Phytochrome A: Structure/Function and Signaling Pathways

描述（由申请人提供）：感觉光感受器的五成员光敏色素（phy）家族（phyA至phyE）对信息光信号的感知启动了细胞内转导过程，该过程最终导致核基因表达的改变，该核基因指导适应性反应以适应主要环境。这项研究计划的长期目标是确定这一过程发生的分子，细胞和生化机制。现有的数据表明，phy信号涉及光感受器分子从细胞质到细胞核的快速光激活易位，在细胞核中，它与包括转录因子的bHLH家族成员的信号伴侣物理相互作用，导致靶基因的转录调控。最近的证据已经导致了一个范式转变的潜在机制参与的信号交易。这些数据表明，至少有两个这些bHLH因子，PIF 1和PIF 3，phy结合诱导其快速降解，可能通过泛素蛋白酶体系统（UPS）。然而，这种现象的分子基础和调控后果尚未完全确定。我们建议使用phyA，最好的特点和实验最听话的家庭成员来解决这些不足之处。本提案的具体目标是：（a）鉴定和表征phyA信号转导中的分子组分;（B）确定phyA诱导的bHLH转录因子PIF 1和PIF 3降解的分子机制;（c）确定实施phyA诱导的基因表达程序的主要靶基因和转录网络。实验方法将包括：（a）克隆在拟南芥中phyA信号中间体的遗传筛选中鉴定的组分;（B）酵母双杂交筛选潜在的初级phy信号配偶体;（c）分子遗传学和生物化学研究，以检查潜在的UPS参与PIF 1和PIF 3降解，以鉴定潜在的E3泛素连接酶，并确定phy信号传递的机制;（d）phyA信号传导缺陷突变体的基于微阵列的表达谱分析，以鉴定早期应答基因并绘制通过phyA调节的转录网络的信号通道;和（e）染色质免疫沉淀，以鉴定作为光感受器和/或其bHLH信号传导配偶体的潜在直接靶标的启动子。了解真核细胞感知和处理细胞外信息信号的分子和细胞机制的全谱仍然是生物医学研究的中心目标。这里提出的实验系统和策略有可能为实现这一目标做出重大贡献。

DESCRIPTION (provided by applicant): The perception of informational light signals by the five-member phytochrome (phy) family of sensory photoreceptors (phyA to phyE) initiates an intracellular transduction process that culminates in the altered expression of nuclear genes that direct adaptational responses appropriate to the prevailing environment. The long-term goal of this research program is to define the molecular, cellular and biochemical mechanisms by which this process occurs. The available data indicate that phy signaling involves rapid light-activated translocation of the photoreceptor molecule from the cytoplasm to the nucleus where it interacts physically with signaling partners that include members of the bHLH family of transcription factors, resulting in transcriptional regulation of target genes. Recent evidence has resulted in a paradigm shift regarding the potential mechanism involved in the signaling transaction. The data suggest that for at least two of these bHLH factors, PIF1 and PIF3, phy binding induces their rapid degradation, possibly via the ubiquitin proteosome system (UPS). However, the molecular basis and regulatory consequences of this phenomenon are yet to be fully defined. We propose to address these deficiencies using phyA, the best characterized and experimentally most tractable member of the family. The specific objectives of this proposal are: (a) to identify and characterize molecular components in phyA signal transduction; (b) to define the molecular mechanism of phyA-induced degradation of the bHLH transcription factors, PIF1 and PIF3; and (c) to define the primary target genes and transcriptional networks that implement the phyA-induced gene expression program. The experimental approaches will include: (a) cloning of components identified in genetic screens for phyA signaling intermediates in Arabidopsis; (b) yeast two-hybrid screens for potential primary phy signaling partners; (c) molecular-genetic and biochemical studies to examine potential UPS involvement in PIF1 and PIF3 degradation, to identify the potential E3 ubiquitin ligase involved, and to define the mechanism of phy signal transfer; (d) microarray-based expression profiling of phyA-signaling-defective mutants to identify early-response genes and to map signal channeling through the phyA-reglated transcriptional network; and (e) chromatin immunoprecipitation to identify promoters that are potential direct targets of the photoreceptor and/or its bHLH signaling partners. Understanding the full spectrum of molecular and cellular mechanisms by which eukaryotic cells perceive and transduce extracellular informational signals remains a central goal of biomedical research. The experimental system and strategies proposed here have the potential to contribute significantly to this goal.