Multiple roles and interaction factors of an E-box element in Chlamydomonas reinhardtii

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.