Hox Control of Cell-Specific EGF Signaling During Development

发育过程中细胞特异性 EGF 信号传导的 Hox 控制

• 批准号: 9068974
• 负责人: BRIAN GEBELEIN
• 金额: $ 29.07万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9068974
• 关键词: Abdomen; Affinity; Algorithms; Animals; Binding; Binding Sites; Biochemical; Biochemistry; Bioinformatics; Biological Assay; Biological Process; Biological Testing; Blood Cells; Cell Culture Techniques; Cell Proliferation; Cells; Chest; Comparative Study; Complex; Coupling; DNA; DNA Binding; Data; Development; Dorsal; Drosophila genus; Drosophila melanogaster; EGF gene; Embryo; Embryonic Development; Ensure; Event; Funding; Gene Expression; Gene Expression Regulation; Gene Transfer Techniques; Genetic; Genetic Transcription; Genomic Segment; Genomic approach; Genomics; Goals; Grant; Growth; Guidelines; Health; Hepatocyte; Human; Human Development; Kidney; Logic; Mammals; Mediating; Metabolic; Modeling; Morphogenesis; Mus; Mutagenesis; Neurons; Organ; Organism; Outcome; Peptide Hydrolases; Phenotype; Process; Proteins; Regulation; Regulator Genes; Reporter; Research; Research Proposals; Resolution; Role; Sensory; Signal Pathway; Signal Transduction; Specific qualifier value; Specificity; Speed; System; Testing; Tissues; To specify; Transcriptional Activation; Transgenic Organisms; Vertebrates; base; body system; cell type; cofactor; fly; gene interaction; insight; intercellular communication; leukemia; non-genetic; novel; paralogous gene; precursor cell; pressure; relating to nervous system; rhomboid; transcription factor

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.

描述（由申请人提供）：复杂的动物使用数百个转录因子（TF）在每个器官内专用细胞类型的分化过程中准确控制细胞特异性基因表达。尽管基因组方法表明许多TF结合了数千个重叠区域，但DNA结合事件和TF相互作用在生物学上有意义仍然是一个主要挑战。该应用的长期目标是获得对TF，转录机制和顺式调节逻辑的高分辨率理解，用于确保果蝇发育过程中强大的细胞特异性EGF信号传导。我们的实验系统是菱形（Rho）蛋白酶的转录激活，从而触发了特定的腹部感觉器官前体细胞（SOP）的EGF分泌，以诱导动物生长和生存性所需的代谢细胞（Oenocytes）。由于只有一部分腹部和胸腔SOP的一部分激活了RHO，而Rho的转录水平决定了指定的烯型烯烃的数量，因此，RHO的调节是一个很好的模型，可以理解区域和组织施法的转录因子如何整合以控制可靠的细胞特异性基因表达和现象型型均匀。我们在该赠款的第一个资金周期中的发现表明：a）RHO包含多个激活腹部SOP基因表达的顺式调节模块（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五个TF，包括腹部A（ABD-A）HOX复合物，其中包含内部和同胸HOX辅助因子和两个神经元转录因子（无感和PAX2）； c）ABDA毫无疑问的拮抗作用是一种新型保守的HOX转录机制，它控制着小鼠的EGF信号传导，血细胞增殖和白血病进展。在这些发现的基础上，该应用程序具有三个目的：1）确定如何将区域ABD-A HOX因子与神经限制的PAX2因子集成在一起，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SOP子集中调节RHO的其他神经元转录输入的作用。 3）使用基本的顺式调节逻辑来开发生物信息学算法以预测其他RHO 确保鲁棒表达水平和表型的CRM。我们的方法结合了果蝇遗传学，无偏见的诱变报告基因测定法和BAC基因组救援测定法与细胞培养，生物化学和生物信息学的速度相结合。这些目标的成功完成具有很高的潜力，可以揭示新的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救援测定法相结合，我们将获得有关CRM和转录因子如何控制复杂动物中强大细胞特异性基因表达的新见解。由于所研究的TF和生物学过程在苍蝇和哺乳动物之间是高度保守的，因此我们乐观，我们的机械研究将继续发现与人类健康和发展有关的新基因调节机制。