Showing papers by "Michael Snyder published in 2003"

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

Abstract: 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.

Protein analysis on a proteomic scale.

Abstract: 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.

C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression.

Abstract: To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.

Distribution of NF-κB-binding sites across human chromosome 22

Abstract: We have mapped the chromosomal binding site distribution of a transcription factor in human cells. The NF-κB 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-κB 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-κB binding was also found at genes whose expression was regulated by tumor necrosis factor α, a known inducer of NF-κB-dependent gene expression, as well as adjacent to genes whose expression is not affected by tumor necrosis factor α. Many of these latter genes are either known to be activated by NF-κB under other conditions or are consistent with NF-κB's role in the immune and apoptotic responses. Our results suggest that binding is not restricted to promoter regions and that NF-κB binding occurs at a significant number of genes whose expression is not altered, thereby suggesting that binding alone is not sufficient for gene activation.

The transcriptional activity of human Chromosome 22

Abstract: A DNA microarray representing nearly all of the unique sequences of human Chromosome 22 was constructed and used to measure global-transcriptional activity in placental poly(A) + RNA. We found that many of the known, related and predicted genes are expressed. More importantly, our study reveals twice as many transcribed bases as have been reported previously. Many of the newly discovered expressed fragments were verified by RNA blot analysis and a novel technique called differential hybridization mapping (DHM). Interestingly, a significant fraction of these novel fragments are expressed antisense to previously annotated introns. The coding potential of these novel expressed regions is supported by their sequence conservation in the mouse genome. This study has greatly increased our understanding of the biological information encoded on a human chromosome. To facilitate the dissemination of these results to the scientific community, we have developed a comprehensive Web resource to present the findings of this study and other features of human Chromosome 22 at http://array.mbb.yale.edu/chr22.

Analyzing antibody specificity with whole proteome microarrays

Abstract: Although approximately 10,000 antibodies are available from commercial sources, antibody reagents are still unavailable for most proteins1. Furthermore, new applications such as antibody arrays2,3,4,5 and monoclonal antibody therapeutics6,7 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 ∼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.

Molecular dissection of a yeast septin: distinct domains are required for septin interaction, localization, and function.

Abstract: The septins are a family of cytoskeletal proteins present in animal and fungal cells. They were first identified for their essential role in cytokinesis, but more recently, they have been found to play an important role in many cellular processes, including bud site selection, chitin deposition, cell compartmentalization, and exocytosis. Septin proteins self-associate into filamentous structures that, in yeast cells, form a cortical ring at the mother bud neck. Members of the septin family share common structural domains: a GTPase domain in the central region of the protein, a stretch of basic residues at the amino terminus, and a predicted coiled-coil domain at the carboxy terminus. We have studied the role of each domain in the Saccharomyces cerevisiae septin Cdc11 and found that the three domains are responsible for distinct and sometimes overlapping functions. All three domains are important for proper localization and function in cytokinesis and morphogenesis. The basic region was found to bind the phosphoinositides phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate. The coiled-coil domain is important for interaction with Cdc3 and Bem4. The GTPase domain is involved in Cdc11-septin interaction and targeting to the mother bud neck. Surprisingly, GTP binding appears to be dispensable for Cdc11 function, localization, and lipid binding. Thus, we find that septins are multifunctional proteins with specific domains involved in distinct molecular interactions required for assembly, localization, and function within the cell.

Microarrays to characterize protein interactions on a whole-proteome scale.

Abstract: Protein microarrays contain a defined set of proteins spotted and analyzed at high density, and can be generally classified into two categories; protein profiling arrays and functional protein arrays. Functional protein arrays can be made up of any type of protein, and therefore have a diverse set of useful applications. Advantages of these arrays include low reagent consumption, rapid interpretation of results, and the ability to easily control experimental conditions. The ultimate form of a functional protein array consists of all of the proteins encoded by the genome of an organism; such an array would be the whole proteome equivalent of the whole genome DNA arrays that are now available. While proteome microarrays may not have reached the stage of maturity of DNA microarrays, recent developments have shown that many of the barriers holding back the technology can be overcome. Arrays of this type have already been used to rapidly screen large numbers of proteins simultaneously for biochemical activities, protein-protein interactions, protein-lipid interactions, protein-nucleic acid interactions, and protein-small molecule interactions. Eventually, functional protein arrays will be used to facilitate various steps in the drug discovery and early development processes that are currently bottlenecks in the drug development continuum.

