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Michael Snyder

Publications and source records attributed to Michael Snyder.

At least 55 records · Page 3Linked to original sources
PubMed2004

Finding new components of the target of rapamycin (TOR) signaling network through chemical genetics and proteome chips.

The TOR (target of rapamycin) proteins play important roles in nutrient signaling in eukaryotic cells. Rapamycin treatment induces a state reminiscent of the nutrient starvation response, often resulting in growth inhibition. Using a chemical genetic modifier screen, we identified two classes of small molecules, small-molecule inhibitors of rapamycin (SMIRs) and small-molecule enhancers of rapamycin (SMERs), that suppress and augment, respectively, rapamycin's effect in the yeast Saccharomyces cerevisiae. Probing proteome chips with biotinylated SMIRs revealed putative intracellular target proteins, including Tep1p, a homolog of the mammalian PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumor suppressor, and Ybr077cp (Nir1p), a protein of previously unknown function that we show to be a component of the TOR signaling network. Both SMIR target proteins are associated with PI(3,4)P2, suggesting a mechanism of regulation of the TOR pathway involving phosphatidylinositides. Our results illustrate the combined use of chemical genetics and proteomics in biological discovery and map a path for creating useful therapeutics for treating human diseases involving the TOR pathway, such as diabetes and cancer.

Humans↗
PubMed2004

Global identification of human transcribed sequences with genome tiling arrays.

Elucidating the transcribed regions of the genome constitutes a fundamental aspect of human biology, yet this remains an outstanding problem. To comprehensively identify coding sequences, we constructed a series of high-density oligonucleotide tiling arrays representing sense and antisense strands of the entire nonrepetitive sequence of the human genome. Transcribed sequences were located across the genome via hybridization to complementary DNA samples, reverse-transcribed from polyadenylated RNA obtained from human liver tissue. In addition to identifying many known and predicted genes, we found 10,595 transcribed sequences not detected by other methods. A large fraction of these are located in intergenic regions distal from previously annotated genes and exhibit significant homology to other mammalian proteins.

Animals↗
PubMed2004

Regulation of gene expression by a metabolic enzyme.

Gene expression in eukaryotes is normally believed to be controlled by transcriptional regulators that activate genes encoding structural proteins and enzymes. To identify previously unrecognized DNA binding activities, a yeast proteome microarray was screened with DNA probes; Arg5,6, a well-characterized mitochondrial enzyme involved in arginine biosynthesis, was identified. Chromatin immunoprecipitation experiments revealed that Arg5,6 is associated with specific nuclear and mitochondrial loci in vivo, and Arg5,6 binds to specific fragments in vitro. Deletion of Arg5,6 causes altered transcript levels of both nuclear and mitochondrial target genes. These results indicate that metabolic enzymes can directly regulate eukaryotic gene expression.

Aldehyde Oxidoreductases↗
PubMed2004

Genomic analysis of regulatory network dynamics reveals large topological changes.

Network analysis has been applied widely, providing a unifying language to describe disparate systems ranging from social interactions to power grids. It has recently been used in molecular biology, but so far the resulting networks have only been analysed statically. Here we present the dynamics of a biological network on a genomic scale, by integrating transcriptional regulatory information and gene-expression data for multiple conditions in Saccharomyces cerevisiae. We develop an approach for the statistical analysis of network dynamics, called SANDY, combining well-known global topological measures, local motifs and newly derived statistics. We uncover large changes in underlying network architecture that are unexpected given current viewpoints and random simulations. In response to diverse stimuli, transcription factors alter their interactions to varying degrees, thereby rewiring the network. A few transcription factors serve as permanent hubs, but most act transiently only during certain conditions. By studying sub-network structures, we show that environmental responses facilitate fast signal propagation (for example, with short regulatory cascades), whereas the cell cycle and sporulation direct temporal progression through multiple stages (for example, with highly inter-connected transcription factors). Indeed, to drive the latter processes forward, phase-specific transcription factors inter-regulate serially, and ubiquitously active transcription factors layer above them in a two-tiered hierarchy. We anticipate that many of the concepts presented here--particularly the large-scale topological changes and hub transience--will apply to other biological networks, including complex sub-systems in higher eukaryotes.

