LUX ARRHYTHMO encodes a nighttime repressor of circadian gene expression in the Arabidopsis core clock - PubMed (original) (raw)

LUX ARRHYTHMO encodes a nighttime repressor of circadian gene expression in the Arabidopsis core clock

Anne Helfer et al. Curr Biol. 2011.

Abstract

Circadian clocks provide an adaptive advantage by allowing organisms to anticipate daily and seasonal environmental changes [1, 2]. Eukaryotic oscillators rely on complex hierarchical networks composed of transcriptional and posttranslational regulatory circuits [3]. In Arabidopsis, current representations of the circadian clock consist of three or four interlocked transcriptional feedback loops [3, 4]. Although molecular components contributing to different domains of these circuits have been described, how the loops are connected at the molecular level is not fully understood. Genetic screens previously identified LUX ARRHYTHMO (LUX) [5], also known as PHYTOCLOCK1 (PCL1) [6], an evening-expressed putative transcription factor essential for circadian rhythmicity. We determined the in vitro DNA-binding specificity for LUX by using universal protein binding microarrays; we then demonstrated that LUX directly regulates the expression of PSEUDO RESPONSE REGULATOR9 (PRR9), a major component of the morning transcriptional feedback circuit, through association with the newly discovered DNA binding site. We also show that LUX binds to its own promoter, defining a new negative autoregulatory feedback loop within the core clock. These novel connections between the archetypal loops of the Arabidopsis clock represent a significant advance toward defining the molecular dynamics underlying the circadian network in plants and provide the first mechanistic insight into the molecular function of the previously orphan clock factor LUX.

Copyright © 2011 Elsevier Ltd. All rights reserved.

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Figures

Figure 1

Figure 1. LUX is a sequence-specific DNA-binding protein

(A) LUX DNA binding site motif determined by universal protein binding microarray (PBM) experiments. (B) Effect of point mutations on DNA binding affinity. The scatter plot shows enrichment scores (E-scores) for two “all 10-mer” microarrays of different design; for each design, the E-scores from 3 replicates were averaged. The E-score correlates with the binding affinity of LUX for the sequence, and is measured on a scale of −0.5 (worst) to 0.5 (best). Spots containing the 6-mers GATACG and GATTCG are marked in red and orange, respectively; spots containing the variants G

G

TACG and GAT

G

CG are marked in green and blue, respectively. Below the scatter plot, E-scores are shown for variants at each position of the most preferred 8-mer AGATACGC. (C) LUX binding to synthetic multimers of the binding motif in a yeast one-hybrid system. Perfect match or mutant versions of the binding motif were multimerized and cloned upstream of a minimal promoter::LacZ transcriptional fusion. Bars represent the fold of induction in β-galactosidase activity in the presence of LUX-GAL4AD over control plasmid (means ± SEM, n=6 independent experiments). The selected mutations were predicted to abolish binding, based on PBM E-Scores. See also Tables S1–S3 and Figure S1.

Figure 2

Figure 2. LUX binds to PRR9 and LUX promoters in vivo

(A) Schematic of the PRR9 promoter (+1 is the transcriptional start site). The black arrowhead indicates the LBS; white arrowheads indicate degenerate binding sites (GATWKG or GATWCY where K indicates C or T, and Y indicates G or T). White rectangles represent the promoter fragments used for yeast one-hybrid assays in (B), with numbers relative to the transcriptional start site. Grey rectangles show the amplicons used in the ChIP experiments (C) and are numbered using the LBS as a reference (positions relative to the transcriptional start site are detailed in Table S4). (B) Binding of LUX to PRR9 promoter in yeast. Bars represent the fold of induction in β-galactosidase activity in the presence of LUX-GAL4AD over control plasmid (n=4 independent experiments). −243/+225(LBSm) is the −243/+225 fragment with a mutated LBS (GATTCG ->TCGGAT). (C) Binding of LUX to the PRR9 promoter in vivo. ChIP assays were performed with wild-type CAB2::LUC (wt) or lux-4 LUX::LUX-GFP (LUX::LUX-GFP) seedlings. Plants were grown under 12 h light/12 h dark (LD) cycles and transferred to continuous light (LL). Samples were collected from two independent lines (#43 and 49) at Zeitgeber Time 14 (ZT14) during the first day in LL, and processed for ChIP using an anti-GFP antibody. The immunoprecipitated DNA was quantified using real-time polymerase chain reaction (RTPCR) with primers specific for the amplicons represented in (A). UBQ, UBIQUITIN; CS coding sequence. Results were normalized to the input DNA (n=3 independent experiments). (D) Schematic of the LUX promoter. Black and white arrowheads indicate the LBS and degenerate LBS sequences, respectively, as described in (A). The grey rectangle shows the amplicon centered on the LBS used for ChIP assays. (E) Binding of LUX to its own promoter in vivo. The ChIP assays were performed as described in (C), with regions of the UBQ promoter or LUX CS as negative controls. Values represent means ± SEM in (B), (C), and (E). All primer sequences are detailed in Table S4. See also Figure S2.

