The role of an E-box element: multiple frunctions and interacting partners.

Circadian clocks can be entrained by light-dark or temperature cycles. In the green alga Chlamydomonas reinhardtii, 12h changes in temperature between 18°C and 28°C synchronize its clock. Both subunits of the circadian RNA-binding protein CHLAMY1, named C1 and C3, are able to integrate temperature information. C1 gets hyper-phosphorylated in cells grown at 18°C and the level of C3 is up-regulated at this temperature. In the long period mutant per1, where temperature entrainment is disturbed, the temperature-dependent regulation of C1 and C3 is altered. Up-regulation of C3 at the low temperature is mediated predominantly by an E-box element situated in its promoter region. This cis-acting element is also relevant for circadian expression of c3 as well as of its up-regulation in cells, where C1 is overexpressed. Among the few identified factors interacting with the E-box region, C3 is also present, suggesting that it feedbacks on its own transcription.

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.

Both subunits of the circadian RNA-binding protein CHLAMY1 can integrate temperature information.

The circadian RNA-binding protein CHLAMY1 from the green alga Chlamydomonas reinhardtii consists of two subunits named C1 and C3. Changes in the C1 level cause arrhythmicity of the phototaxis rhythm, while alterations in the level of C3 lead to acrophase shifts. Thus, CHLAMY1 is involved in maintaining period and phase of the circadian clock. Here, we analyzed the roles of the two subunits in the integration of temperature information, the basis for other key properties of circadian clocks, including entrainment by temperature cycles and temperature compensation. Applied temperatures (18 degrees C and 28 degrees C) were in the physiological range of C. reinhardtii. While C1 is hyperphosphorylated at low temperature, the C3 expression level is up-regulated at 18 degrees C. An inhibitor experiment showed that this up-regulation occurs at the transcriptional level. Promoter analysis studies along with single promoter element mutations revealed that individual replacement of two DREB1A-boxes lowered the amplitude of c3 up-regulation at 18 degrees C, while replacement of an E-box abolished it completely. Replacement of the E-box also caused arrhythmicity of circadian-controlled c3 expression. Thus, the E-box has a dual function for temperature-dependent up-regulation of c3 as well as for its circadian expression. We also found that the temperature-dependent regulation of C1 and C3 as well as temperature entrainment are altered in the clock mutant per1, indicating that a temperature-controlled network of C1, C3, and PER1 exists.