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，对于动物发育过程中大多数细胞和组织的正确命运规范至关重要。这些古老且高度保守的途径主要通过改变转录因子（tf）的活性来调节发育细胞的命运和成人的健康，而转录因子反过来又控制靶基因的表达。位于通路靶基因附近的信号调节增强子（或顺式调节元件）含有信号调节tf和其他组织或细胞类型特异性tf的结合位点；这些因素之间的相互作用决定了靶基因的表达模式。然而，在任何多细胞生物中，没有任何信号调节增强子被充分表征，从某种意义上说，它所有必要的直接调节输入都被解释了。此外，在机制水平上，没有任何已知的与转录激活有关的生化活动被证明足以（单独或共同）驱动动物发育过程中的基因表达。这些和其他我们认识上的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”两个信号调节的发育增强因子；也就是说，定义和功能表征所有必需的监管子元素。我们的研究结果以及其他研究明确表明，简单地结合已知的具有良好特征的增强子的调节TF位点不足以刺激体内转录。该项目将体内功能报告基因分析与已知转录控制机制的研究、新调控因子的鉴定以及果蝇遗传学的优势相结合。该项目有两个主要目标：(1)鉴定气泡（spa）和dppVM增强子中的DNA元素，这些元素是体内基因激活所必需的；确定这些元素排列的拓扑/结构规则。(2)确定spa和dppVM中已知和新的调控元件在转录控制机制中的作用，如局部染色质修饰、DNA合作结合、核小体定位和核内组织；在这些增强子中分离并表征与新调控DNA元件结合的因子。

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.