| Property | Value |
|---|---|
| BioDare ID | 13401436566946 |
| Author | Norihito Nakamichi |
| Institution | RIKEN Plant Science Center |
| License | CC_BY |
Literature data from: 'PSEUDO-RESPONSE REGULATORS 9, 7, and 5 are transcriptional repressors in the Arabidopsis circadian clock.' by: Norihito Nakamichi.
An interlocking transcriptional-translational feedback loop of clock-associated genes is thought to be the central oscillator of the circadian clock in plants. TIMING OF CAB EXPRESSION1 (also called PSEUDO-RESPONSE REGULATOR1 [PRR1]) and two MYB transcription factors, CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), play pivotal roles in the loop. Genetic studies have suggested that PRR9, PRR7, and PRR5 also act within or close to the loop; however, their molecular functions remain unknown. Here, we demonstrate that PRR9, PRR7, and PRR5 act as transcriptional repressors of CCA1 and LHY. PRR9, PRR7, and PRR5 each suppress CCA1 and LHY promoter activities and confer transcriptional repressor activity to a heterologous DNA binding protein in a transient reporter assay. Using a glucocorticoid-induced PRR5-GR (glucorticoid receptor) construct, we found that PRR5 directly downregulates CCA1 and LHY expression. Furthermore, PRR9, PRR7, and PRR5 associate with the CCA1 and LHY promoters in vivo, coincident with the timing of decreased CCA1 and LHY expression. These results suggest that the repressor activities of PRR9, PRR7, and PRR5 on the CCA1 and LHY promoter regions constitute the molecular mechanism that accounts for the role of these proteins in the feedback loop of the circadian clock.
Demonstration that PRRs are transcriptional repressors. Very rough curation by Andrew Millar, needs original numerical data, curation of ChIP timeseries and LHY, CCA1 mRNA profiles from same samples.
Protein abundance profiles for the Flag-tagged PRR proteins, in WT (Col-0) and prr mutants, compared to LHY and CCA1 mRNA and ChIP to LHY/CCA1 promoters. Biological replicates were tested, data very clean, but error bars not curated.
To detect FLAG-PRR-GFP proteins (Figure 5), 10 mL of the chromatin complexes was mixed in a 1:1 ratio (v/v) with 2x lithium dodecyl sulfate sample (SM) buffer (Kiba et al., 2007), boiled at 95 deg C for 5 min, loaded in a Super Sep Ace 10 to ;20% gradient gel (Wako), and blotted onto an Immobilon-P membrane (Millipore).
Western blot (tagged proteins detected with anti-FLAG antibody, via alkaline phosphatase.)
Literature data from: '' by: .
To detect FLAG-PRR-GFP proteins (Figure 5), 10 mL of the chromatin
complexes was mixed in a 1:1 ratio (v/v) with 2x lithium dodecyl sulfate sample (SM) buffer (Kiba et al., 2007), boiled at 95 deg C for 5 min, loaded in a Super Sep Ace 10 to ;20% gradient gel (Wako), and blotted onto an Immobilon-P membrane (Millipore). The membrane was incubated with monoclonal anti-FLAG antibody (F3165; Sigma-Aldrich). Goat anti-mouse IgG conjugated with alkaline phosphatase (170-6520; Bio-Rad) was used as the secondary antibody, and protein signals were detected using the NBT/BCIP system (Roche). To detect native PRR5 and PRR9, frozen plantmaterials were ground to a fine powder and suspended in a 1:1 ratio (w/v) with 2x SM buffer and incubated for 5 min at 95 deg C. Rabbit anti-PRR5 or anti-PRR9 antibodies were used for the primary antibody. Goat anti-rabbit IgG conjugated with alkaline phosphatase (170-6518; Bio-Rad) was used as the secondary antibody. Quantitation of immunodetected proteins was performed using ImageJ software (http://rsb.info.nih.gov/ij/).
The paper also describes and affinity-purified anti-PRR9 native antibody. Data were normalised to the peak. Biological replicates were tested but error bars were not curated
Growth on Agar 1.2% MS 1 2% Sucrose for 14.0 days (white LD).
| Type | Duration (days) | Cycle (h) | Start | Duration | Spectrum | Source | Intensity |
|---|---|---|---|---|---|---|---|
| diurnal light | 14 | 24 | 0:00 | 12:00 | white | tube | 80-100 |
| Type | Duration (days) | Cycle (h) | Base (°C) | Warm (°C) | Warm start | Warm duration |
|---|---|---|---|---|---|---|
| constant temperature | 14 | 24 | 22 | -- | -- | -- |
Growth on Agar 1.2% MS 1 2% Sucrose for 1.0 days (white LD).
| Type | Duration (days) | Cycle (h) | Start | Duration | Spectrum | Source | Intensity |
|---|---|---|---|---|---|---|---|
| diurnal light | 1 | 24 | 0:00 | 12:00 | white | tube | 80-100 |
| Type | Duration (days) | Cycle (h) | Base (°C) | Warm (°C) | Warm start | Warm duration |
|---|---|---|---|---|---|---|
| constant temperature | 1 | 24 | 22 | -- | -- | -- |