Algorithms↗
PubMed2004

Regulation of polarized growth initiation and termination cycles by the polarisome and Cdc42 regulators.

The dynamic regulation of polarized cell growth allows cells to form structures of defined size and shape. We have studied the regulation of polarized growth using mating yeast as a model. Haploid yeast cells treated with high concentration of pheromone form successive mating projections that initiate and terminate growth with regular periodicity. The mechanisms that control the frequency of growth initiation and termination under these conditions are not well understood. We found that the polarisome components Spa2, Pea2, and Bni1 and the Cdc42 regulators Cdc24 and Bem3 control the timing and frequency of projection formation. Loss of polarisome components and mutation of Cdc24 decrease the frequency of projection formation, while loss of Bem3 increases the frequency of projection formation. We found that polarisome components and the cell fusion proteins Fus1 and Fus2 are important for the termination of projection growth. Our results define the first molecular regulators that control the timing of growth initiation and termination during eukaryotic cell differentiation.

Cell Cycle↗
PubMed2004

Major molecular differences between mammalian sexes are involved in drug metabolism and renal function.

Many anatomical differences exist between males and females; these are manifested on a molecular level by different hormonal environments. Although several molecular differences in adult tissues have been identified, a comprehensive investigation of the gene expression differences between males and females has not been performed. We surveyed the expression patterns of 13,977 mouse genes in male and female hypothalamus, kidney, liver, and reproductive tissues. Extensive differential gene expression was observed not only in the reproductive tissues, but also in the kidney and liver. The differentially expressed genes are involved in drug and steroid metabolism, osmotic regulation, or as yet unresolved cellular roles. In contrast, very few molecular differences were observed between the male and female hypothalamus in both mice and humans. We conclude that there are persistent differences in gene expression between adult males and females. These molecular differences have important implications for the physiological differences between males and females.

Animals↗
PubMed2004

Fast optimal genome tiling with applications to microarray design and homology search.

In this paper, we consider several variations of the following basic tiling problem: given a sequence of real numbers with two size-bound parameters, we want to find a set of tiles of maximum total weight such that each tiles satisfies the size bounds. A solution to this problem is important to a number of computational biology applications such as selecting genomic DNA fragments for PCR-based amplicon microarrays and performing homology searches with long sequence queries. Our goal is to design efficient algorithms with linear or near-linear time and space in the normal range of parameter values for these problems. For this purpose, we first discuss the solution to a basic online interval maximum problem via a sliding-window approach and show how to use this solution in a nontrivial manner for many of the tiling problems introduced. We also discuss NP-hardness results and approximation algorithms for generalizing our basic tiling problem to higher dimensions. Finally, computational results from applying our tiling algorithms to genomic sequences of five model eukaryotes are reported.

Algorithms↗
PubMed2004

Large-scale mutagenesis of the yeast genome using a Tn7-derived multipurpose transposon.

We present here an unbiased and extremely versatile insertional library of yeast genomic DNA generated by in vitro mutagenesis with a multipurpose element derived from the bacterial transposon Tn7. This mini-Tn7 element has been engineered such that a single insertion can be used to generate a lacZ fusion, gene disruption, and epitope-tagged gene product. Using this transposon, we generated a plasmid-based library of approximately 300,000 mutant alleles; by high-throughput screening in yeast, we identified and sequenced 9032 insertions affecting 2613 genes (45% of the genome). From analysis of 7176 insertions, we found little bias in Tn7 target-site selection in vitro. In contrast, we also sequenced 10,174 Tn3 insertions and found a markedly stronger preference for an AT-rich 5-base pair target sequence. We further screened 1327 insertion alleles in yeast for hypersensitivity to the chemotherapeutic cisplatin. Fifty-one genes were identified, including four functionally uncharacterized genes and 25 genes involved in DNA repair, replication, transcription, and chromatin structure. In total, the collection reported here constitutes the largest plasmid-based set of sequenced yeast mutant alleles to date and, as such, should be singularly useful for gene and genome-wide functional analysis.

Amino Acid Sequence↗
PubMed2004

CREB binds to multiple loci on human chromosome 22.

