ABSTRACT
Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.
Subject(s)
Computational Biology/methods , Gene Expression Profiling/methods , Gene Regulatory Networks , RNA, Messenger/metabolism , Software , Genomics/methods , Proteomics/methodsABSTRACT
Nuclear pore complexes (NPCs) govern macromolecular transport between the nucleus and cytoplasm and serve as key positional markers within the nucleus. Several protein components of yeast NPCs have been implicated in the epigenetic control of gene expression. Among these, Nup2p is unique as it transiently associates with NPCs and, when artificially tethered to DNA, can prevent the spread of transcriptional activation or repression between flanking genes, a function termed boundary activity. To understand this function of Nup2p, we investigated the interactions of Nup2p with other proteins and with DNA using immunopurifications coupled with mass spectrometry and microarray analyses. These data combined with functional assays of boundary activity and epigenetic variegation suggest that Nup2p and the Ran guanylyl-nucleotide exchange factor, Prp20p, interact at specific chromatin regions and enable the NPC to play an active role in chromatin organization by facilitating the transition of chromatin between activity states.
Subject(s)
Chromatin/metabolism , DNA-Binding Proteins/metabolism , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription, Genetic/physiology , Active Transport, Cell Nucleus/physiology , Chromatin/genetics , DNA-Binding Proteins/genetics , Gene Silencing/physiology , Guanine Nucleotide Exchange Factors , Histones/genetics , Histones/metabolism , Microarray Analysis , Models, Biological , Nuclear Pore/genetics , Nuclear Pore Complex Proteins/genetics , Nuclear Proteins/genetics , Nucleosomes/metabolism , Open Reading Frames/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Telomere/genetics , Telomere/metabolismABSTRACT
We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.
Subject(s)
Actins/metabolism , Intracellular Membranes/metabolism , Peroxisomes/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , rho GTP-Binding Proteins/metabolism , Actins/analysis , GTP Phosphohydrolases/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Theoretical , Peroxins , Peroxisomes/genetics , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism , rho GTP-Binding Proteins/geneticsABSTRACT
Yeast cells were induced to proliferate peroxisomes, and microarray transcriptional profiling was used to identify PEX genes encoding peroxins involved in peroxisome assembly and genes involved in peroxisome function. Clustering algorithms identified 224 genes with expression profiles similar to those of genes encoding peroxisomal proteins and genes involved in peroxisome biogenesis. Several previously uncharacterized genes were identified, two of which, YPL112c and YOR084w, encode proteins of the peroxisomal membrane and matrix, respectively. Ypl112p, renamed Pex25p, is a novel peroxin required for the regulation of peroxisome size and maintenance. These studies demonstrate the utility of comparative gene profiling as an alternative to functional assays to identify genes with roles in peroxisome biogenesis.