Defining Genes in the Genomics Era

Abstract: Even with the availability of the genome sequences of many different organisms, we are still left wondering about the definition of a true gene. In their Perspective, Snyder and Gerstein discuss different criteria that can be used to define what a gene is in the era of genomics.

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

Abstract: 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.

ExpressYourself: a modular platform for processing and visualizing microarray data

Abstract: 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 multistep 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.

Specific protein targeting during cell differentiation: polarized localization of Fus1p during mating depends on Chs5p in Saccharomyces cerevisiae.

Abstract: In budding yeast, chs5 mutants are defective in chitin synthesis and cell fusion during mating. Chs5p is a late-Golgi protein required for the polarized transport of the chitin synthase Chs3p to the membrane. Here we show that Chs5p is also essential for the polarized targeting of Fus1p, but not of other cell fusion proteins, to the membrane during mating.

Recent developments in analytical and functional protein microarrays.

Abstract: In recent years, the genomes of many different organisms have been fully sequenced and annotated. As a consequence of this information, a number of methods have emerged to study the function of many genes and proteins in parallel. One recent approach for the large-scale analysis of proteins is the use of protein microarrays in which hundreds to thousands of proteins are arrayed and assayed simultaneously. Protein arrays can be used for assessing protein levels and following disease markers, identifying biochemical activities, analyzing post-translational modifications, building interaction networks, and for drug discovery and development. In this review, we discuss the construction of different types of protein arrays, and their numerous and diverse applications.

Identification of novel functional elements in the human genome

Abstract: a nearly complete draft of the human genome has been determined, producing an enormous wealth of information (Olivier et al. 2001). However, the sequence by itself reveals little about the critical elements encoded in the DNA, and consequently, it is paramount to identify the functional elements encoded in the 3 billion base pairs and to determine how they work together to mediate complex processes such as development and responses to environmental alterations. Two essential tasks toward this goal are the identification of coding and transcriptionally active regions in the human genome and determining how they are regulated. The identification of these regions is an essential first step for the comprehensive and systematic analysis of gene and protein function. Thus far, a variety of different approaches have been used for identification of coding sequences and other functional elements in genomic DNA (Snyder and Gerstein 2003). Genes have been identified by generating and sequencing of cDNAs, expressed sequence tags (ESTs), and related approaches , and then mapping the mRNA coding sequences onto genomics DNA (Lander et al. 2001). Genes have also been identified by computational methods such as motif searches, identification of long open reading frames, and comparative genomic studies to identify conserved sequences, particularly those predicted to encode proteins (Lander et al. 2001; Venter et al. 2001; Water-ston et al. 2002). The availability of the full genomic DNA sequence allows the direct identification of transcribed sequences by globally interrogating all regions of the genome using genomic DNA microarrays. In addition to identification of genes, it is also of high interest to identify the elements that regulate their expression. Such information is crucial for understanding how the activity of genes is controlled and thereby what is essential for understanding cell proliferation and differentiation. Approaches to analyze gene regulation in the past have been hampered by the fact that the approaches are either not comprehensive or are indirect. For example, comparative analysis of gene expression using DNA mi-croarrays in lines expressing or lacking a factor of interest is indirect—changes in gene expression may be due to downstream effects of the factor. Recently, we have developed an approach for identifying the binding sites of transcription factors on a global scale (Iyer et al. 2001; Horak et al. 2002). This procedure involves immunoprecipitation of chromatin (ChIP) associated with a transcription factor of interest and using the associated DNA to probe a genomic DNA array containing …