Cell-cycle-gated feedback control mediates desensitization to interferon stimulation
- Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, United States
- Department of Bioengineering, University of California, San Diego, United States
- Department of Chemistry and Biochemistry, University of California, San Diego, United States
- Department of Pathology, Moores UCSD Cancer Center, University of California, San Diego, United States
Figures
Figure 1 with 4 supplements
IFN-α pretreatments with different durations lead to opposite effects to the second stimulation.
(A) Schematic of HeLa reporter cell line engineered using CRISPR/Cas9 (top). STAT1 was tagged with mCherry at C-terminus to monitor the translocation and expression. The coding sequence for P2A-YFP …
Figure 1—figure supplement 1
Cell line construction and validation.
(A) Illustration of cell line construction steps. Full detail was described in Materials and methods. Fluorescent reporters introduced and the targeted genes are shown. In each step, homogenous …
Figure 1—figure supplement 2
Dose-dependent responses to IFN-α treatment.
Time traces of nuclear to cytoplasmic ratio for STAT1-mCherry, STAT1-mCherry fluorescence, and PIRF9-YFP fluorescence in response to different concentrations (ng/ml) of IFN-α, as indicated. Averages …
Figure 1—figure supplement 3
Dose dependence of desensitization to IFN-α treatment.
Bar graphs showing the amounts of PIRF9-YFP induction to the second IFN input (100 ng/ml) in WT (A - C) and USP18-KD (D – F), pretreated with different concentrations of IFN-α for different …
Figure 1—figure supplement 4
Dependence of desensitization effects on the break time.
Bar graphs showing the amounts of PIRF9-YFP induction to the second IFN input followed by different break time durations. Cells were pretreated with 100 ng/ml IFN-α for 24 hrs or not pretreated …
Figure 2 with 4 supplements
USP18 mediates desensitization induced by the prolonged IFN-α pretreatment.
(A) Representative time-lapse images of STAT1 nuclear translocation in response to the second IFN-α treatment under different pretreatment conditions in WT cells (top) and USP18-KD (bottom). (B) …
Figure 2—figure supplement 1
Validation of the USP18-KD cell line.
(A) Western blots of USP18 expression in WT and USP18-KD cells. Cells were treated with 100 ng/ml IFN-α for indicated times, harvested and lysed for immunoblotting with the USP18 antibody. Time …
Figure 2—figure supplement 2
Quantification of USP18-mediated desensitization in STAT1 nuclear translocation.
(A) Averaged time traces of nuclear/cytoplasmic STAT1-mCherry in WT in response to the second IFN-α treatment under different pretreatment conditions (depicted in Figure 1D). (B) Violin plot shows …
Figure 2—figure supplement 3
SOCS1 does not mediate the desensitization of STAT1 nuclear translocation upon IFN stimulation.
(A) Quantitative PCR results comparing SOCS1 expression in WT (back) and SOCS1-KD (red). The data was normalized to WT without IFN-α treatment. The knockdown efficiency (73.71%) was calculated as …
Figure 2—figure supplement 4
Vesicular stomatitis virus (VSV) replication in WT and USP18-KD cells with and without 24-hr IFN-α pretreatment.
Cells with or without a 24-hr pretreatment of 100 ng/ml IFN-α and an 8-hr break time were infected with 2500 PFU (plaque forming units) of VSV. After 18 hrs, viral supernatant was collected and …
Figure 3 with 2 supplements
A kinetic model suggests a delayed negative feedback loop through USP18.
(A) A diagram for the simple kinetic model of the IFN-driven gene regulatory network. (B) Fitting errors between simulations and the data with different assigned values of the delay time in USP18 …
Figure 3—figure supplement 1
Model fitting results and the parameter analysis.
(A) A diagram for the simple kinetic model of the IFN-driven gene regulatory network with reaction parameters labeled. (B) Fitting results with different delay times. The model fitting results …
Figure 3—figure supplement 2
Pulsatile IFN-α treatment induces higher ISG expression in single cells.
(A) The violin plots showing single-cell distributions of PIRF9-YFP induction upon 5 × 8-hr pulsatile (blue) or 40-hr sustained (red) IFN-α treatments in WT (top) and USP18-KD (bottom) cells. The …
Figure 4 with 3 supplements
Heterogeneous delays in USP18 upregulation by IFN were observed in single cells.
(A) Schematic of the dual reporter cell line. A coding sequence for NLS-CFP-P2A was inserted endogenously into the N-terminus of USP18 coding sequence of the previous cell line. IFN-α induces …
Figure 4—figure supplement 1
Construction of the cell line with PUSP18-CFP reporter.
(A) Illustration showing the introduction of PUSP18-CFP into the dual reporter cell line in Figure 1A. NLS-CFP-P2A coding sequence was inserted between the promoter and coding sequence of the USP18 …
Figure 4—figure supplement 2
Relationships between variations in USP18 upregulation and the heterogeneity in ISGF3 components.
