Phytochrome A: Structure/Function and Signaling Pathways

描述（由申请人提供）：定义真核细胞感知、转导和响应细胞外信号的全谱机制仍然是当代生物医学研究的中心目标。这里的程序集中在细胞跟踪和响应来自环境的信息光信号的机制上。细胞质定位的光敏色素（phy）光敏感受器对这些信号的感知启动了一个转导过程，最终导致核基因表达的改变，从而指导一系列形态发生反应。这项研究的长期目标是确定这一过程发生的分子机制。目前的证据表明，bHLH转录因子的一个小亚家族，称为pif（物理相互作用因子），在黑暗中促进脑形态形成的发展，并且这种活性被光激活的物理分子直接结合逆转，在它们的光诱导易位到细胞核中。这一过程中的信号传递涉及生理诱导的磷酸化、泛素化和pif的降解，随之而来的转录变化驱动向光形态形成的转变。尽管最近取得了进展，但物理- pif和pif -基因组界面的分子和生化交易的机制基础仍未完全确定。这一A1建议的具体目的是：(A)定义物理- pif界面信号传导的分子和生化机制，重点关注新发现的蛋白激酶和泛素连接酶的偶联活性，这些蛋白激酶和泛素连接酶被激活的phyB（一种假激酶）与PIF3募集成一个多蛋白复合物，同时将光信号转导和衰减到直接靶基因（DTGs）。(B)定义新发现的PIF转录激活DTGs双峰调控的机制基础，包括PIF启动子占用的差异和dna结合PIF内在活性的定量局部调节。将使用多轨道策略，包括质谱分析、酵母1-、2-和3-杂交以及蛋白质微阵列筛选，以确定物理pif信号复合物和/或结合pif转录活性的分子修饰剂中的候选相互作用物，并对这些候选物的体内功能相关性进行反向遗传评估；体外生化重构实验将用于剖析信号复合体内的活动；dna亲和纯化测序（DAP-seq）程序将用于确定pif中负责目标启动子选择性的序列；和PIF- dtg启动子的表观基因组图谱将生成，以检查PIF-染色质相互作用在调节差异的PIF转录活性中的作用。

DESCRIPTION (provided by applicant): Defining the full spectrum of mechanisms used by eukaryotic cells to perceive, transduce and respond to extracellular signals remains a central goal of contemporary biomedical research. The program here is focused on the mechanisms by which cells track and respond to informational light signals from the environment. Perception of such signals by the cytoplasmically-localized, phytochrome (phy) photosensory receptors initiates a transduction process that culminates in the altered expression of nuclear genes that direct an array of morphogenic responses. The long-term goal of this research is to define the molecular mechanisms by which this process occurs. Current evidence indicates that a small subfamily of bHLH transcription factors, termed PIFs (for phy-Interacting Factors), promote skotomorphogenic development in darkness, and that this activity is reversed by direct binding of photoactivated phy molecules, following their light-induced translocation into the nucleus. Signal transfer in this process involves phy-induced phosphorylation, ubiquitylation and degradation of the PIFs, with consequent transcriptional changes that drive a transition to photomorphogenic development. Despite recent progress, the mechanistic bases of the molecular and biochemical transactions at the phy-PIF and PIF-genome interfaces remain to be fully defined. The specific aims of this A1 proposal are: (A) To define the molecular and biochemical mechanisms underlying signaling at the phy-PIF interface, focused on the coupled activities of newly-identified protein kinases and ubiquitin ligases, that are recruited, with PIF3 by activated phyB (a pseudokinase), into a multiprotein complex, that concomitantly transduces and attenuates light signals to direct-target genes (DTGs). (B) To define the mechanistic basis of a newly identified bimodal regulation of PIF transcriptional activation of DTGs, involving both differential between-PIF promoter occupancy and quantitative local modulation of the intrinsic activity of DNA-bound PIFs. A multi-track strategy will be used involving mass-spectrometry, yeast 1-, 2- and 3-hybrid, and protein-microarray screens to identify candidate interactors in phy-PIF signaling complexes and/or molecular-modifiers of bound-PIF transcriptional activity, coupled with reverse-genetic assessment of the in vivo functional relevance of such candidates; in vitro biochemical reconstitution experiments will be used to dissect the activities within the signaling complex; a DNA-affinity-purification-sequencing (DAP-seq) procedure will be used to define sequences responsible for target-promoter selectivity among the PIFs; and epigenome maps of the PIF-DTG promoters will be generated to examine the role of PIF-chromatin interactions in regulating differential PIF transcriptional activity.