Genome-wide identification of CCA1 targets uncovers an expanded clock network in Arabidopsis

Abstract

The circadian clock in Arabidopsis exerts a critical role in timing multiple biological processes and stress responses through the regulation of up to 80% of the transcriptome. As a key component of the clock, the Myb-like transcription factor CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is able to initiate and set the phase of clock-controlled rhythms and has been shown to regulate gene expression by binding directly to the evening element (EE) motif found in target gene promoters. However, the precise molecular mechanisms underlying clock regulation of the rhythmic transcriptome, specifically how clock components connect to clock output pathways, is poorly understood. In this study, using ChIP followed by deep sequencing of CCA1 in constant light (LL) and diel (LD) conditions, more than 1,000 genomic regions occupied by CCA1 were identified. CCA1 targets are enriched for a myriad of biological processes and stress responses, providing direct links to clock-controlled pathways and suggesting that CCA1 plays an important role in regulating a large subset of the rhythmic transcriptome. Although many of these target genes are evening expressed and contain the EE motif, a significant subset is morning phased and enriched for previously unrecognized motifs associated with CCA1 function. Furthermore, this work revealed several CCA1 targets that do not cycle in either LL or LD conditions. Together, our results emphasize an expanded role for the clock in regulating a diverse category of genes and key pathways in Arabidopsis and provide a comprehensive resource for future functional studies.

Keywords: circadian clock; clock-controlled outputs; genome-wide; transcriptional regulation.

Fig. S1.

(A) Correlation of sequence tags between each of three CCA1 ChIP-Seq experiments performed at ZT26 in LL and one ChIP-Seq experiment performed at ZT38 in LL. R² values of correlation between the three ZT26 replicates are LL1–LL2, 0.93; LL2–LL3, 0.77; and LL1–LL3, 0.82. (B) Histogram showing distances between the identified peaks of CCA1 occupancy in LL and the TSS of the nearest gene (bin size = 0–500 bp; range, 0–3,000 bp). (C) Phase enrichment of cycling CCA1-occupied targets. The expression datasets include transcripts that display >0.5 MBPMA in LL (LL23_LDHH). Genes were grouped by phase and MBPMA score (strength of cycling) into 36 groups. The ratio of CCA1 occupancy to the number of genes in each group is indicated by the intensity of the heat map (red and blue colors indicate maximum and minimum ratios of putative targets in the bin). (D) Additional motifs enriched in CCA1-occupied targets in LL. (E) Functional enrichment analysis with full GO biological process category of the 1,100 CCA1 targets identified in LL with a peak within 1 kb of the TSS. Circle size is proportional to gene numbers, and the color of each circle represents the enrichment P value for the GO term label on that circle, with orange representing highest enrichment and yellow the lowest enrichment above the cutoff (FDR corrected 0.01). Distance between nodes was arranged manually to optimize readability. The graph was generated using BINGO software (63).

Fig. 1.

(A) Genomic distribution of CCA1-occupied sites in LL. Promoter/TSS is defined as the region from −1,000 to +1 from the TSS. (B) The Venn diagram shows the overlap of the presence of an EE in a region 1-kb upstream of genes closest to CCA1-occupied peaks relative to the genome. (C) Functional enrichment analysis against GO Slim of the 1,100 putative CCA1 targets identified in LL with a peak within 1 kb of the TSS. The sizes of circles are based on the gene numbers. The color of each circle represents the enrichment P value for the GO term label on that circle, with orange representing highest enrichment and yellow the lowest enrichment above the cutoff [false-discovery rate (FDR) corrected 0.01]. The distance between nodes was arranged manually to optimize readability. The graph was generated using BINGO software (63). (D) Phase enrichment of cycling CCA1-occupied targets. The expression datasets include transcripts that display >0.5 MBPMA in constant light [LL12_LDHH, entrained in 12-h:12-h LD cycles at constant temperature (HH) and then subjected to LL; data from the Kay laboratory] (3). Genes were grouped by phase and MBPMA score (strength of cycling) into 36 groups. The ratio of CCA1 occupancy to the number of genes in each group is indicated by the intensity of the heat map (red and blue colors indicate the maximum and minimum ratios of putative targets in the bin, respectively. (E) Example of an EE-containing, evening-phased, and cycling CCA1 target, EARLY-RESPONSIVE TO DEHYDRATION 7 (ERD7, AT2G17840). (Upper) Normalized tag counts by location in the genome for each of the three replicate ChIP-Seq experiments in LL. Genome annotation shows the ERD7 gene visualized using IGB (55). (Lower) ERD7 shows rhythmic expression in two of the previously published time series experiments in constant conditions, LL12_LDHH and LL23_LDHH [entrained in 12-h:12-h LD cycles at constant temperature (HH) and then subjected to LL; data are from the Millar laboratory (4)]. (F) The putative CCA1 targets were grouped based on their rhythmic expression in previous studies. Genes were classified as noncycling in either of the time series experiments performed in LL, as cycling in both experiments, or as cycling in only one experiment or the other. (G) Example of a CCA1 target, Ras-related small GTP-binding family protein (AT1G02620), which does not cycle in either published LL time series. (H) CCA1 targets, selected as having strong peaks of CCA1 occupancy within 1 kb of the TSS, were examined for their phase of peak expression in two published LL time series. Here we classified evening genes as those with peak phase of expression between ZT10 and ZT16.