The cyclic AMP-responsive element-binding protein (CREB) is an important transcription factor that can be activated by hormonal stimulation and regulates neuronal function and development. An unbiased, global analysis of where CREB binds has not been performed. We have mapped for the first time the binding distribution of CREB along an entire human chromosome. Chromatin immunoprecipitation of CREB-associated DNA and subsequent hybridization of the associated DNA to a genomic DNA microarray containing all of the nonrepetitive DNA of human chromosome 22 revealed 215 binding sites corresponding to 192 different loci and 100 annotated potential gene targets. We found binding near or within many genes involved in signal transduction and neuronal function. We also found that only a small fraction of CREB binding sites lay near well-defined 5' ends of genes; the majority of sites were found elsewhere, including introns and unannotated regions. Several of the latter lay near novel unannotated transcriptionally active regions. Few CREB targets were found near full-length cyclic AMP response element sites; the majority contained shorter versions or close matches to this sequence. Several of the CREB targets were altered in their expression by treatment with forskolin; interestingly, both induced and repressed genes were found. Our results provide novel molecular insights into how CREB mediates its functions in humans.

Binding Sites↗
PubMed2004

Microbial synergy via an ethanol-triggered pathway.

We have discovered a microbial interaction between yeast, bacteria, and nematodes. Upon coculturing, Saccharomyces cerevisiae stimulated the growth of several species of Acinetobacter, including, A. baumannii, A. haemolyticus, A. johnsonii, and A. radioresistens, as well as several natural isolates of Acinetobacter. This enhanced growth was due to a diffusible factor that was shown to be ethanol by chemical assays and evaluation of strains lacking ADH1, ADH3, and ADH5, as all three genes are involved in ethanol production by yeast. This effect is specific to ethanol: methanol, butanol, and dimethyl sulfoxide were unable to stimulate growth to any appreciable level. Low doses of ethanol not only stimulated growth to a higher cell density but also served as a signaling molecule: in the presence of ethanol, Acinetobacter species were able to withstand the toxic effects of salt, indicating that ethanol alters cell physiology. Furthermore, ethanol-fed A. baumannii displayed increased pathogenicity when confronted with a predator, Caenorhabditis elegans. Our results are consistent with the concept that ethanol can serve as a signaling molecule which can affect bacterial physiology and survival.

Acinetobacter↗
PubMed2003

Analyzing antibody specificity with whole proteome microarrays.

Although approximately 10,000 antibodies are available from commercial sources, antibody reagents are still unavailable for most proteins. Furthermore, new applications such as antibody arrays and monoclonal antibody therapeutics have increased the demand for more specific antibodies to reduce cross-reactivity and side effects. An array containing every protein for the relevant organism represents the ideal format for an assay to test antibody specificity, because it allows the simultaneous screening of thousands of proteins for possible cross-reactivity. As an initial test of this approach, we screened 11 polyclonal and monoclonal antibodies to approximately 5,000 different yeast proteins deposited on a glass slide and found that, in addition to recognizing their cognate proteins, the antibodies cross-reacted with other yeast proteins to varying degrees. Some of the interactions of the antibodies with noncognate proteins could be deduced by alignment of the primary amino acid sequences of the antigens and cross-reactive proteins; however, these interactions could not be predicted a priori. Our findings show that proteome array technology has potential to improve antibody design and selection for applications in both medicine and research.

Antibodies↗
PubMed2003

Negative regulation of calcineurin signaling by Hrr25p, a yeast homolog of casein kinase I.

Calcineurin is a Ca2+/calmodulin-regulated protein phosphatase required for Saccharomyces cerevisiae to respond to a variety of environmental stresses. Calcineurin promotes cell survival during stress by dephosphorylating and activating the Zn-finger transcription factor Crz1p/Tcn1p. Using a high-throughput assay, we screened 119 yeast kinases for their ability to phosphorylate Crz1p in vitro and identified the casein kinase I homolog Hrr25p. Here we show that Hrr25p negatively regulates Crz1p activity and nuclear localization in vivo. Hrr25p binds to and phosphorylates Crz1p in vitro and in vivo. Overexpression of Hrr25p decreases Crz1p-dependent transcription and antagonizes its Ca2+-induced nuclear accumulation. In the absence of Hrr25p, activation of Crz1p by Ca2+/calcineurin is potentiated. These findings represent the first identification of a negative regulator for Crz1p, and establish a novel physiological role for Hrr25p in antagonizing calcineurin signaling.

