Dissecting Promoter Function in Vivo

许多基本的细胞过程在转录水平上受到控制。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。以往对玉米类黄酮生物合成调控的研究为研究正常体内环境下启动子结构提供了独特的工具。作为了解体内启动子功能的模型，该途径的结构基因（A1）的调控区域已经在体外被广泛解剖，并且是至少两个明确定义的调控系统的目标，将被研究。A1启动子中顺式调控元件的组织将通过体内足迹策略进一步研究，以确定正常染色质环境下调控因子识别的序列。将利用独特的遗传资源在体内研究A1转录上顺式作用序列缺失的结果。点突变和调控元件的小插入在体内的影响已经折射到体外分析将被分析。在两个非常好的遗传特征的情况下，A1的调节被转移到转座元件。此外，转座子的表观遗传修饰直接影响A1的转录。我们对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。转座子是否能够以不依赖于调控因子的方式促进A1转录，以及转座子的元件是否完全取代基因正常的顺式作用调控元件，这些都将被研究。最后，通过研究A1基因启动子受转座元件插入控制时的体内甲基化状态，研究转座子对基因表达的表观遗传影响。总之，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。

Many 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. Previous studies on the control of maize flavonoid biosynthesis have provided unique tools to investigate promoter structure in its 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 organization of cis-acting regulatory elements in the A1 promoter will be further-investigated by using in vivo footprinting strategies to identify the sequences recognized by the regulators in normal chromatin environments. Unique genetic resources will be used to study in vivo the result of deletions of cis-acting sequences on A1 transcription. The in vivo effect of point mutations and small insertions in regulatory elements that have been refractive to in vitro analysis will be analyzed. In two very well genetically-characterized cases, the regulation of A1 is transferred to transposable elements. Moreover, epigenetic modifications of the transposons directly affect A1 transcription. Our unique understanding of the regulation of A1 makes it ideal to interpret transposable element gene control systems providing important insights into the molecular bases of epigenetic phenomena. Whether transposons can promote A1 transcription in a regulator-independent fashion, and whether elements of the transposon entirely replace the normal cis-acting regulatory elements of the gene will be examined. Finally, the epigenetic effect that transposons often have on gene expression, by studying the in vivo methylation status of the A1 gene promoter when its expression is controlled by transposable element insertions will be investigatedTaken 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.