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James W Posakony

Publications and source records attributed to James W Posakony.

8 recordsLinked to original sources
PubMed2005

Lateral inhibition in proneural clusters: cis-regulatory logic and default repression by Suppressor of Hairless.

Lateral inhibition, wherein a single cell signals to its neighbors to prevent them from adopting its own fate, is the best-known setting for cell-cell communication via the Notch (N) pathway. During peripheral neurogenesis in Drosophila, sensory organ precursor (SOP) cells arise within proneural clusters (PNCs), small groups of cells endowed with SOP fate potential by their expression of proneural transcriptional activators. SOPs use N signaling to activate in neighboring PNC cells the expression of multiple genes that inhibit the SOP fate. These genes respond transcriptionally to direct regulation by both the proneural proteins and the N pathway transcription factor Suppressor of Hairless [Su(H)], and their activation is generally highly asymmetric; i.e. only in the inhibited (non-SOP) cells of the PNC, and not in SOPs. We show that the substantially higher proneural protein levels in the SOP put this cell at risk of inappropriately activating the SOP-inhibitory genes, even without input from N-activated Su(H). We demonstrate that this is prevented by direct ;default' repression of these genes by Su(H), acting through the same binding sites it uses for activation in non-SOPs. We show that de-repression of even a single N pathway target gene in the SOP can extinguish the SOP cell fate. Finally, we define crucial roles for the adaptor protein Hairless and the co-repressors Groucho and CtBP in conferring repressive activity on Su(H) in the SOP. Our work elucidates the regulatory logic by which N signaling and the proneural proteins cooperate to create the neural precursor/epidermal cell fate distinction during lateral inhibition.

Animals↗
PubMed2005

An ancient transcriptional regulatory linkage.

Changes in gene regulatory networks are a major engine for creating developmental novelty during evolution. Conversely, regulatory linkages that survive for very long evolutionary periods might be characteristic of ancient and abstract functions of fundamental utility to all metazoans. The proneural genes, which encode a distinctive family of basic helix-loop-helix (bHLH) transcriptional activators, act to promote neural cell fates in the ectoderm of diverse species. Here we report that these genes have been associated for at least 600-700 million years--since before the cnidarian/bilaterian divergence--with a high-affinity binding site for Hairy/Enhancer of split (Hes) repressor proteins. We suggest that the systematic identification of such ancient and conserved connections will be a powerful means of uncovering the primordial functions of transcription factors and signaling systems.

Animals↗
PubMed2005

Genetic programs activated by proneural proteins in the developing Drosophila PNS.

Neurogenesis depends on a family of proneural transcriptional activator proteins, but the "proneural" function of these factors is poorly understood, in part because the ensemble of genes they activate, directly or indirectly, has not been identified systematically. We have taken a direct approach to this problem in Drosophila. Fluorescence-activated cell sorting was used to recover a purified population of the cells that comprise the "proneural clusters" from which sensory organ precursors of the peripheral nervous system (PNS) arise. Whole-genome microarray analysis and in situ hybridization was then used to identify and verify a set of genes that are preferentially expressed in proneural cluster cells. Genes in this set encode proteins with a diverse array of implied functions, and loss-of-function analysis of two candidate genes shows that they are indeed required for normal PNS development. Bioinformatic and reporter gene studies further illuminate the cis-regulatory codes that direct expression in proneural clusters.

Animals↗
PubMed2004

GenePalette: a universal software tool for genome sequence visualization and analysis.

To make effective use of the growing host of complete genome sequences, biologists must have easy-to-use software tools that allow them to visualize, analyze, and modify genome data in an interactive and generalized manner. In an effort to bridge the gap between genome and researcher, we have created GenePalette (www.genepalette.org), a desktop application that can access any genome sequence and display the positions of various features [e.g., transcription factor binding sites (TFBSs)] relative to the introns and exons of annotated genes. Written in Java, GenePalette can run on all Java-supporting operating systems (Mac, PC, Unix, Linux). Annotated sequence encompassing the majority of public genome data is rapidly retrieved from GenBank or Ensembl. The software provides intuitive access to the selected genomic region through three interface components: a colorful graphical display showing a schematic of genes and features; an annotated sequence view in which features and genes are highlighted directly on the sequence; and the selectable raw sequence. The three interface components are fully integrated and presented on one page, permitting the user to move easily between representations at different levels of resolution, ranging from kilobases to individual nucleotides. GenePalette is a particularly powerful platform for analyzing the organization of cis-regulatory elements and designing wet-lab experiments to investigate them.

