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: 8850709
• 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 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8850709
• 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）：复杂动物在每个器官内专门细胞类型的分化过程中使用数百种转录因子（TF）来精确控制细胞特异性基因表达。虽然基因组方法表明许多 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。该应用的长期目标是获得对转录因子、转录机制和顺式调控逻辑的高分辨率理解，以确保果蝇发育过程中细胞特异性 EGF 信号传导的稳健。我们的实验系统是菱形 (rho) 蛋白酶的转录激活，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）。由于只有腹部 SOP 的一个子集而不是胸部 SOP 激活 rho，并且 rho 的转录水平决定了指定的卵细胞数量，因此 rho 的调节可以作为一个很好的模型来了解区域和组织限制性转录因子如何整合以控制强大的细胞特异性基因表达和表型结果。我们在本次资助的第一个资助周期中的研究结果表明：A) rho 包含多个顺式调节模块 (CRM)，可激活腹部 SOP 基因表达； B) rho CRM 包含许多重叠的 TF 结合位点，可直接整合 5 个 TF，包括包含 Extradenticle 和 Homothorax Hox 辅因子的 Abdominal-A (Abd-A) Hox 复合物以及两个神经元转录因子（Senseless 和 Pax2）； C) AbdA-Senseless 拮抗作用是一种新型保守的 Hox 转录机制，可控制果蝇中的 EGF 信号传导以及小鼠中的血细胞增殖和白血病进展。基于这些发现，该应用程序具有三个目标：1) 确定区域 Abd-A Hox 因子如何与神经限制性 Pax2 因子整合以激活 rho 并评估哪些其他 Hox 因子使用 Pax2 作为辅助因子。 2) 定义在 SOP 的特定子集中调节 rho 的额外神经元转录输入的作用。 3) 使用底层顺式调控逻辑开发生物信息学算法来预测额外的 rho 确保稳定表达水平和表型的 CRM。我们的方法将果蝇遗传学、无偏诱变报告分析和 BAC 基因组拯救分析的优点与细胞培养、生物化学和生物信息学的速度结合起来。成功完成这些目标很有可能发现新颖的 TF 相互作用，从而开辟新的研究途径。此外，通过将高分辨率诱变研究与提供具有生物学意义的读数的基因组拯救测定相结合，我们将获得关于 CRM 和转录因子如何控制复杂动物内强大的细胞特异性基因表达的新见解。由于所研究的转录因子和生物过程在果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展相关的新基因调控机制。