Calcineurin↗
PubMed2003

A Bayesian networks approach for predicting protein-protein interactions from genomic data.

We have developed an approach using Bayesian networks to predict protein-protein interactions genome-wide in yeast. Our method naturally weights and combines into reliable predictions genomic features only weakly associated with interaction (e.g., messenger RNAcoexpression, coessentiality, and colocalization). In addition to de novo predictions, it can integrate often noisy, experimental interaction data sets. We observe that at given levels of sensitivity, our predictions are more accurate than the existing high-throughput experimental data sets. We validate our predictions with TAP (tandem affinity purification) tagging experiments. Our analysis, which gives a comprehensive view of yeast interactions, is available at genecensus.org/intint.

Bayes Theorem↗
PubMed2003

Distribution of NF-kappaB-binding sites across human chromosome 22.

We have mapped the chromosomal binding site distribution of a transcription factor in human cells. The NF-kappaB family of transcription factors plays an essential role in regulating the induction of genes involved in several physiological processes, including apoptosis, immunity, and inflammation. The binding sites of the NF-kappaB family member p65 were determined by using chromatin immunoprecipitation and a genomic microarray of human chromosome 22 DNA. Sites of binding were observed along the entire chromosome in both coding and noncoding regions, with an enrichment at the 5' end of genes. Strikingly, a significant proportion of binding was seen in intronic regions, demonstrating that transcription factor binding is not restricted to promoter regions. NF-kappaB binding was also found at genes whose expression was regulated by tumor necrosis factor alpha, a known inducer of NF-kappaB-dependent gene expression, as well as adjacent to genes whose expression is not affected by tumor necrosis factor alpha. Many of these latter genes are either known to be activated by NF-kappaB under other conditions or are consistent with NF-kappaB's role in the immune and apoptotic responses. Our results suggest that binding is not restricted to promoter regions and that NF-kappaB binding occurs at a significant number of genes whose expression is not altered, thereby suggesting that binding alone is not sufficient for gene activation.

Binding Sites↗
PubMed2003

ExpressYourself: A modular platform for processing and visualizing microarray data.

DNA microarrays are widely used in biological research; by analyzing differential hybridization on a single microarray slide, one can detect changes in mRNA expression levels, increases in DNA copy numbers and the location of transcription factor binding sites on a genomic scale. Having performed the experiments, the major challenge is to process large, noisy datasets in order to identify the specific array elements that are significantly differentially hybridized. This normally requires aggregating different, often incompatible programs into a multi-step pipeline. Here we present ExpressYourself, a fully integrated platform for processing microarray data. In completely automated fashion, it will correct the background array signal, normalize the Cy5 and Cy3 signals, score levels of differential hybridization, combine the results of replicate experiments, filter problematic regions of the array and assess the quality of individual and replicate experiments. ExpressYourself is designed with a highly modular architecture so various types of microarray analysis algorithms can readily be incorporated as they are developed; for example, the system currently implements several normalization methods, including those that simultaneously consider signal intensity and slide location. The processed data are presented using a web-based graphical interface to facilitate comparison with the original images of the array slides. In particular, Express Yourself is able to regenerate images of the original microarray after applying various steps of processing, which greatly facilities identification of position-specific artifacts. The program is freely available for use at http://bioinfo.mbb.yale.edu/expressyourself.

Algorithms↗
PubMed2003

Protein analysis on a proteomic scale.

The long-term challenge of proteomics is enormous: to define the identities, quantities, structures and functions of complete complements of proteins, and to characterize how these properties vary in different cellular contexts. One critical step in tackling this goal is the generation of sets of clones that express a representative of each protein of a proteome in a useful format, followed by the analysis of these sets on a genome-wide basis. Such studies enable genetic, biochemical and cell biological technologies to be applied on a systematic level, leading to the assignment of biochemical activities, the construction of protein arrays, the identification of interactions, and the localization of proteins within cellular compartments.

Animals↗