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The Candida albicans transcription factor Cas5 couples stress responses, drug resistance and cell cycle regulation.

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The Candida albicans transcription factor Cas5 couples stress responses, drug resistance and cell cycle regulation.

Nat Commun. 2017 Sep 11;8(1):499

Authors: Xie JL, Qin L, Miao Z, Grys BT, Diaz JC, Ting K, Krieger JR, Tong J, Tan K, Leach MD, Ketela T, Moran MF, Krysan DJ, Boone C, Andrews BJ, Selmecki A, Ho Wong K, Robbins N, Cowen LE

Abstract
The capacity to coordinate environmental sensing with initiation of cellular responses underpins microbial survival and is crucial for virulence and stress responses in microbial pathogens. Here we define circuitry that enables the fungal pathogen Candida albicans to couple cell cycle dynamics with responses to cell wall stress induced by echinocandins, a front-line class of antifungal drugs. We discover that the C. albicans transcription factor Cas5 is crucial for proper cell cycle dynamics and responses to echinocandins, which inhibit β-1,3-glucan synthesis. Cas5 has distinct transcriptional targets under basal and stress conditions, is activated by the phosphatase Glc7, and can regulate the expression of target genes in concert with the transcriptional regulators Swi4 and Swi6. Thus, we illuminate a mechanism of transcriptional control that couples cell wall integrity with cell cycle regulation, and uncover circuitry governing antifungal drug resistance.Cas5 is a transcriptional regulator of responses to cell wall stress in the fungal pathogen Candida albicans. Here, Xie et al. show that Cas5 also modulates cell cycle dynamics and responses to antifungal drugs.

PMID: 28894103 [PubMed - in process]



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Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide Variety of Non-canonical Specificities.

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Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide Variety of Non-canonical Specificities.

Structure. 2017 Oct 03;25(10):1598-1610.e3

Authors: Teyra J, Huang H, Jain S, Guan X, Dong A, Liu Y, Tempel W, Min J, Tong Y, Kim PM, Bader GD, Sidhu SS

Abstract
SH3 domains are protein modules that mediate protein-protein interactions in many eukaryotic signal transduction pathways. The majority of SH3 domains studied thus far act by binding to proline-rich sequences in partner proteins, but a growing number of studies have revealed alternative recognition mechanisms. We have comprehensively surveyed the specificity landscape of human SH3 domains in an unbiased manner using peptide-phage display and deep sequencing. Based on ∼70,000 unique binding peptides, we obtained 154 specificity profiles for 115 SH3 domains, which reveal that roughly half of the SH3 domains exhibit non-canonical specificities and collectively recognize a wide variety of peptide motifs, most of which were previously unknown. Crystal structures of SH3 domains with two distinct non-canonical specificities revealed novel peptide-binding modes through an extended surface outside of the canonical proline-binding site. Our results constitute a significant contribution toward a complete understanding of the mechanisms underlying SH3-mediated cellular responses.

PMID: 28890361 [PubMed - in process]



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Variant Interpretation: Functional Assays to the Rescue.

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Variant Interpretation: Functional Assays to the Rescue.

Am J Hum Genet. 2017 Sep 07;101(3):315-325

Authors: Starita LM, Ahituv N, Dunham MJ, Kitzman JO, Roth FP, Seelig G, Shendure J, Fowler DM

Abstract
Classical genetic approaches for interpreting variants, such as case-control or co-segregation studies, require finding many individuals with each variant. Because the overwhelming majority of variants are present in only a few living humans, this strategy has clear limits. Fully realizing the clinical potential of genetics requires that we accurately infer pathogenicity even for rare or private variation. Many computational approaches to predicting variant effects have been developed, but they can identify only a small fraction of pathogenic variants with the high confidence that is required in the clinic. Experimentally measuring a variant's functional consequences can provide clearer guidance, but individual assays performed only after the discovery of the variant are both time and resource intensive. Here, we discuss how multiplex assays of variant effect (MAVEs) can be used to measure the functional consequences of all possible variants in disease-relevant loci for a variety of molecular and cellular phenotypes. The resulting large-scale functional data can be combined with machine learning and clinical knowledge for the development of "lookup tables" of accurate pathogenicity predictions. A coordinated effort to produce, analyze, and disseminate large-scale functional data generated by multiplex assays could be essential to addressing the variant-interpretation crisis.

