DESCRIPTION (provided by applicant): Complex animals use hundreds of transcription factors (TFs) to accurately control cell-specific gene expression during the differentiation of specialized cell types within each organ. While genomic approaches have shown that many TFs bind thousands of overlapping regions, deciphering which DNA binding events and TF interactions are biologically meaningful remains a major challenge. The long-term goal of this application is to obtain a high-resolution understanding of the TFs, transcriptional mechanisms, and cis-regulatory logic used to ensure robust cell-specific EGF signaling during Drosophila development. Our experimental system is the transcriptional activation of the rhomboid (rho) protease that triggers EGF secretion from specific abdominal sensory organ precursor cells (SOPs) to induce metabolic cells (oenocytes) needed for animal growth and viability. Since only a subset of abdominal but not thoracic SOPs activate rho and the transcriptional levels of rho dictate the number of oenocytes specified, the regulation of rho serves as a great model to understand how regional- and tissue-restricted transcription factors are integrated to control robust cell-specific gene expression and phenotypic outcomes. Our findings during the first funding cycle of this grant revealed that: A) rho contains multiple cis-regulatory modules (CRMs) that activate abdominal SOP gene expression; B) A rho CRM contains numerous overlapping TF binding sites that directly integrate five TFs including an Abdominal-A (Abd-A) Hox complex containing the Extradenticle and Homothorax Hox cofactors and two neuronal transcription factors (Senseless and Pax2); C) AbdA-Senseless antagonism is a novel conserved Hox transcriptional mechanism that controls both EGF signaling in flies and blood cell proliferation and leukemia progression in mice. Building on these findings, this application has three aims: 1) Determine how the regional Abd-A Hox factor is integrated with the neural-restricted Pax2 factor to activate rho and assess which other Hox factors use Pax2 as a cofactor. 2) Define the role of additional neuronal transcriptional inputs that regulate rho in a specific subset of SOPs. 3) Use the underlying cis-regulatory logic to develop a bioinformatics algorithm to predict additional rho CRMs that ensure robust expression levels and phenotypes. Our approach combines the advantages of Drosophila genetics, non-biased mutagenesis reporter assays, and BAC genomic rescue assays with the speed of cell culture, biochemistry and bioinformatics. The successful completion of these aims has a high potential to uncover novel TF interactions that will open up new avenues of research. In addition, by coupling high-resolution mutagenesis studies with genomic rescue assays that provide a biologically meaningful readout, we will obtain new insights into how CRMs and transcription factors control robust cell-specific gene expression within a complex animal. Since the TFs and biological processes studied are highly conserved between flies and mammals, we are optimistic our mechanistic studies will continue to uncover new gene regulatory mechanisms relevant to human health and development.

PUBLIC HEALTH RELEVANCE: The precise control of gene expression by transcription factors is essential for the proper formation of specialized cell types within each organ system. Unfortunately, we lack an understanding of how transcription factors are integrated to yield cell-specific gene expression within a complex organism. The goal of this research proposal is to determine how region-specific Hox transcription factors interact with tissue-specific transcription factors to regulate cell-specific gene expression during development. Our proposed studies utilize powerful genetic and biochemical approaches in the fruit- fly Drosophila melanogaster, and we have already used this approach to uncover novel gene interactions relevant to human development and leukemia. Thus, since both the processes and the transcription factors studied are highly conserved from flies to mammals, our findings will reveal fundamental new insights relevant to vertebrate development and human health. __SpecificAimsTextDelimiter__ 1. SPECIFIC AIMS: Cell signaling pathways are often transiently activated in localized embryonic regions to specify diverse cell types during development. The goal of this grant is to use Drosophila genetics, transgenic reporter assays, and biochemical approaches to obtain a high-resolution understanding of the cis-regulatory logic and transcriptional mechanisms used to ensure robust cell-cell signaling and the specification of an essential cell type. Our experimental system is the cell-specific activation of the rhomboid (rho) protease that triggers EGF release from a subset of abdominal