CK1/Doubletime activity delays transcription activation in the circadian clock
- The Rockefeller University, United States
- Cornell University, United States
Figures
Figure 1 with 1 supplement
Residue substitutions of S589 differently affect behavioral rhythmicity.
(A) Transgenic wild-type per (black line), S589A per (red line) or S589D per (blue line) were expressed in a per0 genetic background and monitored for behavior in a 12:12 light dark cycle followed …
Figure 1—figure supplement 1
Residue substitutions of S589 do not genetically interact with O-Glc-NAc Transferase (OGT).
The behavioral period of heterozygous flies carrying the transgenic per variant (as indicated) in the absence of DBT overexpression (grey bars, per0; [per] / +; +) or with DBT overexpression in …
Figure 2 with 1 supplement
Residue substitutions of S589 do not affect nuclear entry, advance PER degradation in small ventral lateral neurons in vivo.
(A) Fly brains were collected at the indicated zeitgeber time points (ZT) and fixed. Representative images of s-LNvs stained for PER (green) and PDF (blue) are shown. The scale bar represents 5 μm. …
Figure 2—figure supplement 1
Residue substitutions of S589 do not affect nuclear accumulation in the large ventral lateral neurons in vivo.
Stained fly brains were visually scored to determine PER/TIM nuclear accumulation. PER or TIM staining that is predominantly nuclear or cytoplasmic in the small LNvs were valued 1 or 0, …
Figure 3 with 1 supplement
Residue substitutions of S589 determine PER/TIM stability in cultured cells.
S2 cells expressing per driven by a heat-shock promoter, and dbt and tim driven by actin promoter were collected each hour post heat shock (h PHS) in a pulse-chase experiment using cycloheximide. (A)…
Figure 3—figure supplement 1
PER/TIM nuclear accumulation in S2 cells is promoter-dependent, PER stability is plotted as a PER/DBT ratio.
(A) S2 cells were transfected with plasmids that express per-mCherry (PER) or tim-YFP (TIM) fusion genes by actin promoter (pAc>) or heat shock promoter (pHS>) as indicated. Cells were imaged 2 hr …
Figure 4 with 1 supplement
589 substitutions differently regulate per promoter activity in vivo.
(A) Flies expressing the indicated transgenic per variant in a per0 background and the luciferase gene driven by the per promoter were measured for bioluminescence activity in constant darkness. …
Figure 4—figure supplement 1
Realignment of bioluminescence period.
Bioluminescence data in Figure 4A replotted to align the start of each crest on each day to emphasize the differences in crest and trough width each day. Solid lines depict the bounds of a full …
Figure 5 with 1 supplement
Fluorescence based transcription assay allows simultaneous measurement of transcription inhibition and protein stability.
(A) Flow chart of the principles behind the fluorescence-based transcription assay. (1) Actin promoter (grey) driven clk-cfp (blue) and per promoter (light red) driven yfp (yellow) is cloned into …
Figure 5—figure supplement 1
Protein stability and nuclear accumulation in the transcription inhibition assay.
(A) Scatter plot of S2 cells that were gated for CFP expression (CFP+), and re-plotted to demonstrate YFP and mCherry expression. (B) Cells analyzed in Figure 4 were plotted for mCherry fluorescence …
Figure 6 with 1 supplement
S589 and the PER Short Downstream Domain (PER-SD) cooperate to regulate transcriptional inhibition and PER protein stability.
(A) The phosphorylation cascade model of PER. Phosphorylation of the PER-SD region blocks S589 phosphorylation through an intermediary site (S596) while promoting S47 phosphorylation. S589 …
Figure 6—figure supplement 1
Residue substitutions of DBT target sites do not affect global conformational differences in purified PER fragment, but affect complex assembly.
(A) SEC-MALS traces of wild type (blue line) or S589D/4D (red line) PER fragments (amino acids 1–700). Absorbance is measured at 280 nm across time, peaking at ~0.6. The purple peak represents the …
Figure 7
A proposed model for DBT-mediated PER regulation.
Non-phosphorylated PER is stable, but unable to inhibit dCLK-mediated transcription. DBT binds PER and phosphorylates the PER-SD to activate PER inhibition of dCLK (Step 1). DBT phosphorylates S589 …
Author response image 1
PER stability in steady state conditions in cultured cells.
Wild type PER and PER variants S589A, S589D, 4A and 4D were exogenously expressed in cultured cells, or co-expressed with wild type DBT (C) or DBT-K38R (K). Protein extracts from cells were analyzed …
Additional files
-
Supplementary file 1
Fly behavioral data.
Behavioral periods of flies charted in Figure 1B, C and D are detailed. Tau: behavioral period, SD: standard deviation, No. Arr.: Number of arrhythmic flies, Total Number: Total number of flies tested. fs: figure supplement.
- https://doi.org/10.7554/eLife.32679.016
-
Transparent reporting form
- https://doi.org/10.7554/eLife.32679.017
