Quantitative analysis of transcription factor binding and expression using Calling Cards Reporter Arrays

We report a tool, Calling Cards Reporter Arrays (CCRA), that measures transcription factor (TF) binding and the consequences on gene expression for hundreds of synthetic promoters in yeast. Using Cbf1p and MAX, we demonstrate that the CCRA method is able to detect small changes in binding free energy with a sensitivity comparable to in vitro methods, enabling the measurement of energy landscapes in vivo. We then demonstrate the quantitative analysis of cooperative interactions by measuring Cbf1p binding at synthetic promoters with multiple sites. We find that the cooperativity between Cbf1p dimers varies sinusoidally with a period of 10.65 bp and energetic cost of 1.37 KBT for sites that are positioned \u27out of phase\u27. Finally, we characterize the binding and expression of a group of TFs, Tye7p, Gcr1p and Gcr2p, that act together as a \u27TF collective\u27, an important but poorly characterized model of TF cooperativity. We demonstrate that Tye7p often binds promoters without its recognition site because it is recruited by other collective members, whereas these other members require their recognition sites, suggesting a hierarchy where these factors recruit Tye7p but not vice versa. Our experiments establish CCRA as a useful tool for quantitative investigations into TF binding and function

Regulatory Signaling Networks Governing Budding Yeast Filamentous Growth.

In response to specific stresses, the budding yeast Saccharomyces cerevisiae undergoes a morphogenetic program wherein cells elongate and interconnect, forming pseudohyphal filaments. This filamentous growth transition has been studied extensively as a model signaling system with relevance to fungal pathogenicity. Classic studies have identified core pseudohyphal growth signaling modules in yeast; however, the scope of regulatory networks that control yeast filamentation is broad and incompletely defined. In this work, we address the genetic basis of yeast pseudohyphal growth by implementing a systematic analysis of 4909 genes for overexpression phenotypes in a filamentous strain of S. cerevisiae. Our results identify 551 genes conferring exaggerated invasive growth upon overexpression under normal vegetative growth conditions. In particular, overexpression screening suggests that nuclear export of the osmoresponsive MAPK Hog1p may enhance pseudohyphal growth. The function of nuclear Hog1p is unclear from previous studies, but our analysis using a nuclear-depleted form of Hog1p is consistent with a role for nuclear Hog1p in repressing pseudohyphal growth. In a second study, we interrogate the kinase signaling network regulating filamentous growth using a quantitative phosphoproteomic approach. The filamentous growth transition is controlled by at least three kinase signaling pathways; however, the global scope of filamentous growth kinase signaling networks is not presently understood. We engineered kinase-dead mutations in a core set of eight regulatory protein kinases and identified differentially phosphorylated proteins relative to wild type by SILAC-based mass spectrometry. Our analysis reveals 752 significantly differentially phosphorylated phosphopeptides, including many that are previously unsurveyed in any yeast strain. From this set of significantly differentially abundant phosphopeptides, we identify novel functional regulatory phosphorylation events crucial for proper filamentation. This collective phosphoproteomic data also reveals novel contributions of two cellular processes during filamentous growth: First, genetic analysis suggests that the components of a translationally repressive mRNA decay complex regulate MAPK signaling downstream of the MAPKKK Stellp. Second, null mutants of the inositol kinase pathway genes indicate an unexpected regulatory role for soluble inositol polyphosphates in the regulation of filamentous growth. In sum, this collective work more completely defines the genomic complement and kinase signaling networks contributing to this model stress response.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/108918/1/chashive_1.pd

Zinc cluster transcription factors frequently activate target genes using a non-canonical half-site binding mode

Gene expression changes are orchestrated by transcription factors (TFs), which bind to DNA to regulate gene expression. It remains surprisingly difficult to predict basic features of the transcriptional process, including in vivo TF occupancy. Existing thermodynamic models of TF function are often not concordant with experimental measurements, suggesting undiscovered biology. Here, we analyzed one of the most well-studied TFs, the yeast zinc cluster Gal4, constructed a Shea-Ackers thermodynamic model to describe its binding, and compared the results of this model to experimentally measured Gal4p binding in vivo. We found that at many promoters, the model predicted no Gal4p binding, yet substantial binding was observed. These outlier promoters lacked canonical binding motifs, and subsequent investigation revealed Gal4p binds unexpectedly to DNA sequences with high densities of its half site (CGG). We confirmed this novel mode of binding through multiple experimental and computational paradigms; we also found most other zinc cluster TFs we tested frequently utilize this binding mode, at 27% of their targets on average. Together, these results demonstrate a novel mode of binding where zinc clusters, the largest class of TFs in yeast, bind DNA sequences with high densities of half sites

Pycallingcards: An integrated environment for visualizing, analyzing, and interpreting Calling Cards data

