(l to r) Anna Puszynska, Joseph Piechura, and Erin O’Shea (not shown Joseph Markson)
Organisms from humans to bacteria utilize endogenous timing mechanisms called circadian clocks to coordinate the timing of physiological processes with the predictable day/night cycle. All circadian clocks are composed of a core oscillator, which provides timing information, and an output pathway, which decodes information from the core oscillator to control physiological responses such as gene expression. For example, the cyanobacterium Synechococcus elongatus contains a circadian clock that generates genome-wide oscillations in gene expression, with oscillations of individual genes displaying a variety of phases.
In S. elongatus, the core oscillator of the clock is composed of a three protein post-translational oscillator (PTO). The PTO separately controls both a kinase and a phosphatase, which together act on the putative transcription factor RpaA to generate circadian oscillations in RpaA phosphorylation. Genetic studies have shown that RpaA is required for circadian oscillations in expression of a handful of genes, but the mechanistic role for RpaA in promoting global circadian gene expression was unknown.
In a recent paper published in Cell, we show that RpaA is the master regulator of genome-wide circadian gene expression in S. elongatus. First, we show that when rpaA is deleted, global circadian gene expression arrests in a state similar to that of wild-type cells at dawn, even if the PTO continues to oscillate. This suggests that RpaA is indeed an essential node through which the core oscillator controls global gene expression.
Next, we demonstrate that RpaA binds to roughly 100 sites in the genome in vivo with a circadian periodicity in phase with oscillations in RpaA phosphorylation. Furthermore, we show that RpaA binds to several of these sites in vitro in a phosphorylation-dependent manner, suggesting that clock control of RpaA phosphorylation directly regulates its activity. Genes apparently controlled directly by RpaA include regulators of cell division, translation, carbon metabolism, and transcription, as well as components of the clock itself, demonstrating the pervasive control of physiology by the clock in cyanobacteria.
Finally, we show that induction of a constitutively activated RpaA variant in cells that lack RpaA can switch cells from a dawn-like expression state to a dusk-like expression state, causing changes in expression of roughly 700 circadian genes. Surprisingly, gene expression phasing in wild-type cells is recapitulated upon induction of the RpaA phosphomimetic, suggesting that expression phases are hard-wired downstream of RpaA phosphorylation. Together, our results demonstrate that by generating circadian oscillations in phosphorylation of a single transcription factor, a circadian clock can produce genome-wide multiphasic gene expression oscillations.
