Gene Networks and Chromatin Regulation of Phaseolin Transcription

种子贮藏蛋白质是人类和牲畜重要的食物来源。菜豆蛋白是储存在菜豆种子中最丰富的蛋白质。从遗传学上讲，它是由一个小家族的相似但不相同的基因编码的，其中一个（phas）已经被非常详细地研究过。这项由NSF资助的研究的长期目标是了解特定基因在特定组织中的特定时间是如何被激活的，并导致了植物分子生物学的重大进展。这些包括植物mRNA的分离和体外翻译，植物基因中内含子的首次展示，以及发育调控基因（phas）从一个物种到另一个物种（豆类到烟草）的首次功能转移。phas的转录调控在空间和时间上都受到限制。结果发现，抑制性染色质结构和旋转定位的核小体的phas启动子的三个阶段的TATA盒的建立是负责缺乏phas在营养组织中的表达。然而，使用雌二醇诱导系统PvALF生产和外部提供的脱落酸（阿坝），它是可能的诱导激活的phas启动子驱动的GUS报告基因表达的拟南芥植株纯合的两个转基因（pER 8/XVEHA.PvALF）的叶子。这些实验表明，激活的phas是一个两步的过程，涉及核小体重塑启动种子特异性B3结构域转录激活因子，PvALF，然后脱落酸（阿坝）驱动的激活强大的GUS转录从phas启动子，很容易识别的蓝色叶片组织化学染色。该项目的具体目标1是鉴定与PvALF效应子相互作用以实现染色质重塑的蛋白质。除了酵母双杂交筛选和抗HA标签抗体免疫沉淀外，一种新的方法将是在增强和激活步骤期间的特定时间点从拟南芥叶片中提取RNA。从RNA样品逆转录的cDNA的高通量测序将允许鉴定在这些步骤中打开的基因以及它们出现的顺序。在目标2中，我们将进一步研究phas启动子增强和激活的各种事件。具体地，将确定组蛋白H3消耗或取代是否是效应子介导的phas表达激活所固有的。PvALF和相关效应子在启动种子特异性启动子转录中的关键作用使得寻找诱导PvALF及其亲属转录的关键事件势在必行（目标3）。更广泛的影响除了获得基因表达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节的有关。该项目将为博士后研究助理，研究生提供培训，并将为本科生提供实验室经验的机会。几个方面的拟议研究（如cDNA克隆，酵母单/双杂交筛选和T-DNA诱变实验）预计将提供令人兴奋的机会，本科生参与。拟南芥转基因株系或从拟议的研究中产生的突变体（例如表达Myc标记的组蛋白H3或H3.3的转基因株系）也将提供给植物界的研究人员。将鼓励不同文化背景和性别的学生参加。

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.