Dissecting Promoter Function in Vivo

许多基本的细胞过程是在转录水平上控制的。过去几年对反式作用调控元件体内特性的广泛了解，与对顺式作用调控元件的体内组织、进化和功能知之甚少形成鲜明对比。以往对玉米类黄酮生物合成调控的研究已经为研究其体内正常环境中的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，将研究在体外被广泛解剖的途径(A1)的结构基因的调节区，该调节区是至少两个明确的调节系统的靶标。A1启动子中顺式作用调控元件的组织将通过使用体内足迹策略来进一步研究，以确定在正常染色质环境中由调节器识别的序列。独特的遗传资源将被用于体内研究顺式作用序列缺失对A1转录的结果。将分析在体外分析中折射的调节元件中的点突变和小插入的体内效应。在两个非常好的遗传特征的案例中，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元件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。转座子是否可以以一种不依赖于调节器的方式促进A1转录，以及转座子的元件是否完全取代了基因的正常顺式作用调节元件。最后，转座子对基因表达的表观遗传效应，通过研究A1基因启动子在体内的甲基化状态，当其表达受转座元件插入控制时，将被调查。这些研究加在一起，应该提供对植物基因调节区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，这些研究得出的结果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.