sensory organ precursor cells (SOPs) during embryonic development. Neighboring cells that receive sufficient EGF form hepatocyte-like cells called oenocytes (Oes) and the number of Oes produced correlates with the amount of EGF secreted. Since a critical number of Oes are required for animal growth and viability, the activation of rho in abdominal SOPs is under strong selective pressure. Thus, the robust cell-specific transcriptional activation of rho provides a useful paradigm to understand how localized EGF secretion specifies an essential cell type in a complex organism.!!! During the first funding cycle of this grant, we achieved the following goals: A) Identified multiple rho cis- regulatory modules (CRMs) that drive abdominal SOP gene expression; B) Demonstrated the Abdominal-A (Abd-A) Hox factor and the Extradenticle (Exd) and Homothorax (Hth) Hox co-factors directly regulate rho through an antagonistic transcriptional mechanism with the Senseless (Sens) transcription factor (TF); C) Found that the neuronal-restricted Pax2 protein forms a large TF complex with Abd-A/Exd/Hth to directly activate rho in abdominal SOPs. D) Demonstrated that the Hox-Sens antagonism mechanism is conserved in mammalian blood cells, a finding with novel implications for the study of human leukemia. Building on these data, this application has three broad objectives: First, we aim to determine how region- and tissue-restricted TFs form higher-order protein/DNA complexes that yield robust cell-specific EGF secretion. Second, we will develop bioinformatics algorithms and use transgenic reporter assays to test additional genomic regions within the rho locus for abdominal SOP activity. Third, we will use genomic rescue assays to test the biological significance of each rho CRM to induce Oes. Our approach, which combines the advantages of rapid Drosophila genetics and transgenesis with cell culture, biochemistry and bioinformatics, has a high potential to uncover novel TF interactions and a deeper understanding of CRM function. Moreover, since the TFs and biological processes studied are conserved between flies and mammals, we are optimistic our mechanistic studies will continue to uncover new gene regulatory mechanisms relevant to human health and development. Aim1: Assess the role of Pax2 as a Hox cofactor. In addition to our findings that Abd-A and Pax2 cooperate to activate SOP transcription, studies in mice revealed the abdominal Hox11 factors interact with Pax2 to regulate gene expression during kidney morphogenesis. Since Pax2 is expressed in specific organs in flies and vertebrates, the direct integration of abdominal Hox and Pax2 factors yields region- and cell-specific gene expression. However, we currently do not understand which Hox factors synergize with Pax2 and how Hox- Pax2 interactions activate transcription. In this aim, we will test the hypothesis that Pax2 functions as an abdominal-specific Hox cofactor that activates transcription by: A) Determining the molecular interactions mediating Pax2/Abd-A/Hth/Exd complex formation. B) Identifying the transcriptional activation mechanisms mediated by the Pax2/Abd-A/Hth/Exd complex. C) Assessing Pax2-Hox specificity by performing comparative studies with Hox paralogs. Aim2: Define the role of neuronal and dorsal-ventral TFs in generating SOP-specific rho activation. To date, we have shown that the Abd-A, Exd, Hth, Sens, and Pax2 TFs directly regulate a rho CRM in a specific abdominal SOP. However, these five TFs are expressed in many abdominal SOPs that do not activate rho transcription, indicating additional factors are required for cell-specific gene regulation. In this aim, we will test the hypothesis that two additional TFs are integrated by a rho CRM to yield SOP-specific transcription. A) Determine the role of the Cut TF in restricting rho CRM activity to a specific subtype of SOPs. B) Determine the role of the Spalt TFs in activating a rho CRM in dorsal but not ventral SOPs. Aim3: Determine the cis-regulatory logic underlying robust cell-specific rho activation. A primary goal in the field of gene regulation is to determine how TF binding sites are organized within CRMs to promote robust cell-specific gene expression. Defining the degree to which TF binding sites are constrained and/or flexibly arranged within CRMs would provide key guidelines for the prediction of CRMs based on genomic sequence. In this aim, we combine transgenic reporter assays, bioinformatics, and genomic rescue assays to: A) Explore the role of TF binding site order, orientation, spacing, and affinity in determining how TF complexes mediate rho CRM activation in abdominal SOPs. B) Use the underlying cis-regulatory logic to develop a comprehensive experimental approach to predict and functionally test additional potential CRMs from the rho locus.