Hox Control of Cell-Specific EGF Signaling During Development

描述(由申请人提供)：复杂的动物使用数百种转录因子(TF)在每个器官内特定细胞类型的分化过程中精确控制细胞特定基因的表达。虽然基因组学方法表明许多转录因子结合了数千个重叠的区域，但破译哪些DNA结合事件和转录因子相互作用具有生物学意义仍然是一个主要挑战。这一应用的长期目标是获得对果蝇发育过程中用于确保强大的细胞特异性EGF信号的转录因子、转录机制和顺式调控逻辑的高分辨率理解。我们的实验系统是菱形(Rho)蛋白酶的转录激活，它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)。由于只有一部分腹部而不是胸部的SOP激活Rho，并且Rho的转录水平决定了指定的卵母细胞数量，因此Rho的调控是一个很好的模型，可以理解区域和组织限制的转录因子是如何整合在一起的，以控制强大的细胞特异性基因表达和表型结果。我们在这笔赠款的第一个资金周期内的发现揭示：a)Rho包含多个可激活腹部SOP基因表达的顺式调节模块(CRM)；b)Rho CRM包含许多重叠的TF结合位点，可直接整合五个TF：腹部A(ABD-A)Hox复合体，其包含牙外环和同胸HOX共因子以及两个神经元转录因子(Sensless和Pax2)；c)Abda无意义拮抗是一种新的保守的Hox转录机制，可以控制果蝇中的EGF信号以及小鼠的血细胞增殖和白血病的进展。在这些发现的基础上，这项应用有三个目标：1)确定区域性Abd-A HOX因子如何与神经限制性Pax2因子整合来激活Rho，并评估哪些其他Hox因子使用Pax2作为辅助因子。2)确定额外的神经元转录输入在特定SOP子集中调节Rho的作用。3)使用基本的顺式调节逻辑来开发生物信息学算法来预测额外的RHO 确保稳健的表达水平和表型的CRM。我们的方法结合了果蝇遗传学、无偏向突变报告分析和BAC基因组拯救分析的优点，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很有可能发现新的TF相互作用，这将开辟新的研究途径。此外，通过将高分辨率突变研究与基因组拯救分析相结合，提供具有生物学意义的读数，我们将对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有新的见解。由于所研究的转录因子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，我们的机械研究将继续揭示与人类健康和发育相关的新的基因调控机制。

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.