Hox Control of Cell-Specific EGF Signaling During Development

描述（由申请人提供）：复杂动物使用数百个转录因子（tf）来精确控制每个器官内特化细胞类型分化过程中细胞特异性基因的表达。虽然基因组方法表明许多TF结合了数千个重叠区域，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主要挑战。该应用程序的长期目标是获得高分辨率的tf、转录机制和顺式调控逻辑，以确保果蝇发育过程中细胞特异性EGF信号的健壮性。我们的实验系统是rhomboid （rho）蛋白酶的转录激活，该酶触发特定腹部感觉器官前体细胞（SOPs）分泌EGF，以诱导动物生长和生存所需的代谢细胞（卵泡细胞）。由于只有一小部分腹部而非胸部的SOPs激活rho，并且rho的转录水平决定了指定的卵泡细胞的数量，因此rho的调控可以作为一个很好的模型来理解如何整合区域和组织限制性转录因子来控制稳健的细胞特异性基因表达和表型结果。我们在该资助的第一个资助周期的研究结果显示：A) rho含有多个激活腹部SOP基因表达的顺式调控模块（CRMs）；B) rho CRM包含许多重叠的TF结合位点，这些位点直接整合了5个TF，包括一个腹部-A (Abd-A) Hox复合物，该复合物含有外膜和同源胸膜Hox辅助因子以及两个神经元转录因子（无意义和Pax2）；C) abda -无意义拮抗是一种新的保守的Hox转录机制，它既控制果蝇的EGF信号传导，也控制小鼠的血细胞增殖和白血病进展。基于这些发现，本应用程序有三个目的：1)确定区域Abd-A Hox因子如何与神经限制性Pax2因子结合以激活rho，并评估哪些其他Hox因子使用Pax2作为辅助因子。2)定义在特定SOPs子集中调节rho的额外神经元转录输入的作用。3)使用潜在的顺式调控逻辑开发生物信息学算法来预测额外的rho

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.