Clock transcription factor CCA1 is regulated through sumoylation
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Date
07/07/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Hansen, Louise Lipczak
Metadata
Abstract
The circadian clock is an endogenous timekeeper that synchronises biological processes with
daily external rhythms such as light and temperature cycles. It provides organisms with a
competitive advantage by allowing anticipation of daily events. The circadian clock
encompasses a network of transcription-translational feedback loops (TTFLs) that
orchestrates rhythmic expression of a large part of the genome. This network is regulated at
post-transcriptional and post-translational level. Post-translational regulation of clock
proteins is essential to ensure stable rhythms and robust timekeeping. Unlike the genes in
the TTFL network, modifiers of clock proteins at post-translational level are conserved
across taxa.
SUMO, a small ubiquitin-related post-translational modifier, regulates timekeeping in
mammals through modification of the clock transcription factor BMAL. In this study, SUMO
is shown to contribute to oscillator function in Arabidopsis plants. Methods have been
developed to prove that mutant lines defective in SUMO machinery, including SUMO-ligase
and -protease mutants, display long circadian rhythms. Additionally, sumoylation on the
crucial plant clock transcription factor CCA1 is observed in vivo. A fraction of the protein is
sumoylated across the expression window of CCA1, with the phase of peak sumoylation in
advance of peak total CCA1. The effect of sumoylation of CCA1 was investigated with
respect to localisation, stability and DNA binding affinity of the protein, as these are
previously described possible effects of sumoylation. The subcellular location of CCA1-YFP
fusions in protoplasts was not altered in mutant lines of the SUMO machinery. In vitro
experiments show that sumoylation negatively affects the affinity of CCA1 to its cognate
promotor element, suggesting that SUMO could act as a reversible attenuator of CCA1
activity. Furthermore, effects of SUMO machinery mutations appear to be differential across
a range of physiologically relevant temperatures, implying that sumoylation could be
involved in the response to or buffering against fluctuating ambient temperatures.
There is an increasing amount of evidence to suggest that metabolic oscillations are not only
driven by transcriptional outputs of the clock, but are to some extent self-sustained and can
feed timing information back into the clock. Glutathione was investigated as a possible
metabolic feedback signal. Expression of clock gene CCA1 was found to be abolished in a
mutant of the rate-limiting enzyme for glutathione synthesis (pad2-1). Surprisingly however,
the amount of glutathione was not found to oscillate.
Combined, the results discussed in this thesis provide a substantial advance on our
understanding of post-translational regulation and the integration of metabolic and
environmental information into the plant circadian clock.