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

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 clockcontrolled 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.genome-wide | circadian clock | clock-controlled outputs | transcriptional regulation I n anticipation of the daily changes in light and temperature, the plant's internal circadian clock regulates a large portion of the transcriptome, nearly 80% in rice, poplar, and Arabidopsis (1, 2). The clock provides temporal coordination of multiple biological functions to ensure optimal efficiency. For example, before dawn, photosynthetic transcripts accumulate in anticipation of the expected sunlight. Even when shifted to constant light (LL) and temperature, in the absence of day/night cues, transcripts associated with photosynthesis are still up-regulated before the subjective day (1-4). Not only are light-and energyrelated activities restricted to particular times, but other aspects of plant growth, including water use, hormone activity, UVB response, and low-temperature response, show this gating effect (5-9). This temporal partitioning of biological responses ultimately provides an optimal integration of the plant's organismal functions with the environment.The circadian clock in plants involves a posttranscriptional component and a well-studied transcriptional-translation feedback regulation between the Myb-like transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and a member of the PSEUDO RESPONSE REGULATOR (PRR) family, TIMING OF CAB2 EXPRESSION 1 (TOC1) (10-16). Together these three components contribute t...

Circadian Clock and Photoperiodic Flowering in Arabidopsis: CONSTANS Is a Hub for Signal Integration

“…1). At dawn, two MYB transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), repress evening-phased genes (Nagel et al, 2015;Kamioka et al, 2016). This repression is partly dependent on the function of the CONSTITUTIVE PHOTOMORPHOGENIC10 (COP10)-DE-ETIOLATED1-DAMAGED DNA BINDING1 complex, a negative regulator for photomorphogenesis (Lau et al, 2011).…”

“…Chromatin immunoprecipitation sequencing analyses brought us, to our knowledge, a new insight regarding potential variation in CCA1 binding sites. In addition to Evening Element and the CCA1 Binding Site, DNA motifs that contain G-box or CT repeats are significantly enriched in the CCA1 binding regions (Nagel et al, 2015;Kamioka et al, 2016), indicating that CCA1 may form complexes with different transcription factors that alter the DNA binding preferences of complexes that contain CCA1. Previously, genetic results implied that CCA1 and LHY directly act as transcriptional activators for the morningphased genes PSEUDO RESPONSE REGULATOR9 (PRR9) and PRR7 (Farré et al, 2005).…”

Blue Light- and Low Temperature-Regulated COR27 and COR28 Play Roles in the Arabidopsis Circadian Clock

“…In addition, levels of both COR27 and COR28 were also regulated by the clock, both peaking in abundance in the afternoon (ZT15) in LD conditions ( Figure 5B). Given that there are EE (AAAATATCT) and EE-like elements (AATATCT) in the COR27 promoter region (Mikkelsen and Thomashow, 2009) and that it was reported that the promoter of COR27 was occupied by CCA1 in a ChIP-seq analysis of plants expressing CCA1-GFP (Nagel et al, 2015), we measured transcription of COR27 and COR28 in the cca1 lhy double mutant. ACTIN7 (ACT7), ASPARTIC PROTEINASE A1 (APA1), and ISOPENTENYL PYROPHOSPHATE: DIMETHYLALLYL PYROPHOSPHATEISOMETASE2 (IPP2) (Endo et al, 2014) were used as internal controls for normalization in our qPCR analysis.…”

EARLY FLOWERING3 Redundancy Fine-Tunes Photoperiod Sensitivity

“…In turn, the expression of EC genes and formation of the EC itself are also tightly regulated by the circadian clock through multiple regulatory mechanisms (Herrero et al, 2012;Choudhary et al, 2015). The ELF3, ELF4, and LUX promoters are bound by the key clock protein CCA1, a morning phased myb transcription factor that acts together with its paralog LATE ELONGATED HYPOCOTYL (LHY) to repress evening-phased genes during the day (Lu et al, 2012;Adams et al, 2015;Nagel et al, 2015;Kamioka et al, 2016). Autoregulation of the EC may also be achieved through binding of the LUX protein to its own promoter .…”