PMID: 28886340 [PubMed - indexed for MEDLINE]



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Non-base-contacting residues enable kaleidoscopic evolution of metazoan C2H2 zinc finger DNA binding.

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Non-base-contacting residues enable kaleidoscopic evolution of metazoan C2H2 zinc finger DNA binding.

Genome Biol. 2017 Sep 06;18(1):167

Authors: Najafabadi HS, Garton M, Weirauch MT, Mnaimneh S, Yang A, Kim PM, Hughes TR

Abstract
BACKGROUND: The C2H2 zinc finger (C2H2-ZF) is the most numerous protein domain in many metazoans, but is not as frequent or diverse in other eukaryotes. The biochemical and evolutionary mechanisms that underlie the diversity of this DNA-binding domain exclusively in metazoans are, however, mostly unknown.
RESULTS: Here, we show that the C2H2-ZF expansion in metazoans is facilitated by contribution of non-base-contacting residues to DNA binding energy, allowing base-contacting specificity residues to mutate without catastrophic loss of DNA binding. In contrast, C2H2-ZF DNA binding in fungi, plants, and other lineages is constrained by reliance on base-contacting residues for DNA-binding functionality. Reconstructions indicate that virtually every DNA triplet was recognized by at least one C2H2-ZF domain in the common progenitor of placental mammals, but that extant C2H2-ZF domains typically bind different sequences from these ancestral domains, with changes facilitated by non-base-contacting residues.
CONCLUSIONS: Our results suggest that the evolution of C2H2-ZFs in metazoans was expedited by the interaction of non-base-contacting residues with the DNA backbone. We term this phenomenon "kaleidoscopic evolution," to reflect the diversity of both binding motifs and binding motif transitions and the facilitation of their diversification.

PMID: 28877740 [PubMed - in process]



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A stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning.

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A stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning.

Development. 2017 Dec 01;144(23):4298-4312

Authors: Tewary M, Ostblom J, Prochazka L, Zulueta-Coarasa T, Shakiba N, Fernandez-Gonzalez R, Zandstra PW

Abstract
How position-dependent cell fate acquisition occurs during embryogenesis is a central question in developmental biology. To study this process, we developed a defined, high-throughput assay to induce peri-gastrulation-associated patterning in geometrically confined human pluripotent stem cell (hPSC) colonies. We observed that, upon BMP4 treatment, phosphorylated SMAD1 (pSMAD1) activity in the colonies organized into a radial gradient. We developed a reaction-diffusion (RD)-based computational model and observed that the self-organization of pSMAD1 signaling was consistent with the RD principle. Consequent fate acquisition occurred as a function of both pSMAD1 signaling strength and duration of induction, consistent with the positional-information (PI) paradigm. We propose that the self-organized peri-gastrulation-like fate patterning in BMP4-treated geometrically confined hPSC colonies arises via a stepwise model of RD followed by PI. This two-step model predicted experimental responses to perturbations of key parameters such as colony size and BMP4 dose. Furthermore, it also predicted experimental conditions that resulted in RD-like periodic patterning in large hPSC colonies, and rescued peri-gastrulation-like patterning in colony sizes previously thought to be reticent to this behavior.

PMID: 28870989 [PubMed - indexed for MEDLINE]



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The New RNA World: Growing Evidence for Long Noncoding RNA Functionality.

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The New RNA World: Growing Evidence for Long Noncoding RNA Functionality.

Trends Genet. 2017 Oct;33(10):665-676

Authors: Jandura A, Krause HM

Abstract
The past decade has seen a major increase in the study of noncoding RNAs (ncRNAs). However, there remains a great deal of confusion and debate over the levels of functionality and mechanisms of action of the majority of these new transcripts. This Opinion article addresses several of these issues, focusing particularly on long ncRNAs (lncRNAs). We reemphasize the unique abilities of RNAs to form myriad structures as well as to interact with other RNAs, DNA, and proteins, which provide them with unique and powerful abilities. One of these, the ability to interact sequence specifically with DNA, has been largely overlooked. Accumulating evidence suggests that evolution has taken advantage of RNA's properties via the rapid acquisition of new noncoding genes in testes, with subsequent gains of function in other tissues. This amplification process appears to be one of the major forces driving metazoan evolution and diversity.

