Phytochrome A: Structure/Function and Signaling Pathways

描述（由申请人提供）：光敏色素（phy）家族的五成员感觉光感受器（phyA到phyE）对信息光信号的感知启动细胞内转导过程，最终导致核基因表达的改变，从而指导适应当前环境的适应性反应。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。现有数据表明，物理信号传导涉及光受体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bHLH转录因子家族成员在内的信号传导伙伴相互作用，导致靶基因的转录调节。最近的证据导致了一种范式的转变，即涉及信号交易的潜在机制。这些数据表明，至少有两种bHLH因子PIF1和PIF3，物理结合可能通过泛素蛋白体系统（UPS）诱导其快速降解。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来解决这些缺陷，phyA是家族中最具特征和实验最易于处理的成员。本提案的具体目标是：(a)识别和表征phyA信号转导中的分子成分；(b)确定phya诱导bHLH转录因子PIF1和PIF3降解的分子机制；(c)定义主要靶基因和实现植物诱导基因表达程序的转录网络。实验方法将包括：(a)克隆拟南芥中phyA信号中间体基因筛选中鉴定的成分；(b)酵母双杂交筛选潜在的初级物理信号伙伴；(c)分子遗传学和生化研究，以检查UPS可能参与PIF1和PIF3降解，确定可能参与的E3泛素连接酶，并确定物理信号传递的机制；(d)基于微阵列的phya信号缺陷突变体表达谱，以识别早期反应基因，并通过phya调控的转录网络绘制信号通道；(e)染色质免疫沉淀，以识别光感受器和/或bHLH信号伙伴的潜在直接靶标启动子。了解真核细胞感知和转导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。本文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.