Fig. 2.

(A) Comparison of CCA1-occupied peaks in LL and LD. The Venn diagram shows the number of targets that are LD-specific (455) shaded in blue, the targets that are LL-specific (305) shaded in green, and the overlap between LD and LL targets (1,000 targets). (B) Alignment of the EE motif identified in LL and LD targets. (C) Functional enrichment analysis against GO Slim of the 1,231 CCA1 targets in LD with a peak within 1 kb of the TSS. The sizes of circles are based on the gene numbers. The color of each circle represents the enrichment P value for the GO term label on that circle, with orange representing highest enrichment and yellow the lowest enrichment above the cutoff (FDR corrected 0.01). (D) Phase enrichment of cycling CCA1-occupied targets. The expression datasets include transcripts that display >0.5 MBPMA in LD conditions [LDHH_ST, entrained in 12-h:12-h LD cycles and constant temperature (HH), data are from the Stitt laboratory (ST)] (39). Genes were grouped by phase and MBPMA score. The ratio of CCA1 occupancy to the number of genes in each group is indicated by intensity of the heat map (red and blue colors indicate maximum and minimum ratios of putative targets in the bin, respectively). (E) Example of a CCA1-occupied target in LD visualized using IGB (55). β-Glucosidase, GBA2 type family protein (AT5G49900), shows a rhythmic expression pattern in two of the previously published LD expression experiments (green) but not in LL (blue). (F) An example of a noncycling CCA1-occupied target in LD and LL, AT1G02620, visualized using IGB (55). Genome annotation for target gene structures is shown below. (G) Percentage of the CCA1-occupied targets in LD for each category of expression in each of the four time series datasets. The upper bars represent the percent of CCA1 targets that match the category: red bars represent morning-phased (ZT22–ZT4) genes, blue bars represent evening-phased (ZT10– ZT16) genes, and gray bars represent noncycling genes (MBPMA <0.80). The lower black bars show the total number of genes matching the respective category in that particular dataset.

Fig. S2.

(A) Genomic distribution of CCA1-occupied sites in LD conditions. Promoter/TSS is defined as the region from −1,000 to +1 from the TSS. (B) Histogram showing distances between the identified peaks of CCA1 occupancy in LD and the TSS of the nearest gene (bin size = 0–500 bp; range, 0–3,000 bp). (C) Overlap of EE occurrence in the region 1-kb upstream of genes closest to CCA1-occupied peaks relative to the genome in LD. (D) Comparison between the distribution of the EE motif (AAAATATCT) and the PBX motif (GGGCCCA) in the CCA1-occupied peaks in LL and LD. (E) Additional motifs enriched in CCA1-occupied targets in LD conditions. (F) Functional enrichment analysis with full GO biological process category of the 1,231 CCA1 targets identified in LD with a peak within 1 kb of the TSS. Circle size is proportional to gene numbers, and the color of each circle represents the enrichment P value for the GO term label on that circle, with orange representing highest enrichment and yellow the lowest enrichment above the cutoff (FDR corrected 0.01). The distance between nodes was arranged manually to optimize readability. The graph was generated using BINGO software (63). (G) Phase enrichment of cycling CCA1-occupied targets. The expression datasets include transcripts that display >0.5 MBPMA in LD conditions [LDHH_SM, entrained in 12-h:12-h LD cycles and constant temperature (HH); data are from the Smith laboratory (SM)] (38). Genes were grouped by phase and MBPMA score. The ratio of CCA1 occupancy to the number of genes in each group is indicated by intensity of the heat map (red and blue colors indicate the maximum and minimum ratios of putative targets in the bin, respectively).

Fig. S3.

