SGER: Development of an In Vitro Chromatin Assembly and Transcription System for Plant Genes

对植物和动物基因的研究已经阐明了染色质和染色质调节剂在发育过程中调节基因表达的关键作用。 菜豆蛋白是菜豆中主要的种子贮藏蛋白，其正确的空间和发育表达需要染色体整合。 染色质结构分析已经确定，核小体旋转定位在非种子组织中的菜豆蛋白启动子上，从而阻断转录起始。 这种抑制性的染色质结构在成熟胚胎中β-菜豆蛋白基因激活之前被破坏，这表明了一种在发育过程中可以调节基因表达的机制。 然而，组织特异性转录因子和染色质调节剂在从沉默到活跃的菜豆蛋白基因的转变中所起的作用尚未阐明。 本项目的总体目标是发展一种体外染色质组装和转录检测方法，以研究染色质介导的植物基因调控。 为了实现这一目标，PI将结合联合收割机现有的动物体外染色质组装系统和先前开发的植物体外转录测定与纯化的植物转录因子，以重建菜豆蛋白基因的体外调控。 三个具体目标是1）在体外重建菜豆蛋白基因上的生理染色质，2）使用烟草细胞转录提取物在体外转录菜豆蛋白基因，和3）在体外重建菜豆蛋白基因表达的激素和组织特异性调节。 这些研究的结果将提供明确的证据表明，染色质组装/转录系统忠实地概括了内源性染色体背景和组织特异性表达模式的植物基因在体外。如果成功的话，这种体外系统可能提供一个通用的工具，阐明在植物中驱动染色质介导的基因调控的机制。 对植物发育过程中染色质环境如何影响基因表达的深入了解将极大地促进用于商业和农业用途的稳定转基因植物的工程化。

Studies of both plant and animal genes have illustrated the key role that chromatin and chromatin modulators play in regulating gene expression during development. Correct spatial and developmental expression of phaseolin, the major seed storage protein in bean, requires chromosomal integration. Chromatin structural analyses have established that a nucleosome is rotationally positioned over the phaseolin promoter in non-seed tissue, thereby blocking transcription initiation. This repressive chromatin structure is disrupted prior to beta-phaseolin gene activation in the maturing embryo, suggesting a mechanism by which gene expression can be regulated during development. However, the role that tissue-specific transcription factors and chromatin modulators play in the transition from a silenced to an active phaseolin gene has not been elucidated. The overall aim of this project is to develop an in vitro chromatin assembly and transcription assay to study chromatin-mediated regulation of plant genes. To achieve this goal, the PI will combine existing animal in vitro chromatin assembly systems and a previously developed plant in vitro transcription assay with purified plant transcription factors to reconstitute phaseolin gene regulation in vitro. The three specific aims are 1) to reconstitute physiological chromatin on the phaseolin gene in vitro, 2) to transcribe the phaseolin gene in vitro using a tobacco cell transcription extract, and 3) to reconstitute hormonal and tissue-specific regulation of phaseolin gene expression in vitro. Results from these studies will provide definitive evidence that the chromatin assembly/transcription system faithfully recapitulates the endogenous chromosomal context and tissue-specific expression pattern of a plant gene in vitro. If successful, this in vitro system may provide a universal tool for elucidating the mechanisms driving chromatin-mediated gene regulation in plants. An increased understanding of how chromatin context influences gene expression during plant development will greatly facilitate the engineering of stable transgenic plants for both commercial and agricultural use.