Figure 3

Figure 3. LUX functions as a repressor

(A and B) LUX and PRR9 expression in wild-type plants grown in 12 h light/12 h dark (LD) cycles (A) or constant light (LL) (B). (C) PRR9 expression in wild-type CAB2::LUC (wt) and lux-4 mutant in LD released to LL. (A–C) Seedlings were entrained in LD for 10 days before release to LL. mRNA levels were normalized to IPP2 expression (mean values ± SEM, n=3 independent experiments). (D–G) Effect of the overexpression of LUX fused to either a repression domain (CRES) or an activation domain (VP64) in the lux-4 mutant. (D–F) Bioluminescence assays in wild-type CAB2::LUC (wt), lux-4 mutant, lux-4 35S::LUX-CRES (lux-4 LUX-CRES), and lux-4 35S::LUX-VP64 (lux-4 LUX-VP64) plants. (D) Period length and relative amplitude error (RAE) were calculated using fast Fourier transform nonlinear least-squares analysis (FFT-NLLS). Only plants for which the algorithm retrieves period length and RAE values can be represented on the plot (wt: 8 out of 8; lux-4: 6 out of 8; lux-4 LUX-CRES: 14 out of 14; lux-4 LUX-VP64: 3 out of 16). Individuals with an RAE lower than 0.6 are considered rhythmic. (E, F) Luciferase activity in wt, lux-4, lux-4 LUX-CRES (E), and lux-4 LUX-VP64 (F) lines. Third generation (T3) homozygous plants were entrained in LD for 8 days, then released to LL and imaged every 2.5h for 5 days. Values represent means ± SEM (n=8 for wt and lux-4; n=14 for lux-4 LUX-CRES; n=16 for lux-4 LUXVP64). The experiment was repeated three times with similar results, using two different transgenic lines (data shown for one representative line) selected from an initial screen of 48 primary transformants for each construct (data not shown). (G) Mean hypocotyl lengths of wt, lux-4, lux-4 LUXCRES, and lux-4 LUX-VP64 plants. Seedlings were grown in LD for 10 days before measuring the hypocotyl lengths (means ± SEM, n=20 plants). See also Figure S3.

Figure 4

Figure 4. Model for the proposed role of LUX in the Arabidopsis clock

LUX is responsible for the down-regulation of PRR9 and LUX transcription during late night. Some components of the network were omitted to simplify the model. Morning-expressed genes/proteins are represented in white, while evening-expressed genes/proteins are represented in black.

References

    1. Green RM, Tingay S, Wang ZY, Tobin EM. Circadian rhythms confer a higher level of fitness to Arabidopsis plants. Plant Physiol. 2002;129:576–584. - PMC - PubMed
    1. Dodd AN, Salathia N, Hall A, Kevei E, Toth R, Nagy F, Hibberd JM, Millar AJ, Webb AA. Plant circadian clocks increase photosynthesis, growth, survival, and competitive advantage. Science. 2005;309:630–633. - PubMed
    1. Pruneda-Paz JL, Kay SA. An expanding universe of circadian networks in higher plants. Trends Plant Sci. 2010;15:259–265. - PMC - PubMed
    1. Pokhilko A, Hodge SK, Stratford K, Knox K, Edwards KD, Thomson AW, Mizuno T, Millar AJ. Data assimilation constrains new connections and components in a complex, eukaryotic circadian clock model. Mol Syst Biol. 2010;6:416. - PMC - PubMed
    1. Hazen SP, Schultz TF, Pruneda-Paz JL, Borevitz JO, Ecker JR, Kay SA. LUX ARRHYTHMO encodes a Myb domain protein essential for circadian rhythms. Proc Natl Acad Sci U S A. 2005;102:10387–10392. - PMC - PubMed

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