Investigating Promoter Function in Vivo and the Role of Transposons as Control Elements

大多数基本的细胞过程是在转录水平上控制的。在过去的几年里，人们对反式作用调控元件的体内特性有了广泛的了解，与此形成对比的是，对顺式作用调控元件的体内组织、进化和功能知之甚少。转座子的插入在真核生物启动子的进化中起着至关重要的作用，但其如何影响基因表达的基础尚不清楚。先前对玉米类黄酮生物合成调控的研究为PI在正常体内环境下研究顺式调控元件的结构和功能提供了独特的工具。作为了解体内启动子功能的模型，我们将研究该途径的一个结构基因（A1）的调控区域，该区域在体外已被广泛解剖，并且是至少两个明确定义的调控系统的目标。通过确定这些元素的突变和缺失对植物中A1表达的影响，并利用玉米中天然A1等位基因的多样性，建立对体内A1调控重要的顺式调控序列。至少在两种明确的情况下，A1的调控受到不同转座因子的控制，尽管方式完全相反。对A1调控的独特理解使其成为解释转座因子基因控制系统的理想选择，为表观遗传现象的分子基础提供了重要的见解。PI将调查转座子插入是否覆盖A1的正常调控，并确定转座子是否提供新的顺式调控元件。最后，PI将检查相应转座子的哪些成分参与了包含转座子插入的A1等位基因所显示的新的调控模式。综上所述，这些研究应该是对植物基因调控区域进行的最全面的体内研究之一。由于可用的遗传和分子工具的独特性，从这些研究中得出的发现可能是基因表达调控的一般领域的基础。

Most fundamental cellular processes are controlled at the level of transcription. The extensive knowledge that has been acquired over the past years on the in vivo properties of the trans-acting regulators is contrasted by how little is still known about the in vivo organization, evolution and function of cis-acting regulatory elements. Insertions of transposons have played a crucial role in the evolution of eukaryotic promoters, but the fundaments on how this has impacted gene expression is not known. Previous studies on the control of maize flavonoid biosynthesis have provided the PI with unique tools to investigate the structure and function of cis-acting regulatory elements in their normal in vivo environment. As a model to understand promoter function in vivo, the regulatory region of a structural gene of the pathway (A1) that has been extensively dissected in vitro, and that is a target of at least two well-defined regulatory systems, will be studied. The cis-acting regulatory sequences that are important for the regulation of A1 in vivo will be established, by determining the effect of mutations and deletions of these elements on the expression of A1 in planta, and by exploiting the natural A1 allelic diversity in the Zea. In at least two well-characterized cases, the regulation of A1 becomes under the control of different transposable elements, albeit in exactly opposite ways.The unique understanding of the regulation of A1 makes it ideal to interpret transposable element gene control systems by providing important insights into the molecular bases of epigenetic phenomena. The PI will investigate whether the transposon insertions override the normal regulation of A1, and also determine whether the transposons provide novel cis-regulatory elements. Finally, the PI will examine which components of the corresponding transposases participate in the novel regulatory patterns displayed by the A1 alleles containing the transposon insertions. Taken together, these studies should provide one of the most comprehensive in vivo studies done on the regulatory region of a plant gene. Because of the uniqueness of the genetic and molecular tools available, the findings derived from these studies could be fundamental to the general field of regulation of gene expression.