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.

Public Health Relevance

Understanding the full array of molecular and cellular mechanisms by which cells perceive and transduce external signals remains an important goal of the biomedical sciences. Disruption of cellular signaling circuitry is a major cause of human diseases, such as cancer. The research proposed here will probe the mechanisms by which identified molecules involved in two facets of signaling, known to cause human cancer, work in the cell. Definition of such mechanisms has the potential to provide targets for drug treatments.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM047475-21A1
Application #
8759546
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Maas, Stefan
Project Start
1992-09-30
Project End
2018-06-30
Budget Start
2014-08-15
Budget End
2015-06-30
Support Year
21
Fiscal Year
2014
Total Cost
$380,969
Indirect Cost
$73,964
Name
University of California Berkeley
Department
Other Basic Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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Zhang, Yu; Mayba, Oleg; Pfeiffer, Anne et al. (2013) A quartet of PIF bHLH factors provides a transcriptionally centered signaling hub that regulates seedling morphogenesis through differential expression-patterning of shared target genes in Arabidopsis. PLoS Genet 9:e1003244
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Leivar, Pablo; Tepperman, James M; Cohn, Megan M et al. (2012) Dynamic antagonism between phytochromes and PIF family basic helix-loop-helix factors induces selective reciprocal responses to light and shade in a rapidly responsive transcriptional network in Arabidopsis. Plant Cell 24:1398-419
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Leivar, Pablo; Quail, Peter H (2011) PIFs: pivotal components in a cellular signaling hub. Trends Plant Sci 16:19-28
Franklin, Keara A; Quail, Peter H (2010) Phytochrome functions in Arabidopsis development. J Exp Bot 61:11-24
Quail, Peter H (2010) Phytochromes. Curr Biol 20:R504-7

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