Structure and Function of a Signal-Regulated Developmental Enhancer

描述(由申请人提供)：细胞-细胞信号通路，如Wnt、Notch和MAPK，在动物发育过程中对大多数细胞和组织的正确命运指定至关重要。这些古老而高度保守的途径主要通过改变转录因子(TF)的活性来调控发育细胞的命运和成人的健康，而转录因子又控制着靶基因的表达。位于通路靶基因附近的信号调节增强子(或顺式调节元件)包含信号调节因子和其他特定组织或细胞类型因子的结合位点；这些因子之间的相互作用决定了靶基因的表达模式。然而，在任何多细胞生物体中，没有信号调节的增强子被完全表征，从这个意义上说，它的所有必要的直接调节输入都已经被考虑在内。此外，在机制水平上，没有一种已知的与转录激活有关的生化活动被证明单独或共同足以在动物发育过程中驱动基因表达。这些和我们理解中的其他差距表明，转录激活的重要机制可能仍未被发现。拟议的研究计划致力于“解决”两个信号调控的发育增强剂；也就是定义和描述它们所需的所有调节子成分。我们的结果，以及其他研究，明确地表明，简单地结合已知的具有良好特征的增强剂的调节Tf位点不足以刺激体内转录。在这里提出的项目结合了体内功能报告分析与已知转录调控机制的调查，新调控因子的鉴定，以及果蝇遗传学的优势。该项目有两个主要目标：(1)确定在体内基因激活所必需的SPA和dppVM增强剂中的DNA元件；确定这些元件排列的拓扑/结构规则。(2)确定SpA和dppVM中已知的和新的调控元件在转录调控机制中的作用，如局部染色质修饰、协同DNA结合、核小体定位和核内组织；分离和鉴定这些增强子中与新的调控元件结合的因子。 项目叙事 待研究的信号通路和因子对动物的正常发育至关重要，并与许多发育缺陷和人类疾病有关，最明显的是癌症。更好地从机制上理解信号调节增强子将有助于制定治疗和预防由细胞信号和基因表达异常引起的疾病的策略，并可能进一步开发具有重要临床和研究用途的新的分子工具。

DESCRIPTION (provided by applicant): Cell-cell signaling pathways, such as Wnt, Notch, and MAPK, are critical for the proper fate specification of most cells and tissues during animal development. These ancient and highly conserved pathways regulate developmental cell fate and adult health primarily by altering the activity of transcription factors (TFs), which in turn control the expression of target genes. Signal-regulated enhancers (or cis-regulatory elements) located near pathway target genes contain binding sites for signal-regulated TFs and for other, tissue- or cell-type- specific TFs; interactions among these factors determine the expression pattern of the target gene. However, no signal-regulated enhancer in any multicellular organism has been fully characterized, in the sense that all of its necessary direct regulatory inputs have been accounted for. Furthermore, at the mechanistic level, none of the known biochemical activities implicated in transcriptional activation have been shown to be sufficient, alone or together, to drive gene expression during animal development. These and other gaps in our understanding suggest that important mechanisms of transcriptional activation are likely still undiscovered. The proposed research program is dedicated to "solving" two signal-regulated developmental enhancers; that is, to defining and functionally characterizing all of their required regulatory sub-elements. Our results, along with other studies, show definitively that simply combining the known regulatory TF sites of well-characterized enhancers is not sufficient to stimulate transcription in vivo. The project proposed here combines in vivo functional reporter assays with the investigation of known transcriptional control mechanisms, the identification of novel regulators, and the advantages of Drosophila genetics. The project has two main objectives: (1) Identify the DNA elements within the sparkling (spa) and dppVM enhancers that are necessary for gene activation in vivo; determine the topological/structural rules for arrangement of those elements. (2) Determine the roles of the known and novel regulatory elements within spa and dppVM in mechanisms of transcriptional control such as local chromatin modification, cooperative DNA binding, nucleosome positioning, and intra-nuclear organization; isolate and characterize factors binding to novel regulatory DNA elements within these enhancers. Project Narrative The signaling pathways and factors to be studied are critical for normal animal development, and are implicated in a host of developmental defects and human diseases, most notably cancer. A better mechanistic understanding of signal-regulated enhancers will facilitate the development of strategies for the treatment and prevention of disorders caused by aberrant cell signaling and gene expression, and may further the development of new molecular tools with significant clinical and research utility.