Fig 1.
Binding of LHY and CCA1 to the promoters of clock-associated genes.
(A) In vivo binding of LHY to the promoters of clock-associated genes was tested by ChIP-qPCR analyses of wild-type samples using a polyclonal antibody to the LHY protein. Plants were grown under 12L12D light-dark cycles and tissue was sampled two hours after dawn, corresponding to the time when LHY protein levels are at their maximum [31]. The ACTIN locus was used as a negative control. In order to demonstrate the specificity of the antibody, ChIP enrichments for wild-type (Ws) plants were compared to those for the insertion mutant, lhy-21. Error bars represent standard errors from two independent biological experiments. (B) In vivo binding of CCA1 was tested by performing ChIP-qPCR analyses on cca1-1 CCA1pro::CCA1-HA-YFP or wild-type plants using an antibody to the YFP tag. Enrichment for the ACTIN2-7 locus is shown as a negative control. Error bars represent standard errors from two independent biological experiments. (C) In vitro binding of LHY to purified genomic DNA. Bacterially expressed, His-tagged LHY protein was used to pull down sheared, purified genomic DNA. The resulting enrichment for different target promoter sequences was quantified by real time PCR using the same primers as used for ChIP analyses, and expressed as a percentage of input material. This experiment detected binding to all LHY binding targets identified in ChIP experiments, except for the LHY and CCA1 promoters. Error bars represent standard deviations from two independent binding experiments. (D) Mutations of the G-box and 5A sites disrupt in vivo binding of the LHY protein to its own promoter. The diagram at the top of the panel shows the relative positions of G-box and 5A motifs in the LHY promoter. Positions are numbered relative to the ATG and the arrow represents the transcriptional start site. ChIP experiments were carried out to assay binding of the LHY protein to wild-type and mutated LHY::luc transgenes. In order to account for differences in sample preparation, enrichment for LHY::luc transgene sequence was expressed relative to endogenous LHY promoter sequence. Relative enrichment levels close to 1 were obtained using the wild-type construct (-957/+1), indicating equivalent enrichment for the endogenous and the transgenic copies of the LHY promoter. However, mutation of the G-box flanking regions (ACCACGTGTC to GTCACGTGAC) reduced binding of the LHY protein by over 50%. Mutation of both flanking 5A sites [5A (1) CCAAAAA to TGTCAAA and 5A(2) TTTTTCC to TTTGACA] had a similar effect. Each data point represents the relative enrichment for one transgenic line. Error bars from Q-PCR analyses have been omitted for clarity.
Fig 2.
LHY represses expression of other clock components.
(A) Experimental design. Wild-type and Alcpro::LHY transgenic plants were grown under 12L12D light-dark cycles as illustrated by the white and black bars in the diagram. They were transferred to constant light at the start of the experiment. Expression of the Alcpro::LHY transgene was induced using 6% ethanol (v/v). Different sets of plants were treated at 4-hour intervals over the duration of one circadian cycle, and tissue was harvested 2 hours later. (B) mRNA levels were determined using Nanostring technology and normalized relative to UBC12. Times indicate when the tissue was harvested. (C) shows levels of PRR7 and PRR9 mRNA expression 26 h after induction of the Alcpro::LHY transgene at ZT17. Open bars indicate wild-type data (+EtOH), filled bars Alcpro::LHY data (+EtOH). Transcript levels from Quantitative RT-PCR analyses were normalized relative to ACTIN. Data shown are averages and standard errors from two independent biological replicates. * indicates p <0.05 and ** p<0.01 as determined by t-tests. An additional experiment comparing comparing effects of ethanol on PRR7 and PRR9 expression in Alcpro:::LHY plants, Alcpro::GUS and wild-type plants, 2, 6 and 10 hours after dawn is provided as S1 Fig.
Fig 3.
Induction of the ALCpro::LHY transgene abrogates the peak of LHY and CCA1 expression at dawn.
Ethanol (1% v/v) was added to plants 17 hours after dawn, i.e. just before the normal rise in LHY transcription. (A, B) Immunoblot showing changes in LHY protein levels after ethanol addition, and control experiment showing the specificity of the LHY antibody. The LHY protein is indicated by filled triangles, and a constitutive, cross-reactive band is indicated by open triangles. B indicates bacterially expressed LHY protein. As a loading control, the lower part of the gel was stained with Coomassie blue to reveal the RBCS protein. (C) Quantification of LHY protein levels from (A). LHY protein levels were normalized to the cross-reactive band and expressed relative to wild-type levels at time zero. (D, E) Changes in endogenous LHY and CCA1 mRNA levels as determined by quantitative RT-PCR. Transcript levels were normalized to the ACTIN transcript and to levels in control plants at time zero. Open symbols indicate wild-type and filled symbols, Alcpro::LHY data. Data shown are averages and standard errors from triplicate quantitative RT-PCR analyses. A replicate experiment in shown in S2A–S2C Fig.
Fig 4.
The transcriptional network of the plant circadian clock.
Revised regulatory connections arising from this work are indicated by heavier lines. We add a novel, direct autoregulatory loop from LHY/CCA1 onto their own expression and we introduce a change in the sign of the regulation of PRR5, 7 and 9 transcription by LHY and CCA1. Furthermore, we show that the PRR5 gene is also down-regulated by LHY/CCA1. Each box indicates a gene. Pointed arrows indicate positive regulation and blunt arrows, transcriptional repression. Blue arrows represent regulation by LHY/CCA1, red arrows regulation by the evening complex (EC) and black arrows regulation by the PRRs and TOC1. Orange arrows indicate regulatory interactions between the PRR genes, and dashed arrows indicative positive regulation by the RVE, LNK or LWD proteins.