Ilaria Rebay - US grants

DESCRIPTION (provided by applicant): The long-term objectives of this work are to elucidate the mechanisms whereby cells integrate instructions from multiple signaling pathways and respond in a context-appropriate manner. Reflecting the key role that precisely orchestrated signaling plays in all biological processes, uncontrolled or dysregulated activity of these pathways leads to tumorigenesis and/or developmental defects in humans. Our approach is to exploit the genetically tractable Drosophila system to uncover novel strategies whereby conserved signal transduction cascades converge on nuclear transcription factor networks to direct cell proliferation, fate specification, differentiation, morphogenesis and survival during retinal development. Because developmental signaling mechanisms have been highly conserved in evolution, knowledge of the molecular circuitries used in Drosophila will fundamentally advance our understanding of how cell fates are designated and maintained, and why misregulation results in cancer and disease in mammals. The goal of this proposal is to explore a novel mechanism of signal integration suggested by our ongoing study of transcriptional regulatory networks that orchestrate retinal specification and development. Specifically Eyes absent (Eya), through its unusual dual functionality as transcription factor and protein tyrosine phosphatase (PTP), has the potential both to respond to upstream signaling inputs by directing downstream patterns of gene expression and to influence the activity of these same pathways through its phosphatase function. Although impaired Eya PTP function has been implicated in human disease, including ocular defects, the normal biological function of Eya's PTP activity, and its relationship to Eya-mediated transcriptional regulation, remains poorly understood. This proposal describes a multi-faceted approach combining genetic, biochemical, and cell biological assays to address the hypothesis that proper coordination and regulation of Eya's two essential functions, one as a nuclear transcription factor and one as a component of phosphotyrosine signaling networks, is critical for retinal development. Successful completion of the proposed research plan will provide insight into the cellular and developmental contexts in which Eya's PTP function is required and should uncover novel mechanisms of cross-talk between Eya, the retinal determination gene network and other signaling modules critical for eye development. The specific aims are to elucidate the phosphotyrosine signaling pathways in which Eyes absent (Eya) participates by exploring genetic and biochemical interactions with the Abelson (Abl) and Src64 tyrosine kinases, to investigate the contribution of new binding partners identified in an in vitro expression screen with respect to Eyes absent's two functions as transcription factor and phosphatase, and to test the hypothesis that spatial partitioning of Eyes absent function between nucleus and cytoplasm contributes to proper retinal development. Because the proteins and pathways we are studying have conserved functions in mammals, the new signaling strategies revealed by our work will advance understanding of human development and disease. PUBLIC HEALTH RELEVANCE: Inappropriate regulation of gene function underlies a broad spectrum of human developmental abnormalities and diseases, including cancer. The goal of this research is to reveal the cellular mechanisms that regulate critical decisions during normal development of the eye, as an essential first step toward understanding the diseases and defects that arise when these key controls are lost and eventually toward developing effective therapeutic interventions.

! PROJECT SUMMARY This is a revised proposal for a renewal application for 1R01GM080372 Function and regulation of the ETS transcriptional repressor TEL1/Yan. Developmental programs are driven by transcription factors that coordinate precise patterns of gene expression in response to inductive signaling cues. The goals of this proposal are to uncover the regulatory strategies that keep gene expression programs in progenitors stably off, yet poised for rapid and precise activation in response to the inductive cues that initiate cell fate specification. Specifically we will study how the action of two members of the conserved ETS family of transcription factors, a repressor Yan and an activator Pointed (Pnt), known as TEL1/ETV6 and ETS1/ETS2 in humans, orchestrates the transition from progenitor to specified cell fate in response to receptor tyrosine kinase/Ras/MAPK signaling pathway. The central hypothesis is that the activator Pointed organizes and coordinates the entire sequence of events by first cooperating with Yan to establish the initial repressed state and then working in opposition to switch the system over to activation of gene expression and adoption of cell fate. Aim 1 explores how different Pointed isoforms determine the system's sensitivity and response to MAPK signaling. Aim 2 will elucidate the molecular mechanisms that coordinate Pointed's dual roles as repressor and activator during the cell fate specification process. Because the ideas we are testing address fundamental mechanisms of developmental regulation, and the signaling factors and transcriptional networks we study are conserved, the discoveries that emerge will have broad impact.

? DESCRIPTION (provided by applicant): This proposal is a revised version of a new application entitled A systems-level study of signaling networks and differentiation. The question of how cell fates are accurately specified is of fundamental importance to understanding both normal developmental progression and disease mechanisms that alter cell fates. However we currently have very limited understanding of how gene expression is coordinated across time and space in a multicellular tissue so that cells accurately and reproducibly negotiate the transition from a multipotent to a differentiated state. The goal of thi proposal is to investigate how regulatory networks downstream of receptor tyrosine kinases (RTKs) coordinate expression of key genes needed for multipotency and accurate cell differentiation in the Drosophila eye. The Drosophila visual system provides a superbly tractable and well-defined experimental model with which to address this question. In particular, the stereotyped patterning and architecture of the fly retina facilitates the identification and trackig of individual cell types over space and time. Because signaling mechanisms have proven to be extraordinarily conserved, our exploration of the molecular networks and signaling interactions that drive differentiation in the fly are likely to be relevant to mammalian development and disease. Thus our discoveries could help identify new strategies for therapeutic intervention for diseases such as cancer in which fundamental network properties are disrupted. Aim 1 will explore how stimuli transduced by the EGFR and Notch signaling pathways are integrated with cell autonomous Yan network dynamics to ensure accurate cell differentiation in the developing Drosophila eye. We will quantify and compare the expression dynamics of core Yan network factors in thousands of individual retinal cells at each stage in their development in wild type versus perturbed conditions. The quantitative datasets will be used to construct a stochastic model that integrates information about Yan network molecules across both time and space. Aim 2 will investigate how specific features of Yan network topology contribute to the accuracy of cell differentiation in the developing Drosophila eye. Using a combination of modeling and molecular-genetic experimentation, we will test specific hypotheses regarding how interlocking feed forward and feedback loops can combine to enable accurate differentiation in a multicellular tissue.

! PROJECT SUMMARY Developmental programs are driven by the transcription factors that convert signaling information into the precise patterns of gene expression needed for accurate cell fate transitions. Transcription factor-DNA and transcription factor-transcription factor interactions together produce the necessary regulatory dynamics and precision. Because of the greater complexity and heterogeneity of protein-protein interactions, the field has focused on manipulating DNA sequence to study the mechanisms that determine enhancer specificity and gene expression output. This proposal describes our plan to manipulate transcription factor protein-protein interaction affinity. The experimental plan takes advantage of the deep mechanistic understanding and well- validated in vitro and in vivo assays established over two decades of studying the function and regulation of the conserved ETS family repressor Yan in the developing Drosophila visual system. Aim 1 outlines a multi- disciplinary strategy to engineer a set of yan alleles in which self-association affinity is gradually increased from weak to strong. Using these alleles, Aim 2 tests competing hypotheses for how protein-protein interaction affinity influences Yan function and target gene specificity during photoreceptor specification in the fly retina. Because the ideas we are testing address fundamental mechanisms of transcriptional and developmental regulation, the discoveries that emerge are likely to have impact well beyond our specific model system.