Hox Control of Cell-Specific EGF Signaling During Development

描述（由申请人提供）：复杂动物使用数百种转录因子（TF）来精确控制每个器官内特化细胞类型分化期间的细胞特异性基因表达。虽然基因组方法已经表明许多TF结合数千个重叠区域，但破译哪些DNA结合事件和TF相互作用具有生物学意义仍然是一个重大挑战。本申请的长期目标是获得高分辨率的理解的TF，转录机制，顺式调控逻辑，用于确保强大的细胞特异性EGF信号在果蝇发育。我们的实验系统是菱形（rho）蛋白酶的转录激活，该蛋白酶触发特定腹部感觉器官前体细胞（SOP）分泌EGF，以诱导动物生长和生存所需的代谢细胞（卵母细胞）。由于只有一个子集的腹部，而不是胸部的SOP激活rho和rho的转录水平决定了指定的红细胞的数量，rho的调节作为一个伟大的模型，以了解如何区域和组织限制性转录因子的整合，以控制强大的细胞特异性基因表达和表型结果。我们在该基金的第一个资助周期的研究结果表明：A）rho含有多个激活腹部SOP基因表达的顺式调节模块（CRM）; B）A rho CRM含有许多重叠的TF结合位点，这些结合位点直接整合了5个TF，包括含有外齿和同胸Hox辅因子的Abdominal-A（Abd-A）Hox复合物和两个神经元转录因子（无意义和Pax 2）; C）AbdA-无意义拮抗作用是一种新的保守的Hox转录机制，其控制苍蝇中的EGF信号传导和小鼠中的血细胞增殖和白血病进展。基于这些发现，本申请有三个目的：1）确定区域Abd-A Hox因子如何与神经限制性Pax 2因子整合以激活rho，并评估哪些其他Hox因子使用Pax 2作为辅因子。2)定义额外的神经元转录输入的作用，调节rho在特定子集的SOP。3)使用基本的顺式调控逻辑开发生物信息学算法来预测额外的rho CRM确保稳健的表达水平和表型。我们的方法结合了果蝇遗传学、无偏致突变报告基因测定和BAC基因组拯救测定的优点，以及细胞培养、生物化学和生物信息学的速度。这些目标的成功完成具有很高的潜力，以发现新的TF相互作用，这将开辟新的研究途径。此外，通过将高分辨率诱变研究与提供生物学上有意义的读数的基因组拯救测定相结合，我们将获得对CRM和转录因子如何控制复杂动物中强大的细胞特异性基因表达的新见解。由于研究的TF和生物过程在苍蝇和哺乳动物之间高度保守，我们乐观地认为，我们的机制研究将继续揭示与人类健康和发育相关的新基因调控机制。

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.