Computational Biology↗
PubMed2004

New Drosophila transgenic reporters: insulated P-element vectors expressing fast-maturing RFP.

In vivo green fluorescent protein (GFP)/red fluorescent protein (RFP) double-labeling studies have been hampered by several inconvenient properties of DsRed, the first described RFP. These disadvantages include a very slow (> 24 h) maturation time, emission of contaminating green light, and low solubility. A recently developed variant of DsRed, called DsRed.T4, has a much shorter maturation time, no significant green emission, and improved solubility. We have constructed Drosophila P-element transformation vectors encoding DsRed.T4 for promoter/enhancer analysis, labeling of living cells, or RFP tagging of proteins. These new vectors have all of the features of the widely used Pelican/Stinger GFP vectors, including insulator sequences to reduce position effects, an extensive polylinker, and both cytoplasmic and nuclear-localized forms of the reporter. We have also constructed an upstream activating sequence (UAS)-DsRed.T4 vector, for GAL4 activation of the reporter. We find that DsRed.T4 is very easily detected in transgenic flies without contamination of the GFP signal and that it matures to its fluorescent form nearly simultaneously with GFP. This advance in Drosophila reporter technology makes timed double-labeling experiments in developing transgenic animals possible for the first time.

Animals↗
PubMed2002

Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless.

The DNA-binding transcription factor Suppressor of Hairless [Su(H)] functions as an activator during Notch (N) pathway signaling, but can act as a repressor in the absence of signaling. Hairless (H), a novel Drosophila protein, binds to Su(H) and has been proposed to antagonize N signaling by inhibiting DNA binding by Su(H). Here we show that, in vitro, H directly binds two corepressor proteins, Groucho (Gro) and dCtBP. Reduction of gro or dCtBP function enhances H mutant phenotypes and suppresses N phenotypes in the adult mechanosensory bristle. This activity of gro is surprising, because it is directed oppositely to its traditionally defined role as a neurogenic gene. We find that Su(H)-H complexes can bind to DNA with high efficiency in vitro. Furthermore, a H-VP16 fusion protein causes dominant-negative phenotypes in vivo, a result consistent with the proposal that H functions in transcriptional repression. Taken together, our findings indicate that "default repression" of N pathway target genes by an unusual adaptor/corepressor complex is essential for proper cell fate specification during Drosophila peripheral nervous system development.

Alcohol Oxidoreductases↗
PubMed2002

SCORE: a computational approach to the identification of cis-regulatory modules and target genes in whole-genome sequence data. Site clustering over random expectation.

A large fraction of the information content of metazoan genomes resides in the transcriptional and posttranscriptional cis-regulatory elements that collectively provide the blueprint for using the protein-coding capacity of the DNA, thus guiding the development and physiology of the entire organism. As successive whole-genome sequencing projects--including those of mice and humans--are completed, we have full access to the regulatory genome of yet another species. But our ability to decipher the cis-regulatory code, and hence to link genes into regulatory networks on a global scale, is currently very limited. Here we describe SCORE (Site Clustering Over Random Expectation), a computational method for identifying transcriptional cis-regulatory modules based on the fact that they often contain, in statistically improbable concentrations, multiple binding sites for the same transcription factor. We have carried out a Drosophila genomewide inventory of predicted binding sites for the Notch-regulated transcription factor Suppressor of Hairless [Su(H)] and found that the fly genome contains highly nonrandom clusterings of Su(H) sites over a broad range of sequence intervals. We found that the most statistically significant clusters are very heavily enriched in both known and logical targets of Su(H) binding and regulation. The utility of the SCORE approach was validated by in vivo experiments showing that proper expression of the novel gene Him in adult muscle precursor cells depends both on Su(H) gene activity and sequences that include a previously unstudied cluster of four Su(H) sites, indicating that Him is a likely direct target of Su(H).

Animals↗