PMID: 28870653 [PubMed - in process]



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Metabolomics in Yeast.

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Metabolomics in Yeast.

Cold Spring Harb Protoc. 2017 Sep 01;2017(9):pdb.top083576

Authors: Caudy AA, Mülleder M, Ralser M

Abstract
Budding yeast has from the beginning been a major eukaryotic model for the study of metabolic network structure and function. This is attributable to both its genetic and biochemical capacities and its role as a workhorse in food production and biotechnology. New inventions in analytical technologies allow accurate, simultaneous detection and quantification of metabolites, and a series of recent findings have placed the metabolic network at center stage in the physiology of the cell. For example, metabolism might have facilitated the origin of life, and in modern organisms it not only provides nutrients to the cell but also serves as a buffer to changes in the cellular environment, a regulator of cellular processes, and a requirement for cell growth. These findings have triggered a rapid and massive renaissance in this important field. Here, we provide an introduction to analysis of metabolomics in yeast.

PMID: 28864573 [PubMed - in process]



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Spectrophotometric Analysis of Ethanol and Glucose Concentrations in Yeast Culture Media.

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Spectrophotometric Analysis of Ethanol and Glucose Concentrations in Yeast Culture Media.

Cold Spring Harb Protoc. 2017 Sep 01;2017(9):pdb.prot089102

Authors: Caudy AA

Abstract
Fermentative growth on glucose is one of the most widely studied conditions of yeast growth in the laboratory. The production of ethanol from sugars is relevant to the wine, beer, and bread industries and to production of biofuels. Assaying the levels of glucose and ethanol in yeast growth medium allows the experimenter to determine the consumption of the carbon source glucose and the production of ethanol. This protocol describes enzyme-coupled assays for determination of glucose and ethanol concentrations in a sample of cell-free culture medium. Enzymes convert glucose or ethanol into other compounds through chemical reactions that reduce NAD(P)(+) to NAD(P)H, and the production of NAD(P)H is measured using a spectrophotometer. The methods presented are highly sensitive, with a detection limit of ∼0.4 mg/L of glucose and 50 mg/L of ethanol, and also have the advantage of high specificity. For example, glucose and fructose have identical chemical formulas and thus cannot be distinguished by a mass spectrometer, but the enzyme assay presented here is specific for glucose. The glucose assay can be coupled to other assays to determine the quantity of additional carbohydrates such as fructose, trehalose, and glycogen.

PMID: 28864566 [PubMed - in process]



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Metabolite Extraction from Saccharomyces cerevisiae for Liquid Chromatography-Mass Spectrometry.

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Metabolite Extraction from Saccharomyces cerevisiae for Liquid Chromatography-Mass Spectrometry.

Cold Spring Harb Protoc. 2017 Sep 01;2017(9):pdb.prot089086

Authors: Rosebrock AP, Caudy AA

Abstract
Prior to mass spectrometric analysis, cellular small molecules must be extracted and separated from interfering components such as salts and culture medium. To ensure minimal perturbation of metabolism, yeast cells grown in liquid culture are rapidly harvested by filtration as described here. Simultaneous quenching of metabolism and extraction is afforded by immediate immersion in low-temperature organic solvent. Samples prepared using this method are suitable for a range of downstream liquid chromatography-mass spectrometry analyses and are stable in solvent for >1 yr at -80°C.

PMID: 28864564 [PubMed - in process]



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First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor.

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First critical repressive H3K27me3 marks in embryonic stem cells identified using designed protein inhibitor.

Proc Natl Acad Sci U S A. 2017 Sep 19;114(38):10125-10130

Authors: Moody JD, Levy S, Mathieu J, Xing Y, Kim W, Dong C, Tempel W, Robitaille AM, Dang LT, Ferreccio A, Detraux D, Sidhu S, Zhu L, Carter L, Xu C, Valensisi C, Wang Y, Hawkins RD, Min J, Moon RT, Orkin SH, Baker D, Ruohola-Baker H

Abstract
The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein-protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.

PMID: 28864533 [PubMed - in process]



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