Dissection of a Ciona regulatory element reveals complexity of cross-species enhancer activity.

Vertebrate genomes share numerous conserved non-coding elements, many of which function as enhancer elements and are hypothesised to be under evolutionary constraint due to a need to be bound by combinations of sequence-specific transcription factors. In contrast, few such conserved elements can be detected between vertebrates and their closest invertebrate relatives. Despite this lack of sequence identity, cross-species transgenesis has identified some cases where non-coding DNA from invertebrates drives reporter gene expression in transgenic vertebrates in patterns reminiscent of the expression of vertebrate orthologues. Such instances are presumed to reflect the presence of conserved suites of binding sites in the regulatory regions of invertebrate and vertebrate orthologues, such that both regulatory elements can correctly interpret the trans-activating environment. Shuffling of binding sites has been suggested to lie behind loss of sequence conservation; however this has not been experimentally tested. Here we examine the underlying basis of enhancer activity for the Ciona intestinalis ßγ-crystallin gene, which drives expression in the lens of transgenic vertebrates despite the Ciona lineage predating the evolution of the lens. We construct an interactive gene regulatory network (GRN) for vertebrate lens development, allowing network interactions to be robustly catalogued and conserved network components and features to be identified. We show that a small number of binding motifs are necessary for Ciona ßγ-crystallin expression, and narrow down the likely factors that bind to these motifs. Several of these overlap with the conserved core of the vertebrate lens GRN, implicating these sites in cross species function. However when we test these motifs in a transgenic vertebrate they prove to be dispensable for reporter expression in the lens. These results show that current models depicting cross species enhancer function as dependent on conserved binding sites can be overly simplistic, with sound evolutionary inference requiring detailed dissection of underlying mechanisms.

myGRN: a database and visualisation system for the storage and analysis of developmental genetic regulatory networks.

BACKGROUND: Biological processes are regulated by complex interactions between transcription factors and signalling molecules, collectively described as Genetic Regulatory Networks (GRNs). The characterisation of these networks to reveal regulatory mechanisms is a long-term goal of many laboratories. However compiling, visualising and interacting with such networks is non-trivial. Current tools and databases typically focus on GRNs within simple, single celled organisms. However, data is available within the literature describing regulatory interactions in multi-cellular organisms, although not in any systematic form. This is particularly true within the field of developmental biology, where regulatory interactions should also be tagged with information about the time and anatomical location of development in which they occur. DESCRIPTION: We have developed myGRN (http://www.myGRN.org), a web application for storing and interrogating interaction data, with an emphasis on developmental processes. Users can submit interaction and gene expression data, either curated from published sources or derived from their own unpublished data. All interactions associated with publications are publicly visible, and unpublished interactions can only be shared between collaborating labs prior to publication. Users can group interactions into discrete networks based on specific biological processes. Various filters allow dynamic production of network diagrams based on a range of information including tissue location, developmental stage or basic topology. Individual networks can be viewed using myGRV, a tool focused on displaying developmental networks, or exported in a range of formats compatible with third party tools. Networks can also be analysed for the presence of common network motifs. We demonstrate the capabilities of myGRN using a network of zebrafish interactions integrated with expression data from the zebrafish database, ZFIN. CONCLUSION: Here we are launching myGRN as a community-based repository for interaction networks, with a specific focus on developmental networks. We plan to extend its functionality, as well as use it to study networks involved in embryonic development in the future.

Oligomerization of G-protein-coupled receptors shown by selective co-immunoprecipitation.

Recent studies have shown that G-protein-coupled receptors (GPCRs) can assemble as high molecular weight homo- and hetero-oligomeric complexes. This can result in altered receptor-ligand binding, signaling, or intracellular trafficking. We have co-transfected HEK-293 cells with differentially epitope-tagged GPCRs from different subfamilies and determined whether oligomeric complexes were formed by co-immunoprecipitation and immunoblot analysis. This gave the surprising result that the 5HT(1A) receptor was capable of forming hetero-oligomers with all GPCRs tested including the 5HT(1B), 5HT(1D), EDG(1), EDG(3), GPR(26), and GABA(B2) receptors. The testing of other GPCR combinations showed similar results with hetero-oligomer formation occurring for the 5HT(1D) with the 5HT(1B) and EDG(1) receptor. Control studies showed that these complexes were present in co-transfected cells before the time of lysis and that the hetero-oligomers were comprised of GPCRs at discrete stoichiometries. These findings suggest that GPCRs have a natural tendency to form oligomers when co-transfected into cells. Future studies should therefore investigate the presence and physiological role of GPCR hetero-oligomers in cells in which they are endogenously expressed.