Phytochrome A: Structure/Function and Signaling Pathways

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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.