RNA sequencing reveals two major classes of gene expression levels in metazoan cells

The expression level of a gene is often used as a proxy for determining whether the protein or RNA product is functional in a cell or tissue. Therefore, it is of fundamental importance to understand the global distribution of gene expression levels, and to be able to interpret it mechanistically and functionally. Here we use RNA sequencing (RNA‐seq) of mouse Th2 cells, coupled with a range of other techniques, to show that all genes can be separated, based on their expression abundance, into two distinct groups: one group comprised of lowly expressed and putatively non‐functional mRNAs, and the other of highly expressed mRNAs with active chromatin marks at their promoters. These observations are confirmed in many other microarray and RNA‐seq data sets of metazoan cell types.     

Construction of a Large Extracellular Protein Interaction Network and Its Resolution by Spatiotemporal Expression Profiling

Extracellular interactions involving both secreted and membrane-tethered receptor proteins are essential to initiate signaling pathways that orchestrate cellular behaviors within biological systems. Because of the biochemical properties of these proteins and their interactions, identifying novel extracellular interactions remains experimentally challenging. To address this, we have recently developed an assay, AVEXIS (avidity-based extracellular interaction screen) to detect low affinity extracellular interactions on a large scale and have begun to construct interaction networks between zebrafish receptors belonging to the immunoglobulin and leucine-rich repeat protein families to identify novel signaling pathways important for early development. Here, we expanded our zebrafish protein library to include other domain families and many more secreted proteins and performed our largest screen to date totaling 16,544 potential unique interactions. We report 111 interactions of which 96 are novel and include the first documented extracellular ligands for 15 proteins. By including 77 interactions from previous screens, we assembled an expanded network of 188 extracellular interactions between 92 proteins and used it to show that secreted proteins have twice as many interaction partners as membrane-tethered receptors and that the connectivity of the extracellular network behaves as a power law. To try to understand the functional role of these interactions, we determined new expression patterns for 164 genes within our clone library by using whole embryo in situ hybridization at five key stages of zebrafish embryonic development. These expression data were integrated with the binding network to reveal where each interaction was likely to function within the embryo and were used to resolve the static interaction network into dynamic tissue- and stage-specific subnetworks within the developing zebrafish embryo. All these data were organized into a freely accessible on-line database called ARNIE (AVEXIS Receptor Network with Integrated Expression; www.sanger.ac.uk/arnie) and provide a valuable resource of new extracellular signaling interactions for developmental biology.The individual cells within a multicellular organism communicate with each other to provide coordinated and appropriate cellular responses that ensure the normal development and maintenance of the organism as a whole. Frequently, these intercellular dialogues are initiated by specific binding events mediated by cell surface receptor glycoproteins, the molecular bridges through which cells receive information from their immediate environment and subsequently relay it to cytoplasmic signaling networks. Despite their importance in many different biological contexts, identifying novel extracellular protein interactions remains technically challenging because membrane proteins are difficult to biochemically manipulate, and their interactions are typified by extremely low interaction strengths (1, 2). Several scalable methods to identify this class of interactions have been developed that account for some or all of these challenges: they include approaches based on protein complementation (3), phage display (4), mass spectrometry (5), surface plasmon resonance (6–9), and detection of direct binding between multivalent recombinant proteins (Refs. 10–12; for a review, see Ref. 1). Our own approach, called avidity-based extracellular interaction screen (AVEXIS)1 (see Fig. 1A), involves expression by mammalian cells of soluble recombinant ectodomain regions of cell surface receptor and secreted proteins as either a monobiotinylated bait or a pentamerized β-lactamase-tagged prey. The pentamerization is achieved through a peptide derived from the cartilage oligomeric matrix protein (COMP) (13) and is necessary to increase the local concentration of the ectodomain fragments to effect gains in the overall binding avidity so that even very transient interactions can be detected. We have determined the parameters of the assay and shown that it can reliably detect interactions that are very transient, having monomeric half-lives of ≤0.1 s when subsequently measured in their monomeric state by surface plasmon resonance using purified proteins (10). This assay now permits the systematic screening of thousands of binary interactions and can be used to identify novel receptor-ligand pairs that were previously difficult to detect.Open in a separate windowFig. 1.Summary of AVEXIS method and overall approach to construct and resolve extracellular protein interaction networks. A, the entire ectodomains of endogenous cell surface receptors (pink) are expressed as both monomeric biotinylated baits (B) and pentamerized β-lactamase-tagged (β) preys (P). Bait proteins are immobilized in individual wells of streptavidin-coated microtiter plates and probed with a normalized prey, which, if the two proteins physically interact, is captured within the well. B, a flowchart presenting an overall summary of the work described here, including how interactions from two previous screens (Refs. 10 (Bushell 2008) and 27 (Söllner 2009)) were integrated into the larger network of interactions. C, positive interactions are detected by adding a colorimetric β-lactamase substrate, nitrocefin, which is converted from a yellow to red product. Two typical screening plates are shown illustrating a heterophilic interaction between Cadm3 and Cadm4 that is detected in both bait-prey orientations and a homophilic interaction involving Cadm3. The controls for each prey included a negative bait, the rat Cd4d3+4 protein tag alone (well G6), and a biotinylated anti-rat Cd4 monoclonal antibody (OX68), which captured the Cd4-tagged preys (well G7). Each plate also contained positive control interactions: the rat Cd200R prey was probed against rat Cd200 baits immobilized at the normalized screening threshold and at 1:500 and 1:1,000 dilutions (wells G8–G10, respectively) and against the negative Cd4d3+4 bait (well G12). An additional negative bait (Fgfr1b) was included for both preys (wells G5 and G11). SLRPs, small leucine-rich proteoglycans.To identify novel extracellular receptor-ligand interactions that initiate signaling pathways important for vertebrate development, we have selected two protein families that constitute a significant proportion of the extracellular proteome: the immunoglobulin superfamily (IgSF) (14) and leucine-rich repeat (LRR) (15) families. These two families, which both contain membrane-tethered receptors and secreted proteins, are disproportionately expanded in vertebrates relative to invertebrates and are therefore likely to be involved in initiating vertebrate-specific signaling processes (16). IgSF proteins are used as cell surface recognition molecules in a diverse array of tissues, including the immune and nervous systems (17). They function by forming specific receptor-ligand pairs with other IgSF receptors but also other protein domain families, including the LRR (18). Extracellular LRR domain-containing proteins can be separated into two main families: the small leucine-rich proteoglycans, which are secreted and form a major component of the extracellular matrix (19), and cell surface receptors that are involved in the development and maintenance of the nervous system (20). To elucidate the functional role of novel interactions in vertebrate development, we have chosen to screen for novel receptor-ligand pairs from the zebrafish. This popular model organism has many experimental advantages to study early developmental processes, including the amenability to forward (21–23) and reverse (24, 25) genetics approaches. Importantly, the ready accessibility of large numbers of externally developing translucent embryos and the ability to determine large scale gene expression patterns at different stages of embryonic development make whole organism spatiotemporal gene expression profiling possible (26).Using this approach, we first reported a network of 43 interactions within the zebrafish IgSF proteins (10) and subsequently a neural network of 34 interactions between membrane-tethered receptors from both the IgSF and LRR families (27). Here, we expanded our zebrafish recombinant library by 87 proteins to include many more secreted factors and proteins from other families and discovered 111 new interactions. By combining them with interactions identified in our previous screens, we compiled an expanded static network containing 188 interactions that we analyzed to identify properties of extracellular interaction networks. To begin the functional validation of identified interactions, we determined the developmental expression patterns of all genes encoding proteins within our library and describe here 164 new gene expression patterns, many of which are highly dynamic and tissue-restricted. By integrating the spatiotemporal expression patterns with the interaction network, we were able to determine in which stages and tissues each interaction was likely to function and resolved the aggregate interaction network into stage- and tissue-specific subnetworks. The work described in this study is summarized schematically in Fig. 1B. To facilitate navigation of these data, they were organized into a freely accessible on-line database that provides a valuable and accessible resource of new extracellular signaling interactions for developmental biology.     

