Adult mechanoreceptor bristles in Drosophila develop within undifferentiated epithelial sheets during late larval and early pupal stages. Activity of the achaete and scute proneural transcriptional activators confers on small numbers of cell clusters within this epithelium the competence to adopt the fate of the sensory organ precursor cell (SOP). Within each of these cell cluster, local inhibitory cell-cell interactions mediated by the Notch (N) pathway confine the expression of the SOP fate to a single cell. This determination event is followed by two rounds of patterned cell division that give rise to the 4 kinds of cells that together form the stimulus- receiving apparatus of the bristle. This proposal focuses on components of the Notch cell-cell signalling pathway, which acts in an inhibitory fashion to restrict the number of cells in a cluster that adopt the SOP fate. Delta protein is a transmembrane ligand for the Notch receptor. The Su(H) protein normally binds to an intracellular domain of the Notch receptor, but is released and translocated to the nucleus upon interaction of the Notch and Delta proteins. Su(H) appears to function as a transcription factor; its immediate targets are genes in the E(spl)-C which in turn act as transcriptional repressors at yet other genes. Another protein, Hairless (H), binds to the Su(H) protein and inhibits its DNA-binding activity. Dr. Posakony first proposes to analyze the function of Su(H) and potentially of other proteins in regulating the target genes in the E(spl)-C. He will mutagenize reporter constructs under the control of binding sites for Su(H) to examine the contribution of individual sites and to check for possible synergistic interactions between sites. These results will be verified by in vitro binding studies and tissue culture co-transfection assays using related constructs. Genetic tests will determine whether the musashi gene, which encodes a nuclear RNA-binding protein and which is needed to specify proper bristle cell fates, is a direct target of transcriptional repression by Su(H). This possibility is suggested by the finding that the musashi promoter has 2 sequences matching the consensus for high affinity Su(H) binding sites. In the promoter of many Su(H) target genes, there is a conserved hexameric sequence located between paired Su(H) binding sites. Mutations will be introduced into this site to test its function in vivo. If this hexamer is shown to be of significance, Dr. Posakony will look for hexamer binding activities in extracts of imaginal disks or embryos, and then try to isolate these factors by biochemical purification or by screening expressionm libraries with multimerized hexamer probes. Finally, various assays will be used to see whether the products of two neurogenic genes of currently unknown function, mastermind and neuralized, interact with Su(H) protein. The second specific aim is to investigate the specific function of Bearded and Enhancer of split m4 proteins. These proteins are related in sequence; E(spl)m4 but not Bearded is transcriptionally regulated by Su(H); strong circumstantial evidence suggests that both genes have an important role in peripheral neurogenesis. Antibody will be made against the products of both genes to visualize their cellular and subcellular localization. Other proteins that interact with Bearded and E(spl)m4 proteins will be identified by the yeast two hybrid system or by back-up techniques such as screening expression libraries with labelled proteins. The function of Bearded and E(spl)m4 will be probed by looking at the effects of overexpression of these genes in hsp-70 promoter constructs. As no loss-of-function alleles of E(spl)m4 are currently known, such alleles will be generated by combining small deletions in the E(spl)-C with transgenes of all of the other E(spl)-C genes removed by the deletion. As null mutations in Bearded have no clear phenotype, mutagenesis screens looking for mutations whose phenotypic effects are dependent on Bearded null or gain of function alleles will be initiated. The last specific aim deals with the possible function of a conserved sequence (the Bearded box) in the 3'-untranslated region of Bearded and of the genes in the E(spl)-C. Some very preliminary results suggest that this sequence may act to destabilize transcripts in which it is found. The consequences of introducing the Bearded box into heterologous genes will be tested, as will the effects of mutating the Bearded box in Bearded itself or of these heterologous genes with the introduced sequence. Searches for proteins that interact with the Bearded box will be attempted. This will be done by screening expression libraries with labelled RNA oligonucleotides, by determining whether known Drosophila RNA binding proteins interact with the Bearded box, or by trying to purify Bearded-box binding activities from embryo extracts.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Genetics Study Section (GEN)
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University of California San Diego
Schools of Arts and Sciences
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Miller, Steven W; Posakony, James W (2018) Lateral inhibition: Two modes of non-autonomous negative autoregulation by neuralized. PLoS Genet 14:e1007528
Smith, Andrew F; Posakony, James W; Rebeiz, Mark (2017) Automated tools for comparative sequence analysis of genic regions using the GenePalette application. Dev Biol 429:158-164
Miller, Steven W; Rebeiz, Mark; Atanasov, Jenny E et al. (2014) Neural precursor-specific expression of multiple Drosophila genes is driven by dual enhancer modules with overlapping function. Proc Natl Acad Sci U S A 111:17194-9
Liu, Feng; Posakony, James W (2014) An enhancer composed of interlocking submodules controls transcriptional autoregulation of suppressor of hairless. Dev Cell 29:88-101
Hirono, Keiko; Margolis, Jonathan S; Posakony, James W et al. (2012) Identification of hunchback cis-regulatory DNA conferring temporal expression in neuroblasts and neurons. Gene Expr Patterns 12:11-7
Rebeiz, Mark; Castro, Brian; Liu, Feng et al. (2012) Ancestral and conserved cis-regulatory architectures in developmental control genes. Dev Biol 362:282-94
Liu, Feng; Posakony, James W (2012) Role of architecture in the function and specificity of two Notch-regulated transcriptional enhancer modules. PLoS Genet 8:e1002796
Rebeiz, Mark; Miller, Steven W; Posakony, James W (2011) Notch regulates numb: integration of conditional and autonomous cell fate specification. Development 138:215-25
Miller, Steven W; Avidor-Reiss, Tomer; Polyanovsky, Andrey et al. (2009) Complex interplay of three transcription factors in controlling the tormogen differentiation program of Drosophila mechanoreceptors. Dev Biol 329:386-99
Schweisguth, F; Nero, P; Posakony, J W (1994) The sequence similarity of the Drosophila suppressor of hairless protein to the integrase domain has no functional significance in vivo. Dev Biol 166:812-4

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