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

大多数基本的细胞过程都是在转录水平上控制的。 反式作用调节因子的体内性质在过去几年中已经获得了广泛的知识，但与之形成对比的是，关于顺式作用调节元件的体内组织、进化和功能仍然知之甚少。 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。 以前的研究控制玉米类黄酮的生物合成提供了独特的工具，研究顺式作用的调控元件在其正常的体内环境中的结构和功能的PI。 作为了解体内启动子功能的模型，将研究已经在体外被广泛解剖并且是至少两个明确的调节系统的靶标的途径（A1）的结构基因的调节区。 顺式作用的调节序列，是重要的A1在体内的调节将建立，通过确定这些元件的突变和缺失对A1在植物中的表达的影响，并通过利用天然的A1等位基因的多样性在玉米。 在至少两个特征良好的情况下，A1的调节成为不同的转座因子的控制下，虽然在完全相反的方式。A1的调节的独特理解，使其理想的解释转座因子基因控制系统提供重要的见解的分子基础的表观遗传现象。 PI将研究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将检查相应转座酶的哪些组分参与包含转座子插入的A1等位基因所显示的新调控模式。 总之，这些研究应该提供一个最全面的在体内研究植物基因的调控区。 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.