MOTIVATION: Unraveling the transcriptional programs that control how cells divide, differentiate, and respond to their environments requires a precise understanding of transcription factors\u27 (TFs) DNA-binding activities. Calling cards (CC) technology uses transposons to capture transient TF binding events at one instant in time and then read them out at a later time. This methodology can also be used to simultaneously measure TF binding and mRNA expression from single-cell CC and to record and integrate TF binding events across time in any cell type of interest without the need for purification. Despite these advantages, there has been a lack of dedicated bioinformatics tools for the detailed analysis of CC data. RESULTS: We introduce Pycallingcards, a comprehensive Python module specifically designed for the analysis of single-cell and bulk CC data across multiple species. Pycallingcards introduces two innovative peak callers, CCcaller and MACCs, enhancing the accuracy and speed of pinpointing TF binding sites from CC data. Pycallingcards offers a fully integrated environment for data visualization, motif finding, and comparative analysis with RNA-seq and ChIP-seq datasets. To illustrate its practical application, we have reanalyzed previously published mouse cortex and glioblastoma datasets. This analysis revealed novel cell-type-specific binding sites and potential sex-linked TF regulators, furthering our understanding of TF binding and gene expression relationships. Thus, Pycallingcards, with its user-friendly design and seamless interface with the Python data science ecosystem, stands as a critical tool for advancing the analysis of TF functions via CC data. AVAILABILITY AND IMPLEMENTATION: Pycallingcards can be accessed on the GitHub repository: https://github.com/The-Mitra-Lab/pycallingcards

The yeast Sks1p kinase signaling network regulates pseudohyphal growth and glucose response.

The yeast Saccharomyces cerevisiae undergoes a dramatic growth transition from its unicellular form to a filamentous state, marked by the formation of pseudohyphal filaments of elongated and connected cells. Yeast pseudohyphal growth is regulated by signaling pathways responsive to reductions in the availability of nitrogen and glucose, but the molecular link between pseudohyphal filamentation and glucose signaling is not fully understood. Here, we identify the glucose-responsive Sks1p kinase as a signaling protein required for pseudohyphal growth induced by nitrogen limitation and coupled nitrogen/glucose limitation. To identify the Sks1p signaling network, we applied mass spectrometry-based quantitative phosphoproteomics, profiling over 900 phosphosites for phosphorylation changes dependent upon Sks1p kinase activity. From this analysis, we report a set of novel phosphorylation sites and highlight Sks1p-dependent phosphorylation in Bud6p, Itr1p, Lrg1p, Npr3p, and Pda1p. In particular, we analyzed the Y309 and S313 phosphosites in the pyruvate dehydrogenase subunit Pda1p; these residues are required for pseudohyphal growth, and Y309A mutants exhibit phenotypes indicative of impaired aerobic respiration and decreased mitochondrial number. Epistasis studies place SKS1 downstream of the G-protein coupled receptor GPR1 and the G-protein RAS2 but upstream of or at the level of cAMP-dependent PKA. The pseudohyphal growth and glucose signaling transcription factors Flo8p, Mss11p, and Rgt1p are required to achieve wild-type SKS1 transcript levels. SKS1 is conserved, and deletion of the SKS1 ortholog SHA3 in the pathogenic fungus Candida albicans results in abnormal colony morphology. Collectively, these results identify Sks1p as an important regulator of filamentation and glucose signaling, with additional relevance towards understanding stress-responsive signaling in C. albicans

Inositol polyphosphates regulate and predict yeast pseudohyphal growth phenotypes

<div><p>Pseudohyphal growth is a nutrient-regulated program in which budding yeast form multicellular filaments of elongated and connected cells. Filamentous growth is required for virulence in pathogenic fungi and provides an informative model of stress-responsive signaling. The genetics and regulatory networks modulating pseudohyphal growth have been studied extensively, but little is known regarding the changes in metabolites that enable pseudohyphal filament formation. Inositol signaling molecules are an important class of metabolite messengers encompassing highly phosphorylated and diffusible inositol polyphosphates (InsPs). We report here that the InsP biosynthesis pathway is required for wild-type pseudohyphal growth. Under nitrogen-limiting conditions that can induce filamentation, InsPs exhibit characteristic profiles, distinguishing the InsP₇ pyrophosphate isoforms 1PP-InsP₅ and 5PP-InsP₅. Deletion and overexpression analyses of InsP kinases identify elevated levels of 5PP-InsP₅ relative to 1PP-InsP₅ in mutants exhibiting hyper-filamentous growth. Overexpression of <i>KCS1</i>, which promotes formation of inositol pyrophosphates, is sufficient to drive pseudohyphal filamentation on medium with normal nitrogen levels. We find that the kinases Snf1p (AMPK), Kss1p, and Fus3p (MAPKs), required for wild-type pseudohyphal growth, are also required for wild-type InsP levels. Deletion analyses of the corresponding kinase genes indicate elevated InsP₃ levels and an absence of exaggerated 5PP-InsP₅ peaks in trace profiles from <i>snf1</i>Δ/Δ and <i>kss1</i>Δ/Δ mutants exhibiting decreased pseudohyphal filamentation. Elevated 5PP-InsP₅:1PP-InsP₅ ratios are present in the hyperfilamentous <i>fus3</i> deletion mutant. Collectively, the data identify the presence of elevated 5PP-InsP₅ levels relative to other inositol pyrophosphates as an <i>in vivo</i> marker of hyper-filamentous growth, while providing initial evidence for the regulation of InsP signaling by pseudohyphal growth kinases.</p></div

