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所使用的全光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。通过细胞质定位的光敏色素(PHY)光敏受体感知这些信号，启动了一个转导过程，最终导致指导一系列形态发生反应的核基因表达的改变。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，一个被称为PIF(PHY相互作用因子)的bHLH转录因子小亚家族在黑暗中促进骨形态发生的发育，并且这种活性被光激活的PHY分子直接结合后逆转，这些分子在光诱导下移位到细胞核中。在这个过程中，信号传递涉及PHY诱导的PIF的磷酸化、泛素化和降解，以及随后的转录变化，从而驱动向光形态发生的转变。尽管最近取得了进展，但PHY-PIF和PIF-基因组界面上的分子和生化交易的机制基础仍未完全确定。这一A1建议的具体目标是：(A)确定PHY-PIF界面上潜在的信号转导的分子和生化机制，重点是新发现的蛋白激酶和泛素连接酶的偶联活性，这些酶与PIF3一起被激活的PhyB(一种伪激酶)招募到一个多蛋白复合体中，该复合体同时将光信号传导和衰减到直接靶基因(DTGs)。(B)明确新发现的PIF对DTGs转录激活的双峰调控的机制基础，涉及PIF启动子之间的差异占据和DNA结合的PIF内在活性的定量局部调节。将使用多轨道策略，包括质谱学、酵母1-、2-和3-杂交物和蛋白质微阵列筛选，以确定PHY-PIF信号复合体和/或结合-PIF转录活性的分子修饰物中的候选界面，并对这些候选对象的体内功能相关性进行反向遗传评估；体外生化重建实验将用于剖析信号复合体内的活性；DNA亲和-纯化-测序(DAP-SEQ)程序将用于确定负责PIF中靶标启动子选择性的序列；并将生成PIF-DTG启动子的表观基因组图谱，以检测PIF-染色质相互作用在调节差异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.