A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock

Abstract

Transcriptional feedback loops constitute the molecular circuitry of the plant circadian clock. In Arabidopsis, a core loop is established between CCA1 and TOC1. Although CCA1 directly represses TOC1, the TOC1 protein has no DNA binding domains, which suggests that it cannot directly regulate CCA1. We established a functional genomic strategy that led to the identification of CHE, a TCP transcription factor that binds specifically to the CCA1 promoter. CHE is a clock component partially redundant with LHY in the repression of CCA1. The expression of CHE is regulated by CCA1, thus adding a CCA1/CHE feedback loop to the Arabidopsis circadian network. Because CHE and TOC1 interact, and CHE binds to the CCA1 promoter, a molecular linkage between TOC1 and CCA1 gene regulation is established.

Fig. 1. CHE is a novel CCA1 promoter binding protein

(A and B) Interaction of CHE to different regions of CCA1 (A) and LHY (B) promoters in yeast. Bars represent the fold of induction in β-galactosidase activity for each of the DNA fragments indicated (n=6). (C) Binding of CHE to the TCP-binding site (TBS) in CCA1 promoter determined by electrophoretic mobility shift assay. The DNA/CHE complex (indicated by the arrowhead) was competed by the addition of unlabeled wild-type (TBS) or mutant (mTBS) probes. (D) CHE and CCA1 expression in Col-0 wild-type seedlings growing under constant light (LL). mRNA levels were normalized to IPP2 expression (n=3). (E and F) Subcellular localization of CHE in Arabidopsis protoplasts. Visible light (E), GFP channel (F). (G and H). CHE and CCA1 expression patterns determined by histochemical staining for GUS activity in CHE∷GUS (G) and CCA1∷GUS (H) transgenic seedlings. Scale bars, 0.5 mm. (I) Effect of mutations within the TCP-binding site (mTBS) on CCA1 promoter activity. Promoter∷luciferase constructs [CCA1∷LUC+ and CCA1(mTBS)∷LUC+] were transformed into Col-0 seedlings. Luciferase activity was determined in T1 lines (n=27). (J) Binding of CHE to CCA1 promoter in vivo. ChIP assays were performed with 35S∷CHE-GFP or wild-type CCA1∷LUC+ (wt) seedlings. Immunoprecipitated DNA was quantified by real-time PCR with primers specific for the TCP-binding site in CCA1 promoter (TBS), and for control regions (5′U, 3′D, ACT, UBQ) (12). Results were normalized to the input DNA (n=4). Values represent means ± SEM [(A), (B), (D), (I), (J)].

Fig. 2. CHE is a clock component directly repressing CCA1 promoter activity

(A and C) Bioluminescence analysis of CCA1∷LUC+ expression in CHE overexpression lines (35S∷CHE) (n=20) (A) and che T-DNA insertion lines (che-1 and che-2) (n=45) (C). Wild-type (wt) traces correspond to CCA1∷LUC+ seedlings. (B) Phase change of luciferase expression in wild-type CCA1∷LUC+ (wt) and the 35S∷CHE lines shown in (A). Each symbol represents one seedling. (D) Effect of mutations within the TCP-binding site (mTBS) on CCA1 promoter activity in a che mutant background. Promoter∷luciferase constructs [CCA1∷LUC+ and CCA1(mTBS)∷LUC+] were transformed into che-2 seedlings. Luciferase activity was determined in T1 lines (n=37) (E) Period estimates of luciferase expression in wild-type CCA1∷LUC+ (wt), che and lhy T-DNA insertion lines (che-1, che-2 and lhy-20), and che/lhy double mutants (che-1/lhy-20 and che-2/lhy-20). Each symbol represents one seedling and the line is the average period value (*p<0.0005, **p<0.0001). Values represent means ± SEM [(A), (C), (D)].

Fig. 3. CHE expression is regulated by CCA1 and LHY

(A) Expression of CHE in wild-type Ws (wt) and cca1-11/lhy-21 double mutant seedlings growing in constant light (LL). mRNA levels were normalized to the expression of IPP2 (CHE/IPP2) (n=3). (B) Binding of CCA1 to the CCA1-binding site in CHE promoter determined by electrophoretic mobility shift assay. The DNA/CCA1 complex (indicated by the arrowhead) was competed by the addition of unlabeled wild-type (CBS) or mutant (mCBS) probes. (C) Binding of CCA1 to CHE promoter in vivo. ChIP assays were performed with CCA1∷GFP-CCA1 or wild-type Ws (wt) seedlings. Immunoprecipitated DNA was quantified by real-time PCR with primers specific for the CCA1-binding site in CHE promoter (CBS), and for control regions (ACT, EE, 5′U, 3′D) (12). Results were normalized to the input DNA (n=3). Values represent means ± SEM [(A) and (C)].

Fig. 4. CHE interacts with TOC1

(A) Interaction between CHE and TOC1 proteins in yeast. SD–WL medium is used for the selection of bait and prey proteins where a β-galactosidase overlay assay was performed to visualize the interaction. SD-WLH (3-AT) medium is used for the auxotrophic selection of bait and prey protein interactions. (B) Co-immunoprecipitation assay between TOC1 (YFP-TOC1) and CHE (TAP-CHE) expressed in tobacco leaves. The results are representative of four independent experiments. (C) Binding of TOC1 to the CCA1 promoter in vivo. ChIP assays were performed with TOC1∷YFP-TOC1 or wild-type CAB2∷LUC (wt) seedlings. DNA was quantified by real-time PCR with primers specific for the TCP-binding site in CCA1 promoter (TBS), and for control regions (5′U, 3′D, ACT, UBQ) (12). Results were normalized to the input DNA. Values represent means ± SEM (n=4). (D) Genetic interaction between CHE and TOC1. CHE was overexpressed in the TOC1∷YFP-TOC1 (TMG) background (35S∷CHE/TMG). Period and relative amplitude error estimates of CAB2∷LUC (wt) expression were determined in T3 seedlings (*p<0.0001). Values represent means ± SD (n=20). (E) Model for the proposed role of CHE in the Arabidopsis clock. At dawn high levels of CCA1 and LHY repress CHE and their own expression. CHE levels rise as the day progresses to maintain CCA1 at a minimum. By the end of the day TOC1 antagonizes CHE resetting the cycle.