A G-Box-Like Motif Is Necessary for Transcriptional Regulation by Circadian Pseudo-Response Regulators in Arabidopsis

Abstract

PSEUDO-RESPONSE REGULATORs (PRRs) play overlapping and distinct roles in maintaining circadian rhythms and regulating diverse biological processes, including the photoperiodic control of flowering, growth, and abiotic stress responses. PRRs act as transcriptional repressors and associate with chromatin via their conserved C-terminal CCT (CONSTANS, CONSTANS-like, and TIMING OF CAB EXPRESSION 1 [TOC1/PRR1]) domains by a still-poorly understood mechanism. Here, we identified genome-wide targets of PRR9 using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and compared them with PRR7, PRR5, and TOC1/PRR1 ChIP-seq data. We found that PRR binding sites are located within genomic regions of low nucleosome occupancy and high DNase I hypersensitivity. Moreover, conserved noncoding regions among Brassicaceae species are enriched around PRR binding sites, indicating that PRRs associate with functionally relevant cis-regulatory regions. The PRRs shared a significant number of binding regions, and our results indicate that they coordinately restrict the expression of target genes to around dawn. A G-box-like motif was overrepresented at PRR binding regions, and we showed that this motif is necessary for mediating transcriptional regulation of CIRCADIAN CLOCK ASSOCIATED 1 and PRR9 by the PRRs. Our results further our understanding of how PRRs target specific promoters and provide an extensive resource for studying circadian regulatory networks in plants.

Direct Repression of Evening Genes by CIRCADIAN CLOCK-ASSOCIATED1 in the Arabidopsis Circadian Clock

“…A number of potential regulatory cis-acting elements, including the LUX binding site (GATA/TGC) , TCP binding site (GGNCCCAC) (Pruneda-Paz et al, 2009), protein box (ATGGGCC) (Michael et al, 2008), and morning element (AACCACGAAAA) were absent, whereas a G-box (CACGTG) (Schindler et al, 1992), four EEs (AAAATATCT) (Harmer et al, 2000), and a CCA1 binding site (CBS; AACAATCT or AAAAATCT) (Wang et al, 1997) were found in the region between 21416 and the start codon of PRR5 ( Figure 1A). Genomic sequences that coimmunoprecipitate with PRRs in vivo are enriched for G-boxes Nakamichi et al, 2012;Liu et al, 2013;Liu et al, 2016), and the regulatory elements EE and CBS are directly recognized by RVEs/CCA1/LHY in vitro and by REV8 in vivo (Wang et al, 1997;Alabadí et al, 2001;Rawat et al, 2011), but previous work had left it unclear whether or not CCA1 and LHY associate with PRR5 upstream in vivo.…”

“…Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analyses combined with transcriptomics experiments indicate that clock-associated PRR family proteins directly repress key TFs involved in photoperiodic flowering, hypocotyl elongation, and cold stress responses Nakamichi et al, 2012;Liu et al, 2013;Liu et al, 2016). Genome-wide gene expression analyses using a chemically induced gene expression system revealed the potential targets of RVE8 and TOC1 (Gendron et al, 2012;Hsu et al, 2013).…”

Different Cold-Signaling Pathways Function in the Responses to Rapid and Gradual Decreases in Temperature

“…Moreover, mutations in the genes encoding pseudoresponse regulators (PRR9, PRR7, and PRR5), which are key components of the circadian oscillator, result in the upregulated expression of DREB1s and their downstream genes (Nakamichi et al, 2009(Nakamichi et al, , 2012. These three PRRs and a homolog, TIMING OF CAB EXPRESSION1, repress the expression of CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), generating a negative feedback loop for clock function (Nakamichi et al, 2009;Nagel et al, 2015;Kamioka et al, 2016;Liu et al, 2016). CCA1, which encodes a morning-expressed MYB transcription factor, can bind to the promoter regions of DREB1s, and cold-inducible expression of DREB1s has been reported to be significantly reduced in cca1 lhy double mutant plants (Dong et al, 2011), suggesting that CCA1 and its close homolog, LHY, are also important transcriptional activators in the cold-responsive expression of DREB1s.…”

CONSTANS Imparts DNA Sequence Specificity to the Histone Fold NF-YB/NF-YC Dimer

“…Note that in the NF-CO mutagenesis experiments of Figure 2, a C or a T at this position are essentially equivalent. Finally, the CCACGTG motif, resembling a G-box, and previously described in TOC1 and PRR chromatin immunoprecipitation (ChIP)-seq experiments and in promoters of genes upregulated after TOC1 overexpression (Gendron et al, 2012;Liu et al, 2016), was recovered from the nf-yc3 nf-yc4 nf-yc9 cohort and was the most enriched element in intersections involving this cohort. Collectively, these elements all contain or closely resemble the CCACA core motif identified by in vitro EMSAs as optimal for NF-CO.…”