(A) Functional enrichment analysis against GO Slim of the morning-expressed CCA1 targets identified in LD with a peak within 1 kb of the TSS. (B) Functional enrichment analysis with full GO biological process category of the morning-phased CCA1-occupied targets identified in LD. The circle size is proportional to gene numbers, and the color of each circle represents the enrichment P value for the GO term label on that circle, with orange representing the highest enrichment and yellow representing the lowest enrichment above the cutoff (FDR corrected 0.01). The distance between nodes was arranged manually to optimize readability. The graph was generated using BINGO software (63). (C and D) qRT-PCR of RVE1 (C) and HEMA1 (D) transcript levels in wild-type (Col-0) and cca1-1/lhy-21 plants grown in LL after 10 d entrainment in 12-h:12-h LD cycles or collected after growth only in LD.

Fig. 3.

(A and B) Examples of CCA1-occupied regions in the morning-expressed genes RVE1 (AT5G17300) (A) and HEMA1 (AT1G58290) (B) in LL and LD visualized using IGB (55). Normalized tag counts by location in the genome are shown for each of the three replicate LL ChIP experiments (blue) and for each of the two replicate LD ChIP experiments (green). Genome annotation for RVE1 or HEMA1 gene structures is shown below. (C and D) Real-time qRT-PCR of RVE1 (C) and HEMA1 (D) transcript levels in wild-type (Col-0) and CCA1-OX plants grown in LL after 10 d entrainment in 12-h:12-h LD cycles or only in LD. mRNA levels were normalized to IPP2 and PP2A expression (mean values ± SD, n = 2, two independent experiments. (E) Heatmap comparing the number of reads in this study and published ChIP-Seq experiments for other clock genes: TOC1 (AT5G61380), PRR5 (AT5G24470), and PRR7 (AT5G02810) that map to the 500-bp region surrounding the peaks identified in LD (15, 43, 44). Read counts are normalized to 10 million reads. Normalization factors are LD, 7.48; LL, 3.44 ZT38, 439.75; CCA1_Input, 0.73; TOC1, 6.43; TOC1_Input, 2.24; PRR5, 1.38; PRR5_Input, 5.66; PRR7, 5.10; PRR7_Input, 0.64; and PRR7_Negative control, 2.04. Each row represents a peak in LD, and the number of reads in that peak area in the corresponding ChIP-Seq experiments for each column is indicated from low (blue) to high (green). Read totals less ≤80 are mapped as white. (F) Motifs significantly enriched in CCA1-occupied targets with a morning phase of expression.

Fig. 4.

(A–C) CCA1-occupied peaks for the previously identified CCA1 targets TOC1 (AT5G61380), JMJD5 (AT3G20810), and COR27 (AT5G42900) (A) and uncharacterized CCA1 targets RMA1 (AT4G03510) (B) and DREB2C (AT2G40340) (C). Normalized tag counts by location in the genome are shown for each of the three replicate LL ChIP-Seq experiments (blue) and for each of the two replicate LD ChIP-Seq experiments (green). Genome annotation for each gene structure, visualized using IGB (55), is shown below. (D–G) qRT-PCR of wild-type and CCA1-OX plants grown in LL after 10-d entrainment in 12-h:12-h LD cycles for RMA1 (D), DREB2C (E), AT1G02620 (F), and ABCG2 (AT2G37360) (G). mRNA levels were normalized to IPP2 and PP2A expression (mean values ± SD, n = 2, two independent experiments).

Fig. S4.

(A) qRT-PCR of TOC1 transcript levels in wild-type (Col-0) and CCA1-OX plants grown in LL after 10 d entrainment in 12-h:12-h LD cycles. mRNA levels were normalized to IPP2 and PP2A expression (mean values ± SD, n = 2, two independent experiments). (B–D) Normalized tag counts by location in the genome for CCA1 targets ZAT12 (AT5G59820) (B), CAT3 (AT1G20620) (C), and AT2G37360 (D) for each of the three replicate LL ChIP experiments (blue) and for each of the two replicate LD ChIP experiments (green). Genome annotation for the respective gene structures is shown below.

Fig. S5.

(A and B) Normalized tag counts by location in the genome for CCA1 targets PRR9 (AT2G46790) (A) and LNK3 (AT3G12320) (B) for each of the three replicate LL ChIP experiments (blue) and for each of the two replicate LD ChIP experiments (green). Genome annotation for the respective gene structures is shown below. (C and D) qRT-PCR of LNK3 transcript levels in wild-type (Col-0) (C and D), CCA1-OX (C), and cca1-1/lhy-21 (D) plants grown in LL after 10-d entrainment in 12-h:12-h LD cycles or collected in continuous LD. mRNA levels were normalized to IPP2 and PP2A expression (mean values ± SD, n = 2, two independent experiments).