiquitinated cargo prior to its incorporation into intralumenal vesicles of the multivesicular body (MVB). Importantly, cargoes such as GPI-anchored proteins (GPI-APs) that cannot undergo ubiquitination, rely entirely on Cos proteins for sorting into intralumenal vesicles using the same pathway that depends on ESCRTs and ubiquitin ligases that typical polytopic membrane proteins do.

CLN3 deficient cells display defects in the ARF1-Cdc42 pathway and actin-dependent events.

Juvenile Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL) is a devastating neurodegenerative disease caused by mutations in CLN3, a protein of undefined function. Cell lines derived from patients or mice with CLN3 deficiency have impairments in actin-regulated processes such as endocytosis, autophagy, vesicular trafficking, and cell migration. Here we demonstrate the small GTPase Cdc42 is misregulated in the absence of CLN3, and thus may be a common link to multiple cellular defects.

Roles of rho GTPases in intracellular transport and cellular transformation.

Rho family GTPases belong to the Ras GTPase superfamily and transduce intracellular signals known to regulate a variety of cellular processes, including cell polarity, morphogenesis, migration, apoptosis, vesicle trafficking, viral transport and cellular transformation. The three best-characterized Rho family members are Cdc42, RhoA and Rac1. Cdc42 regulates endocytosis, the transport between the endoplasmic reticulum and Golgi apparatus, post-Golgi transport and exocytosis.

ARF1-regulated coatomer directs the steady-state localization of protein kinase C epsilon at the Golgi apparatus.

Protein kinase C epsilon (PKCε) contributes to multiple signaling pathways affecting human disease. The function of PKCε requires it to undergo changes in subcellular distribution in response to signaling events. While the mechanisms underlying this translocation are incompletely understood, it involves the receptor for activated C kinase protein (RACK2/β'-COP).

Cdc42 regulates microtubule-dependent Golgi positioning.

The molecular mechanisms underlying cytoskeleton-dependent Golgi positioning are poorly understood. In mammalian cells, the Golgi apparatus is localized near the juxtanuclear centrosome via dynein-mediated motility along microtubules. Previous studies implicate Cdc42 in regulating dynein-dependent motility.