Gene Networks and Chromatin Regulation of Phaseolin Transcription

种子贮藏蛋白是人类和牲畜的重要食物来源。菜豆蛋白是四季豆种子中含量最丰富的蛋白质。在遗传学上，它是由一个相似但不相同的小基因家族编码的，其中一个基因(PHAS)已经被非常详细地研究过。这项由美国国家科学基金会资助的研究的长期目标是了解特定基因在特定时间如何在特定组织中激活，并导致植物分子生物学的重大进展。这些技术包括植物信使核糖核酸的分离和体外翻译，植物基因内含子的首次展示，以及发育调节基因(PHA)从一个物种到另一个物种(豆类到烟草)的首次功能性转移。PHAs的转录调控在空间和时间上都受到限制。研究发现，PHAS启动子的三个TATA盒上方的抑制性染色质结构和旋转定位的核小体的建立是导致PHAS在营养组织中缺乏表达的原因。然而，利用雌激素诱导的PvALF产生系统和外源脱落酸(ABA)，可以诱导PHAS启动子驱动的GUS报告基因在两个转基因纯合子(pER8/XVEHA.PvALF)叶片中的表达。这些实验表明，PHAS的激活是一个两步过程，包括由种子特异的B3结构域转录激活剂PvALF启动的核小体重塑，然后由脱落酸(ABA)驱动的PHAS启动子的GUS转录激活，通过组织化学染色很容易被蓝色的叶片识别。该项目的具体目标1是确定与PvALF效应器相互作用的蛋白质，以实现染色质重塑。除了酵母双杂交筛选和抗HA-Tag抗体的免疫沉淀外，一种新的方法将是在增强和激活步骤的特定时间点从拟南芥叶片中提取RNA。对从RNA样本中反转录的cDNA进行高通量测序，将有助于识别在这些步骤中启动的基因以及它们出现的顺序。在目标2中，将进一步剖析控制PHAS启动子增强和激活的各种事件。具体地说，将确定组蛋白H3的缺失或替代是否是效应器介导的PHAS表达激活的内在原因。PvALF及其相关效应子在启动种子特异性启动子转录过程中的关键作用使得寻找诱导PvALF及其近亲转录的关键事件成为当务之急(目标3)。广泛的影响除了获得关于基因表达的分子过程的新信息外，该项目还与了解重要粮食作物种子蛋白的生产是如何调控有关的。该项目将为一名博士后研究助理和一名研究生提供培训，并将为本科生提供实验室经验的机会。拟议研究的几个方面(例如，cDNA克隆、酵母单/双杂交筛选和T-DNA突变实验)有望为本科生提供令人兴奋的参与机会。从拟议的研究中创建的拟南芥转基因株系或突变体(例如，表达Myc标记的组蛋白H3或H3.3的转基因株)也将提供给植物界的研究人员。不同血统和性别的学生将被鼓励参加。

Intellectual meritSeed storage proteins are vital sources of food for mankind and livestock. Phaseolin is the most abundant protein stored in the seeds of the French bean, Phaseolus vulgaris. Genetically, it is encoded by a small family of similar but not identical genes, one of which (phas) has been studied in great detail. This NSF-funded research has the long-term goal of gaining an understanding of how specific genes are activated at certain times in defined tissues, and has led to major advances in plant molecular biology. These include the isolation and in vitro translation of plant mRNA, the first demonstration of introns in a plant gene, and the first functional transfer of a developmentally regulated gene (phas) from one species to another (bean to tobacco). Transcriptional regulation of phas is constrained both spatially and temporally. It was found that the establishment of a repressive chromatin structure and a rotationally positioned nucleosome over three phased TATA boxes of the phas promoter is responsible for the lack of phas expression in vegetative tissue. However, using an estradiol-inducible system for PvALF production and externally supplied abscisic acid (ABA), it was possible to induce activation of phas promoter-driven GUS reporter expression in leaves of Arabidopsis plantlets homozygous for two transgenes (pER8/XVEHA.PvALF). These experiments revealed that the activation of phas is a two-step process that involves nucleosome remodeling initiated by the seed-specific B3 domain transcriptional activator, PvALF, followed by abscisic acid (ABA)-driven activation of robust GUS transcription from the phas promoter that is easily identified by blue-colored leaves upon histochemical staining. Specific objective 1 of this project is to identify proteins that interact with the PvALF effector to achieve chromatin remodeling. In addition to yeast two-hybrid screening and immunoprecipitation with antibody against HA-tag, a novel approach will be to extract RNA from Arabidopsis leaves at specific time points during the potentiation and activation steps. High throughput sequencing of cDNA reverse-transcribed from the RNA samples will allow identification of the genes that are turned on in these steps, and the order in which they appear. In objective 2, further dissection of the various events governing the potentiation and activation of the phas promoter will be undertaken. Specifically, it will be determined if histone H3 depletion or substitution is intrinsic to effector-mediated activation of phas expression. The key role of PvALF and related effectors in initiating transcription from seed-specific promoters makes it imperative to search for the critical event that induces transcription from PvALF and its relatives (objective 3).Broader impactsIn addition to gaining new information on molecular processes of gene expression, this project has relevance to understanding how the production of the seed protein of an important food crop is regulated. This project will provide training for a postdoctoral research associate, a graduate student and will give undergraduate students opportunities for laboratory experience. Several aspects of the proposed studies (e.g. cDNA cloning, yeast one/ two hybrid screening and T-DNA mutagenesis experiments) are expected to provide exciting opportunities for undergraduate participation. Arabidopsis transgenic lines or mutants created from the proposed studies (e.g. transgenic line expressing Myc-tagged histone H3 or H3.3) will also be made available for researchers in the plant community. Students of different heritage and gender will be encouraged to participate.