The impact of gene expression regulation on evolution of extracellular signaling pathways

Extracellular protein interactions are crucial to the development of multicellular organisms because they initiate signaling pathways and enable cellular recognition cues. Despite their importance, extracellular protein interactions are often under-represented in large scale protein interaction data sets because most high throughput assays are not designed to detect low affinity extracellular interactions. Due to the lack of a comprehensive data set, the evolution of extracellular signaling pathways has remained largely a mystery. We investigated this question using a combined data set of physical pairwise interactions between zebrafish extracellular proteins, mainly from the immunoglobulin superfamily and leucine-rich repeat families, and their spatiotemporal expression profiles. We took advantage of known homology between proteins to estimate the relative rates of changes of four parameters after gene duplication, namely extracellular protein interaction, expression pattern, and the divergence of extracellular and intracellular protein sequences. We showed that change in expression profile is a major contributor to the evolution of signaling pathways followed by divergence in intracellular protein sequence, whereas extracellular sequence and interaction profiles were relatively more conserved. Rapidly evolving expression profiles will eventually drive other parameters to diverge more quickly because differentially expressed proteins get exposed to different environments and potential binding partners. This allows homologous extracellular receptors to attain specialized functions and become specific to tissues and/or developmental stages.     

Chloroplast Signaling Gates Thermotolerance in Arabidopsis

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Interfacing systems biology and synthetic biology

A report of BioSysBio 2009, the IET conference on Synthetic Biology, Systems Biology and Bioinformatics, Cambridge, UK, 23-25 March 2009.     

Copy Number Variation in Thai Population

Copy number variation (CNV) is a major genetic polymorphism contributing to genetic diversity and human evolution. Clinical application of CNVs for diagnostic purposes largely depends on sufficient population CNV data for accurate interpretation. CNVs from general population in currently available databases help classify CNVs of uncertain clinical significance, and benign CNVs. Earlier studies of CNV distribution in several populations worldwide showed that a significant fraction of CNVs are population specific. In this study, we characterized and analyzed CNVs in 3,017 unrelated Thai individuals genotyped with the Illumina Human610, Illumina HumanOmniexpress, or Illumina HapMap550v3 platform. We employed hidden Markov model and circular binary segmentation methods to identify CNVs, extracted 23,458 CNVs consistently identified by both algorithms, and cataloged these high confident CNVs into our publicly available Thai CNV database. Analysis of CNVs in the Thai population identified a median of eight autosomal CNVs per individual. Most CNVs (96.73%) did not overlap with any known chromosomal imbalance syndromes documented in the DECIPHER database. When compared with CNVs in the 11 HapMap3 populations, CNVs found in the Thai population shared several characteristics with CNVs characterized in HapMap3. Common CNVs in Thais had similar frequencies to those in the HapMap3 populations, and all high frequency CNVs (>20%) found in Thai individuals could also be identified in HapMap3. The majorities of CNVs discovered in the Thai population, however, were of low frequency, or uniquely identified in Thais. When performing hierarchical clustering using CNV frequencies, the CNV data were clustered into Africans, Europeans, and Asians, in line with the clustering performed with single nucleotide polymorphism (SNP) data. As CNV data are specific to origin of population, our population-specific reference database will serve as a valuable addition to the existing resources for the investigation of clinical significance of CNVs in Thais and related ethnicities.