(A) Scatterplots showing the relationship of PUSP18-CFP expression upon IFN-α treatment with (i) the basal level of PIRF9-YFP, (ii) PIRF9-YFP expression upon IFN-α treatment, (iii) the basal level …
Figure 4—figure supplement 3
Quantifying the percentage of cell cycle progression upon IFN treatment onset in single cells.
(A) Illustration of measurement of cell mitosis times from time-lapse images. (Top) Representative time-lapse images of cells over multiple cell divisions. (Bottom) The size of nucleus (left) and …
Figure 5 with 5 supplements
USP18 expression was differentially regulated by cell cycle phases.
(A) Distributions of delay times in cells treated with different cell cycle perturbations. Cells were serum-starved or treated with lovastatin (5 μM), or with roscovitine (5 μM) for 36 hrs prior to …
Figure 5—figure supplement 1
Distributions of PIRF9 and PUSP18 activation times in cells treated with different cell cycle perturbations.
Cells were serum-starved or treated with lovastatin (5 μM), or with roscovitine (5 μM) for 36 hrs prior to IFN-α treatment. The mean activation times (with 95% confidence interval) of IRF9 were …
Figure 5—figure supplement 2
Cell cycle-dependent USP18 upregulation determined by the FUCCI reporter.
(A) Illustration of how the FUCCI reporter works. The fluorescent signals of chromatin licensing and DNA replication factor 1(Cdt1) and Geminin (Gem) proteins oscillate throughout a cell cycle to …
Figure 5—figure supplement 3
ISG promoters contain a wide range of CpG site numbers and methylation levels.
(A) Histogram showing the numbers of CpG sites at ISG promoters. The promoter region is defined as 1000 bp upstream of the transcription start site. The data are collected from ENCODE database and …
Figure 5—figure supplement 4
Distributions of PIRF9 and PUSP18 activation times in cells treated with decitabine.
Cells were cultured with medium in the absence (control) or presence of 100 μM decitabine for 48 hrs prior to 100 ng/ml IFN-α treatment. The mean activation times (with 95% confidence interval) of IR…
Figure 5—figure supplement 5
The effect of decitabine on cell cycle.
(A) Representative single-cell time traces from decitabine-treated cells. Cells were treated with 100 μM decitabine for 36 hrs prior to being imaged and kept in decitabine throughout the experiment. …
Figure 6
Stochastic simulations with the cell-cycle gated feedback control reproduced single-cell responses to IFN pretreatments with different durations.
(A) A diagram for the simple model of the IFN-driven gene regulatory network that incorporates the cell cycle gating of USP18 upregulation. (B) Cell-cycle-dependent delay times in the model …
Videos
Video 1
Response of the dual reporter cell line to 100 ng/ml IFN-α treatment.
The image sequence was acquired for 50 hrs in which the first 2 hr were before the addition of IFN-α. The NLS-2xiRFP nuclear marker is shown in red and is merged with phase images of the cells. …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB | This paper | NHM003 | Available upon request from Hao lab |
| Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry | This paper | NHM008 | Available upon request from Hao lab |
| Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry, PIRF9-IRF9-P2A-mCitrine | This paper | NHM025 | Available upon request from Hao lab |
| Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry, PIRF9-IRF9-P2A-mCitrine, shRNA USP18 | This paper | NHM026 | Available upon request from Hao lab |
| Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry, PIRF9-IRF9-P2A-mCitrine, shRNA USP18 negative control | This paper | NHM027 | Available upon request from Hao lab |
| Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry, PIRF9-IRF9-P2A-mCitrine, shRNA SOCS1 | This paper | NHM031 | Available upon request from Hao lab |
| Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PSTAT1-STAT1-mCherry, PIRF9-IRF9-P2A-mCitrine, PUSP18-NLS-mCerulean-USP18 | This paper | NHM032 | Available upon request from Hao lab |
| Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PUSP18-NLS-mCerulean-USP18, PCMV-DHB-mCherry | This paper | NHM035 | Available upon request from Hao lab |
| Cell line (Homo sapiens) | HeLa PACTB-NLS-2iRFP-P2A-ACTB, PUSP18-NLS-mCerulean-USP18, PCMV-mCh-Gem1-P2A-CFP-Cdt1 | This paper | NHM036 | Available upon request from Hao lab |
Additional files
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Supplementary file 1
Primers used in this study.
- https://cdn.elifesciences.org/articles/58825/elife-58825-supp1-v1.docx
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Supplementary file 2
Plasmids constructed in this study.
- https://cdn.elifesciences.org/articles/58825/elife-58825-supp2-v1.docx
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Supplementary file 3
Best-fit parameter values of the model.
- https://cdn.elifesciences.org/articles/58825/elife-58825-supp3-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/58825/elife-58825-transrepform-v1.docx
