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Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter
Published on: September 17, 2016
Circadian control of global gene expression by the cyanobacterial master regulator RpaA
Joseph S Markson1, Joseph R Piechura, Anna M Puszynska
1Howard Hughes Medical Institute, Harvard University Faculty of Arts and Sciences Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA; Graduate Program in Biophysics, Harvard University, Cambridge, MA 02138, USA.
Abstract:
The cyanobacterial circadian clock generates genome-wide transcriptional oscillations and regulates cell division, but the underlying mechanisms are not well understood. Here, we show that the response regulator RpaA serves as the master regulator of these clock outputs. Deletion of rpaA abrogates gene expression rhythms globally and arrests cells in a dawn-like expression state. Although rpaA deletion causes core oscillator failure by perturbing clock gene expression, rescuing oscillator function does not restore global expression rhythms. We show that phosphorylated RpaA regulates the expression of not only clock components, generating feedback on the core oscillator, but also a small set of circadian effectors that, in turn, orchestrate genome-wide transcriptional rhythms. Expression of constitutively active RpaA is sufficient to switch cells from a dawn-like to a dusk-like expression state as well as to block cell division. Hence, complex global circadian phenotypes can be generated by controlling the phosphorylation of a single transcription factor.
Insights
The cyanobacterial circadian clock uses the RpaA protein to control genome-wide gene expression rhythms and cell division. Phosphorylation of RpaA acts as a master switch for these essential circadian outputs.
Area of Science:
- Circadian Biology
- Microbiology
- Molecular Biology
Background:
- Cyanobacterial circadian clocks drive daily oscillations in gene expression and cell division.
- The precise molecular mechanisms governing these global rhythms remain largely unknown.
Purpose of the Study:
- To identify the master regulator controlling genome-wide transcriptional oscillations in cyanobacteria.
- To elucidate the role of the response regulator RpaA in the cyanobacterial circadian system.
Main Methods:
- Gene deletion and rescue experiments to assess RpaA function.
- Analysis of genome-wide transcriptional rhythms using transcriptomics.
- Investigation of RpaA phosphorylation states and their impact on gene expression and cell division.
Main Results:
- Deletion of rpaA abolished global gene expression rhythms and arrested cells in a dawn-like state.
- RpaA directly regulates both core clock components and downstream circadian effector genes.
- Phosphorylated RpaA is sufficient to induce a dusk-like expression state and block cell division.
Conclusions:
- The response regulator RpaA is the master regulator of cyanobacterial circadian outputs.
- RpaA integrates feedback on the core oscillator with the orchestration of genome-wide transcriptional rhythms.
- Circadian phenotypes are controlled by the phosphorylation state of the single transcription factor RpaA.
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