Dawn H. Nagel, Colleen J. Doherty, Jose L. Pruneda-Paz, Robert J. Schmitz, Joseph R. Ecker, and Steve A. Kay

Significance

The circadian clock, an endogenous time-keeping mechanism common to most species, allows organisms to coordinate biological processes with specific times of day. In plants, the role of the clock extends to almost every aspect of growth and development, including responses to biotic and abiotic stresses. The core molecular components and circuits of the clock have been well studied in the model organism Arabidopsis thaliana; however, how this mechanism connects to clock-controlled outputs remains poorly understood. Here, we performed a genome-wide characterization of the direct targets of a key clock component in Arabidopsis. Our results emphasize the broad role of the plant clock in regulating multiple biological functions and provide direct links between the oscillator and clock-regulated outputs.

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.

See: http://www.pnas.org/content/112/34/E4802.abstract.html?etoc

PNAS August vol. 112 no. 34 > Dawn H. Nagel,  E4802–E4810, doi: 10.1073/pnas.1513609112

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.