Chromatin Potentiation and ABA Activation of Phaseolin Transcription

HallNSF项目：菜豆蛋白转录的染色质增强和阿坝激活在叶组织中菜豆蛋白（phas）启动子的TATA区域上旋转和旋转定位的核小体赋予转录失活的染色质结构。向转录活性状态的转变需要两个主要步骤：染色质增强和ABA介导的活化。本项目将通过以下具体目标对这些步骤中涉及的过程进行更详细的研究：（1）研究PvALF介导的phas启动子染色质增强的机制：（2）确定PvALF结构域在转录激活中的作用;（3）检测组合顺式元件相互作用及其在ABA介导的phas启动子激活中的作用。在目的（1）中，将在存在和不存在DNA复制抑制剂的情况下，使用分裂和非分裂组织研究DNA复制对phas激活的影响。将使用诱导型启动子来评估DNA复制对PvALF在分别具有活跃或无DNA复制的幼叶或老叶细胞中增强转录的能力的影响。用于探索phas启动子增强和活化的其它系统包括在植物核因子（包括天然和修饰的PvALF）存在和不存在下由非洲爪蟾提取物制备的人工核。在目的（2）中，将使用体外和体内方法评估PvALF的A和B2结构域在转录激活中的作用。PvALF与特定RY元件的相互作用将在营养组织和种子组织中使用带有phas启动子RY元件突变并含有（或缺乏）PvALF的等基因系进行评估。 DNA酶I和DMS足迹法将在体内进行，以确定这些细胞系组织中的phas启动子染色质结构和因子占有率。目标（3）中的实验将允许评价RY元件与相邻元件相互作用以产生功能性转录因子组合的能力。另外的phas启动子构建体将用于探索特定序列元件在ABA介导的转录激活中的参与。phas启动子驱动强的和严格的组织特异性基因表达，被证明在植物生物技术中被广泛使用。因此，该项目的研究结果将具有实际应用价值，并为研究转录的基本生物过程提供一个植物系统。确认复制参与转录激活将代表一个重大的科学进步，因为迄今为止还没有人令人信服地说明复制直接参与高等真核生物基因的转录。

HallNSF project: Chromatin potentiation and ABA activation of phaseolin transcription A rotationally and translationally-positioned nucleosome over the TATA region of the phaseolin (phas) promoter in leaf tissue confers a transcriptionally inactive chromatin structure. Transition to a transcriptionally competent status requires two major steps: chromatin potentiation and ABA-mediated activation. In this project, the processes involved in these steps will be examined in greater detail through the following specific objectives: (1) An investigation of the mechanisms involved in PvALF-mediated chromatin potentiation of the phas promoter; (2) A determination of the role of PvALF domains in transcriptional activation; (3) An examination of combinatorial cis-element interactions and their role in ABA-mediated activation of the phas promoter. In objective (1), the effect of DNA replication on phas activation will be investigated using dividing and non-dividing tissues, in the presence and absence of DNA replication inhibitors. An inducible promoter will be used to evaluate the effect of DNA replication on PvALF's ability to potentiate transcription in cells of young or old leaves which have, respectively, active or no DNA replication. Other systems to be used for exploration of phas promoter potentiation and activation include artificial nuclei made from Xenopus extracts in the presence and absence of plant nuclear factors (including native and modified PvALF). In objective (2), the role of the A and B2 domains of PvALF in transcriptional activation will be evaluated using both in vitro and in vivo approaches. The interaction of PvALF with specific RY elements will be evaluated in vegetative tissues and in seed tissues using isogenic lines bearing phas promoter RY element mutations and containing (or lacking) PvALF. DNase I and DMS footprinting will be undertaken in vivo to determine phas promoter chromatin structure and factor occupancy in tissues of these lines. Experiments in objective (3) will permit evaluation of the ability of RY elements to interact with adjacent elements to yield functional transcription factor combinations. Additional phas promoter constructs will be used to explore the participation of specific sequence elements in ABA-mediated transcriptional activation.The phas promoter, which drives strong and rigorously tissue- (seed) specific gene expression, is proving to be widely used in plant biotechnology. Therefore, findings from this project will have practical application as well as providing a plant system for studying the fundamental biological process of transcription. Confirmation of the participation of replication in transcriptional activation would represent a major scientific advance since no one has thus far illustrated convincingly that replication is directly involved in the transcription of a higher eukaryote gene.