Multiple Roles and Interaction Factors of an E-Box Element in Chlamydomonas reinhardtii

Abstract

The two subunits of the circadian RNA-binding protein CHLAMY1 from Chlamydomonas reinhardtii are involved in maintaining period (C1 subunit) and phase (C3 subunit) of the circadian clock. C1 coregulates the level of C3. Overexpression of C1 causes a parallel increase in C3. Both subunits can also integrate temperature information, resulting in hyperphosphorylation of C1 and up-regulation of C3 at low temperature. Temperature-dependent up-regulation of C3 is mediated predominantly by an E-box element and only partially by two DREB1A-boxes that are situated within the C3 promoter. The E-box element is also involved in circadian C3 expression. Here, we show that the C3 promoter region drives C3 coregulation by C1. We also found that replacement of the E-box prevents the coregulation of C3 in strains overexpressing C1. In contrast, replacement of any of the two DREB1A-boxes does not influence either the coregulation of C3 by increased levels of C1 or circadian C3 expression. Thus, the E-box has multiple key roles, including temperature-dependent up-regulation of C3, its circadian expression, and its coregulation by C1. Using mobility shift assays and DNA-affinity chromatography along with mass spectrometry, we characterized proteins binding specifically to the E-box region and identified five of them. By immunoblotting, we could further show that C3 that was detected in nuclear extracts can be found in the E-box-binding protein complex. Our data indicate a complex transcriptional mechanism of C3 up-regulation and a positive feedback of C3 on its own promoter region.

A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii

“…We observed an ;15-fold increase in the C3 transcript after the cells were exposed to 30 min of either blue or red light, demonstrating that the gene encoding C3 belongs to a group of rapidly induced genes such as VVD in Neurospora; a disruption in VVD also causes phase shifts of circadian rhythms (Heintzen et al, 2001;Lewis et al, 2002). Notably, the level of C3 mRNA is strongly influenced by both light and temperature (Voytsekh et al, 2008;Seitz et al, 2010), suggesting that it links light-and temperature-responsive entrainment pathway(s) with the oscillatory system. Moreover, this protein can influence circadian output as part of the CHLAMY1 complex (Kiaulehn et al, 2007).…”

Novel interaction of two clock‐relevant RNA‐binding proteins C3 and XRN1 in Chlamydomonas reinhardtii

“…The parallel up‐regulation of both proteins, C3 and XRN1, in cells grown at lower ambient temperature (18 °C versus 28 °C) opens up the possibility that XRN1 is a further member of this network and might be involved in entrainment by temperature cycles, as already indicated above. The temperature‐dependent expression of C3 was shown to be under the control of an E‐box element that is situated in the c3 promoter region and targeted by different proteins including also C3 [6]. Related E‐box elements could also be detected up to 1200 bp upstream from the XRN1 start codon.…”

Clocks in Algae

“…An E-box element in the promoter region of c3 is required for the circadian expression and temperature-dependent upregulation of c3 as well as the coregulation of C3 by C1 (65) . C3 interacts strongly with the exoribonuclease XRN1 beginning of the night or at low temperatures (18 °C) and knockouts of XRN1 result in low amplitude rhythms of bioluminescence (66,67) .…”