The Contribution of E2F-Regulated Transcription to Drosophila PCNA Gene Function

E2F proteins control cell cycle progression by predominantly acting as either activators or repressors of transcription [1]. How the antagonizing activities of different E2Fs are integrated by cis-acting control regions into a final transcriptional output in an intact animal is not well understood. E2F function is required for normal development in many species [2–7], but it is not completely clear for which genes E2F-regulated transcription provides an essential biological function. To address these questions, we have characterized the control region of the Drosophila PCNA gene. A single E2F binding site within a 100-bp enhancer is necessary and sufficient to direct the correct spatiotemporal program of G1-S-regulated PCNA expression during development. This dynamic program requires both E2F-mediated transcriptional activation and repression, which, in Drosophila, are thought to be carried out by two distinct E2F proteins [2, 3, 8–11]. Our data suggest that functional antagonism between these different E2F proteins can occur in vivo by competition for the same binding site. An engineered PCNA gene with mutated E2F binding sites supports a low level of expression that can partially rescue the lethality of PCNA null mutants. Thus, E2F regulation of PCNA is dispensable for viability, but is nonetheless important for normal Drosophila development

Mutations of the Drosophila dDP, dE2F, and cyclin E Genes Reveal Distinct Roles for the E2F-DP Transcription Factor and Cyclin E during the G(1)-S Transition

Activation of heterodimeric E2F-DP transcription factors can drive the G(1)-S transition. Mutation of the Drosophila melanogaster dE2F gene eliminates transcriptional activation of several replication factors at the G(1)-S transition and compromises DNA replication. Here we describe a mutation in the Drosophila dDP gene. As expected for a defect in the dE2F partner, this mutation blocks G(1)-S transcription of DmRNR2 and cyclin E as previously described for mutations of dE2F. Mutation of dDP also causes an incomplete block of DNA replication. When S phase is compromised by reducing the activity of dE2F-dDP by either a dE2F or dDP mutation, the first phenotype detected is a reduction in the intensity of BrdU incorporation and a prolongation of the labeling. Notably, in many cells, there was no detected delay in entry into this compromised S phase. In contrast, when cyclin E function was reduced by a hypomorphic allele combination, BrdU incorporation was robust but the timing of S-phase entry was delayed. We suggest that dE2F-dDP contributes to the expression of two classes of gene products: replication factors, whose abundance has a graded effect on replication, and cyclin E, which triggers an all-or-nothing transition from G(1) to S phase

Determinants of leader cells in collective cell migration.

Contains fulltext : 88409.pdf (publisher's version ) (Open Access)Collective migration is a basic mechanism of cell translocation during morphogenesis, wound repair and cancer invasion. Collective movement requires cells to retain cell-cell contacts, exhibit group polarization with defined front-rear asymmetry, and consequently move as one multicellular unit. Depending on the cell type, morphology of the group and the tissue context, distinct mechanisms control the leading edge dynamics and guidance. Leading edge migration may either result from adhesion to ECM and contractile pulling, or from forward pushing. The leading edge consists of either one or few dedicated tip cells or a multicellular leading row that generate adhesion and traction towards the tissue substrate. Alternatively, a multicellular bud consisting of many cells protrudes collectively by proliferation and growth thereby mechanically expanding and pushing towards the tissue stroma. Each type of collective guidance engages distinct spatiotemporal molecular control and feedback towards rearward cells and the adjacent tissue microenvironment; these include intrinsic polarity mechanisms regulated by the interplay between cell-cell and cell-ECM interactions; or the heterotypic integration of stromal cells that adopt leader cell functions. We here classify molecular and mechanical mechanisms of leading function in collective cell migration during morphogenesis and wound repair and discuss how these are recapitulated during collective invasion of cancer cells

Chemical Probe Identification Platform for Orphan GPCRs Using Focused Compound Screening: GPR39 as a Case Example

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Chemical Probe Identification Platform for Orphan GPCRs Using Focused Compound Screening: GPR39 as a Case Example

Orphan G protein-coupled receptors (oGPCRs) are a class of integral membrane proteins for which endogenous ligands or transmitters have not yet been discovered. Transgenic animal technologies have uncovered potential roles for many of these oGPCRs, providing new targets for the treatment of various diseases. Understanding signaling pathways of oGPCRs and validating these receptors as potential drug targets requires the identification of chemical probe compounds to be used in place of endogenous ligands to interrogate these receptors. A novel chemical probe identification platform was created in which GPCR-focused libraries were screened against sets of oGPCR targets, with a goal of discovering fit-for-purpose chemical probes for the more druggable members of the set. Application of the platform to a set of oGPCRs resulted in the discovery of the first reported small molecule agonists for GPR39, a receptor implicated in the regulation of insulin secretion and preservation of beta cells in the pancreas. Compound <b>1</b> stimulated intracellular calcium mobilization in recombinant and native cells in a GPR39-specific manner but did not potentiate glucose-stimulated insulin secretion in human islet preparations