Enhancer trapping reveals widespread circadian clock transcriptional control in Arabidopsis

Abstract

The circadian clock synchronizes the internal biology of an organism with the environment and has been shown to be widespread among organisms. Microarray experiments have shown that the circadian clock regulates mRNA abundance of about 10% of the transcriptome in plants, invertebrates, and mammals. In contrast, the circadian clock regulates the transcription of the virtually all cyanobacterial genes. To determine the extent to which the circadian clock controls transcription in Arabidopsis, we used in vivo enhancer trapping. We found that 36% of our enhancer trap lines display circadian-regulated transcription, which is much higher than estimates of circadian regulation based on analysis of steady-state mRNA abundance. Individual lines identified by enhancer trapping exhibit peak transcription rates at circadian phases spanning the complete circadian cycle. Flanking genomic sequence was identified for 23 enhancer trap lines to identify clock-controlled genes (CCG-ETs). Promoter analysis of CCG-ETs failed to predict new circadian clock response elements (CCREs), although previously defined CCREs, the CCA1-binding site, and the evening element were identified. However, many CCGs lack either the CCA1-binding site or the evening element; therefore, the presence of these CCREs is insufficient to confer circadian regulation, and it is clear that additional elements play critical roles.

Figure 1.

Circadian enhancer trap using luciferase. Seedlings were grown 7 d in 12 h of light and 12 h of dark, and luminescence was measured in continuous light for 5 to 7 d. A, Circadian clock-controlled genes identified by enhancer trapping (CCG-ET) are phased to distinct times of day. Traces were normalized to the average expression over the experiment. All traces are the average of at least 12 seedlings. B, Number of CCG-ET lines with specific circadian phases: circadian time (CT) 0 (0–5.9 h), 6 (6–11.9 h), 12 (12–17.9 h), and 18 (18–23.9 h). All luminescence traces were analyzed for rhythmicity by fast Fourier transform-nonlinear least squares analysis (FFT-NLLS), and circadian phase was determined by normalizing for the period (phase = 24 h × phase/period). C, Expression level of the CCG-ET lines. Expression level was determined directly from the number of photons collected over the sampling interval: 200 (0–199 photons/s), 2,000 (200–1,999 photons/s), 20,000 (2,000–19,999 photons/s), and 200,000 (>20,000 photons/s). All data represent averages from at least two independent experiments. D, Map positions of CCG-ETs on the five Arabidopsis chromosomes.

Figure 2.

Insertions of LUCIFERASE relative to predicted Arabidopsis genes in the enhancer trap lines. T-DNA carrying the LUCIFERASE gene (shaded arrow) inserted as indicated into Arabidopsis genomic sequence in relation to the predicted CCG-ET (black arrow) and the nearest adjacent gene (white arrow). The distance (base pairs) from the ATG (or STOP) of the predicted CCG-ET to the start of the LUCIFERASE gene is indicated. Also indicated, below the horizontal line of the chromosome, is the distance between the predicted CCG-ET and the nearest adjacent gene. Depictions are not to scale.

Figure 3.

CCREs in the promoters of genes encoding clock components. Predicted promoters (–2,000/–1 upstream of the ATG) of CCA1, LHY, APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9 were aligned using AlignACE (http://arep.med.harvard.edu/; Hughes et al., 2000) and RSA (http://embnet.cifn.unam.mx/rsa-tools/). Motif models were created as sequence logos (Schneider and Stephens, 1990) using the program GENIO/logo (http://genio.informatik.unistuttgart.de/GENIO/logo.cgi). At each position, the height of each residue is proportional to its frequency in that position. The total height of all the residues in the position is proportional to the degree of conservation (information content) of the position, presented in bits. Two bits are sufficient to specify a single nucleotide as always present at a given position and, therefore, are the maximum information content possible at any position. Upside-down letters appear if the probability of that nucleotide occupying the position is less than the equally distributed a priori probability of all letters. A, CBS/EE (AAAa/tATCT); B, G box (CACGTG); C, HEX element (TGACGTGG); D, Number of each element and total number of elements in each predicted promoter.

Figure 4.

Occurrence of CBS, EE, and G box in predicted Arabidopsis promoters. Predicted promoters (–2,000/–1 upstream of the ATG) for most Arabidopsis genes (25,545) were searched for the following motifs: CBS (AAAAATCT), EE (AAATATCT), G box (CCACGTGG), and G box core (CACGTG). Venn diagrams illustrate the occurrences of the CBS (upper left), EE (upper right), and G box (bottom). A, Genes from the Arabidopsis genome; B, genes on the Affymetrix Gene Chip (AffyGC); C, genes on the AFGC microarray; D, CCG-ETs; E, clock-controlled genes detected by Harmer et al. (2000); F, clock-controlled genes detected by Schaffer et al. (2001).