nemhauser lab
department of biology
university of washington
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Using well-characterized pathways to study signal integration and dynamics

Summary: If genomic approaches give a ‘bird’s eye’ view of a network, we can also use a ‘worm’s eye’ view to piece together the network from known parts of signaling pathways. Specifically, the Nemhauser Lab has focused on the relationship between two crucial growth-promoting hormones called auxin and brassinosteroids. These studies have raised intriguing questions about the dynamics of cellular responses and highlighted the many levels of interactions that can exist between hormone pathways.

Auxin and Brassinosteroids: Transcriptional profiling revealed a large number of co-regulated genes, and subsequent experiments strongly suggested that auxin and brassinosteroids interact directly on DNA regulatory regions. In a collaboration with Greg Vert and Joanne Chory, we have identified a direct molecular connection between the auxin and brassinosteroid signaling pathways. Phosphorylation by the brassinosteroid-regulated BIN2 kinase results in loss of both DNA binding and repression activities of a member of the Auxin Response Factor family of transcriptional regulators called ARF2. ARF2 is a transcriptional repressor that likely competes for DNA binding with ARFs that activate transcription. We have proposed a model where BIN2 increases expression of auxin-induced genes by directly inactivating repressor ARFs. Recently, we have discovered that the two pathways are completely dependent on one another to trigger expression of at least a subset of target genes.

Auxin and brassinosteroid responses are coordinated. Brassinosteroid-regulated gene expression is mediated by BES1/BZR1 transcription factors. Brassinosteroids inhibit BIN2-mediated phosphorylation of BES1/BZR1 thereby enhancing their activity. Auxin-binding to the TIR1 receptor promotes interaction between Aux/IAA co-repressors and the ubiquitination (U) machinery, leading to degradation. Our recent work suggests that brassinosteroids may facilitate BIN2 phosphorylation of repressor ARFs (yellow), removing them from DNA.
Auxin Synthetic Biology: Direct experimental test of the fundamental assumptions about auxin response dynamics are confounded by the ubiquity of auxin response in plant cells. For example, it is difficult to test our hypothesis that signals from other pathways (i.e., brassinosteroids) modulate a cell’s sensitivity to auxin by changing the levels of repressor ARFs. In collaboration with Eric Klavins in the UW Department of Electrical Engineering, we are systematically transplanting the auxin response pathway from Arabidopsis into the single-celled yeast Saccharomyces cerevisiae. An analogy to our approach is to understand how radios work by removing their components one by one, reconnecting them in a simple setting, and characterizing the resulting circuits in great detail. This approach allows us to (a) isolate parts of auxin response circuits from the rest of the auxin pathway and from the plant itself; (b) perform quantitative assays on the transformed S. cerevisiae cells not possible in plants, such as time-lapse single-cell fluorescence microscopy with controlled auxin pulses; and (c) build entirely novel, auxin response behaviors in S. cerevisiae that could eventually be applied to other organisms.

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