InsP signaling mutants can uncouple the pseudohyphal response from growth in nitrogen-limiting conditions.

<p><b>(A)</b> Pseudohyphal growth phenotypes for InsP signaling mutants grown on standard media with normal levels of ammonium sulfate. The wild-type strain carries the empty pSGP47 vector so that all strains can be cultured uniformly. The wild-type filamentous strain exhibits no pseudohyphal growth under these conditions (“-”). To maintain consistency with other figures, pseudohyphal growth was measured here relative to the corresponding wild-type strain grown under low-nitrogen conditions that induce filamentation. By this comparison, as expected, the wild-type strain under normal nitrogen conditions does not exhibit pseudohyphal filamentation with a colony circumference ratio of roughly 0.6 compared against levels observed in low-nitrogen media. Scale bar, 500 μm. <b>(B)</b> Representative InsP profiles of the indicated strains grown in media with normal levels of ammonium sulfate. Fractions are indicated on the X-axis, with percent CPM shown on the Y-axis. Mean ratios of 5PP-InsP₅:1PP-InsP₅ with standard deviations are shown for the analyzed strains.</p

Overexpression mutants with elevated levels of 5PP-InsP₅ relative to other inositol pyrophosphates exhibit exaggerated pseudohyphal growth.

<p><b>(A)</b> Representative InsP profiles of strains overexpressing <i>VIP1</i> and <i>KCS1</i> grown for eight hours in low-nitrogen minimal media. The ratio of InsP₇ isoforms is indicate as mean values with standard deviations shown. InsP₇ ratios in independent replicates are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007493#pgen.1007493.s011" target="_blank">S7 Table</a>. <b>(B)</b> Representative InsP profile of <i>VIP1</i> overexpression in a <i>siw14</i>Δ/Δ background strain grown in low nitrogen media; a representative InsP profile for <i>siw14</i>Δ/Δ is included also. The InsP trace of the wild-type strain for this analysis is shown in panel A; all strains were grown under identical conditions. Pseudohyphal growth phenotypes are provided for the indicated mutants. Filamentation was quantified as described in Materials and Methods; mean values with standard deviation are indicated. Scale bars, 500 μm.</p

Deletion of the <i>SIW14</i> phosphatase gene results in exaggerated pseudohyphal growth.

<p><b>(A)</b> Surface pseudohyphal filamentation phenotypes of homozygous diploid strains with the indicated InsP phosphatase mutations. Strains were grown on low-nitrogen SLAD medium. Exaggerated pseudohyphal growth in the <i>siw14</i>Δ/Δ mutant is indicated with a “++”; wild-type pseudohypha growth levels are indicated with a “+”. Pseudohyphal growth assays were performed and quantified as described; mean values with standard deviation for three replicates are indicated. Scale bar, 500 μm. <b>(B)</b> Representative InsP profiles of mutated InsP phosphatase strains grown in low-nitrogen minimal media for eight hours. The proportional CPM counts are shown to 1% in order to indicate the changes in IP₆ levels. The InsP trace for a wild-type control strain is shown below the plot. Ratios of 5PP-InsP₅:1PP-InsP₅ are indicated as mean with standard deviation. Ratios of InsP₇ in individual replicates are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007493#pgen.1007493.s011" target="_blank">S7 Table</a>.</p

The pseudohyphal growth regulatory kinases Snf1p, Kss1p, and Fus3p are required for wild-type InsP signaling.

<p><b>(A)</b> Representative InsP profiles of the wild-type filamentous Σ1278b strain and an otherwise isogenic <i>snf1</i>Δ/Δ mutant grown in low-nitrogen media. <b>(B)</b> Representative InsP profiles are indicated for <i>kss1</i>Δ/Δ and <i>fus3</i>Δ/Δ mutants grown in low-nitrogen media; an InsP profile of the wild-type Σ1278b strain under identical growth conditions is provided for comparison. Mean ratios of the InsP₇ isoforms are shown with standard deviation. InsP₇ ratios of independent replicates are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007493#pgen.1007493.s011" target="_blank">S7 Table</a>.</p