A functional link between rhythmic changes in chromatin structure and the Arabidopsis biological clock

Abstract

Circadian clocks rhythmically coordinate biological processes in resonance with the environmental cycle. The clock function relies on negative feedback loops that generate 24-h rhythms in multiple outputs. In Arabidopsis thaliana, the clock component TIMING OF CAB EXPRESSION1 (TOC1) integrates the environmental information to coordinate circadian responses. Here, we use chromatin immunoprecipitation as well as physiological and luminescence assays to demonstrate that proper photoperiodic phase of TOC1 expression is important for clock synchronization of plant development with the environment. Our studies show that TOC1 circadian induction is accompanied by clock-controlled cycles of histone acetylation that favor transcriptionally permissive chromatin structures at the TOC1 locus. At dawn, TOC1 repression relies on the in vivo circadian binding of the clock component CIRCADIAN CLOCK ASSOCIATED1 (CCA1), while histone deacetylase activities facilitate the switch to repressive chromatin structures and contribute to the declining phase of TOC1 waveform around dusk. The use of cca1 late elongated hypocotyl double mutant and CCA1-overexpressing plants suggests a highly repressing function of CCA1, antagonizing H3 acetylation to regulate TOC1 mRNA abundance. The chromatin remodeling activities relevant at the TOC1 locus are distinctively modulated by photoperiod, suggesting a mechanism by which the clock sets the phase of physiological and developmental outputs.

Figure 1.

Circadian Rhythms of H3 Acetylation and SSRP1 Binding to the TOC1 Promoter. (A) Structure of the TOC1 gene. Gray boxes delimitate exons. Arrowheads indicate the regions for PCR amplification in the ChIP assays. (B) Representative PCR bands from ChIP assays in wild-type seedlings entrained under LD cycles and subsequently released to LL conditions. Chromatin was immunoprecipitated as specified in Methods using antiacetylated histone H3 (αAcH3) antibody. Input DNA was used as a control. (C) Relative changes in acetylated H3 and TOC1 mRNA abundance plotted relative to the highest value. Data are represented as mean ± se of three independent experiments. (D) Representative PCR bands from wild-type seedlings entrained under LD cycles and shifted to LL conditions. Samples were processed by ChIP assays with anti-SSRP1 antibody (Duroux et al., 2004). Input DNA was used as a control. (E) TOC1 mRNA expression in wild-type seedlings analyzed by RNA gel blots. Actin mRNA was used as a control. (F) Relative changes in SSRP1 binding and TOC1 mRNA abundance plotted relative to the highest value. Data are representative of at least two independent experiments. Open and dotted boxes indicate the subjective day and subjective night, respectively.

Figure 2.

In Vivo CCA1 Binding, H3 Acetylation, and TOC1 mRNA Accumulation in CCA1-ox Plants. (A) and (B) Representative PCR bands from ChIP assays with CCA1-ox plants using anti-CCA1 antibody (A) or anti-AcH3 antibody (B). Plants were entrained under 12-h-light/12-h-dark cycles and subsequently released to LL conditions. Input DNA was used as control. (C) TOC1 mRNA expression in CCA1-ox plants analyzed by RNA gel blots. rRNA was used as a control. In each case, relative changes were plotted relative to the highest value. Data are representative of at least two independent experiments. Open and dotted boxes indicate the subjective day and subjective night, respectively.

Figure 3.

Effects of TSA Treatment on TOC1 Expression. (A) and (B) Luminescence of transgenic plants carrying the TOC1:LUC transgene examined in the absence or presence of TSA, added at ZT7 (A) or ZT0 (B). (C) Luminescence signals after switching to LL conditions. Experiments were performed as described in Methods. Dotted line indicates the initiation of free-running conditions in LL. Data are the means ± se of the luminescence of 6 to 12 individual seedlings. Arrows indicate the Zeitgeber time of TSA treatment. Data are representative of three independent experiments. Open and closed boxes indicate the light and dark periods, respectively.

Figure 4.

Photoperiodic Regulation of TOC1 Expression. (A) and (B) Luminescence of TOC1:LUC seedlings (A) and in vivo H3 acetylation at the TOC1 promoter (B) after ChIP assays in plants entrained under of 16-h-light/8-h-dark (16:8), 12-h-light/12-h-dark (12:12), or 8-h-light/16-h-dark (8:16) cycles. Samples were processed as described in Methods. (C) Luminescence of TOC1:LUC plants entrained to LgD cycles (16 h light/8 h dark) and subsequently changed to a ShD regime (8 h light/16 h dark). Data are the means ± se of the luminescence of 6 to 12 individual seedlings. Data are representative of at least two independent experiments. Open and closed boxes indicate the light and dark periods, respectively.

Figure 5.

Role of TOC1 in Setting the Phase of CAB2:LUC Expression under Different Photoperiods. (A) and (B) CAB2:LUC luminescence in wild-type and TMG plants entrained to ShD cycles ([A]; 8 h light/16 h dark) and LgD cycles ([B]; 16 h light:8 h dark). Data are the means ± se of the luminescence of 6 to 12 individual seedlings. (C) Phase plot of TOC1:LUC and CAB2:LUC expression in wild-type and TMG plants under the indicated photoperiods. Phases (phase/period × 24 h) were plotted against the strength of the rhythm expressed as relative amplitude error. The rhythm strength is graphed from 0 (center of the plot) to 0.8 (periphery of the circle), which indicates robust and very weak rhythms, respectively. (D) CAB2:LUC expression in TMG and TSA-treated wild-type plants entrained to 16-h-light/8-h-dark cycles. Data are the means ± se of the luminescence of 6 to 12 individual seedlings. Open and closed boxes indicate the light and dark periods, respectively.

Figure 6.

Role of TOC1 in Setting the Phase of Clock-Controlled Developmental Outputs. (A) Hypocotyl lengths of wild-type and TMG plants under ShD (8 h light/16 h dark) conditions. Data are the mean hypocotyl length ± se of 15 to 20 seedlings grown in the presence (+) or absence of TSA. (B) Transition to flowering in wild-type plants in the presence (+) or absence of TSA and in TMG plants maintained under LgDs (16 h light/8 h dark) or ShDs (8 h light/16 h dark). Flowering time is represented as the number of days to flowering (1-cm-high bolt). The experiments were done twice with similar results. (C) and (D) Relative changes of CO expression in wild-type seedlings with or without TSA and in TMG seedlings maintained under ShD ([C]; 8 h light/16 h dark) or LgD ([D]; 16 h light/8 h dark) conditions. Relative changes in CO expression were plotted relative to the highest value. Open and closed boxes indicate the light and dark periods, respectively.

Figure 7.

Schematic Representation Depicting the Rhythmic Regulation of TOC1 Expression. The circadian expression of TOC1 is controlled by dynamic changes in chromatin structure at the TOC1 locus. TOC1 repression depends on circadian binding of CCA1. Decreased CCA1 binding allows transcriptional activation through rhythmic cycles of histone acetylation and binding of SSRP1 (and Spt16) that favor transcriptionally permissive chromatin structures. HDAC activities after TOC1 peak of expression facilitate the switch to repressive chromatin structures and contribute to the declining phase of TOC1 waveform around dusk. Different photoperiodic conditions distinctively modulate these chromatin remodeling activities, defining a mechanism by which plants might synchronize the phase of the biological clock. Nucleosomes are shown as blue circles with the H3 N-terminal tails as curved lines in pale blue; black arrows indicate transcriptional activation, whereas lines ending in perpendicular dashes indicate repression. Open and shaded boxes indicate the light and dark periods, respectively. HAT, histone acetyl-transferase.