Integrating analog and digital modes of gene expression at Arabidopsis FLC
- Department of Computational and Systems Biology, John Innes Centre, United Kingdom
- School of Molecular Biosciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, United Kingdom
- Swedish University of Agricultural Sciences, Plant Biology Department, Sweden
- Department of Cell and Developmental Biology, John Innes Centre, United Kingdom
- EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Australia
- State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China
- Faculty of Medicine and Health Sciences, Norwich Medical School, University of East Anglia, United Kingdom
- College of Life Sciences, Wuhan University, China
- European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, United Kingdom
Figures
Figure 1 with 1 supplement
Schematic of digital and analog gene regulation.
(A) Digital regulation (left) corresponds to loci being in an ‘ON’ state (purple) or ‘OFF’ state (white), where we assume for simplicity that there is only one gene copy per cell. At the tissue …
Figure 1—figure supplement 1
Characterization of fca alleles and FLC-Venus transgene.
(A) Flowering time measured in days from sowing until first appearance of a floral meristem (bolting). 12 plants were measured for each genotype. Boxplots show median and interquartile range (IQR), …
Figure 2 with 5 supplements
FLC expression per cell in fca mutants.
(A) Schematic diagram of FLC-Venus locus with transcript and exonic probe position indicated. A total of 40 probes were designed, 10 against the FLC sequence and 30 for the Venus sequence. (B) …
Figure 2—figure supplement 1
Single-molecule fluorescence in situ hybridization (smFISH) method for FLC-Venus imaging.
(A) Detection of nucleolar signal using FLC-specific probes (magenta) in Ler background fca-1 and fca-3 plants in Arabidopsis thaliana root cells (monolayer of cells derived from root squashes). The …
Figure 2—figure supplement 2
Threshold for ON/OFF state of cells in single-molecule fluorescence in situ hybridization (smFISH) experiments.
Figure 2—figure supplement 1C replotted for different thresholds 0 (A), >1 (B), >2 (C), >3 (D) and >5 (E) mRNAs. Red number indicates the median of the ON cells, while the blue number indicates the …
Figure 2—figure supplement 3
FLC-Venus imaging in fca alleles – root replicates and fca-4.
Representative images of FLC-Venus signal in four genotypes (Ler, fca-3, fca-1, fca-4) from 7 d post sowing. FLC-Venus intensity indicated by color maps; gray shows the propidium iodide (PI) …
Figure 2—figure supplement 4
FLC-Venus imaging in fca mutants and wildtype, in young leaf tissue.
(A) Three representative images of FLC-Venus signal in the three genotypes (Ler, fca-3, fca-1) from 9 d post sowing. FLC-Venus intensity indicated by color map; gray shows the cell wall dye …
Figure 2—figure supplement 5
Segmentation and quantification method for FLC-Venus protein intensity per nucleus.
Custom visualization and segmentation software was used to manually correct automatic segmentations and annotate cells according to cell file. Top: diagram explaining quantification of FLC-Venus …
Figure 3 with 4 supplements
Experimental observation of gradual FLC silencing.
(A) Timeseries of FLC expression in fca mutant alleles transformed with the FLC-Venus construct. Expression is measured by qPCR in whole seedlings relative to the house-keeping gene index (geometric …
Figure 3—figure supplement 1
Degradation rate of FLC mRNA and protein levels.
(A) Histogram of FLC-Venus mRNAs per cell measured by single-molecule fluorescence in situ hybridization (smFISH) after 4 hr or 6 hr of ActD treatment (top) or mock DMSO treatment (bottom). Intronic …
Figure 3—figure supplement 2
FLC and FCA expression in whole seedlings over time .
(A) Timeseries of FLC expression in fca mutant alleles. Expression is measured by qPCR relative to the house-keeping gene index (geometric mean of PP2A and UBC). Error bars show SEM of n = 3 …
Figure 3—figure supplement 3
FLC-Venus time-course replicates in fca alleles.
Additional representative images of FLC-Venus signal in the three genotypes (Ler, fca-3, fca-1) at the three timepoints. FLC-Venus intensity is indicated by color maps; gray shows the PI channel. …
Figure 3—figure supplement 4
Experimental intensity of FLC-Venus in ON cells does not change over time in fca-3.
Histogram of ON cell tail in fca-3 at three timepoints normalized by the total number of cells in each. Only cells with FLC-Venus intensity above 1 are shown. For 7 d n = 109 cells from 17 roots, 15 …
Figure 4 with 1 supplement
Mathematical model captures FLC regulation.
(A) Diagram of mathematical model. Individual FLC gene copies can be ON or OFF, such that a cell can be in one of three states depending on the combination of ON and OFF gene copies within it …
Figure 4—figure supplement 1
Modeling FLC regulation in clonal cell files in the root.
Representative model output for fca-3 root, showing 12 simulated cell files of length 30 cells each, at different timepoints, matching sampling times. (A) The state of the loci (both copies ON – …
Additional files
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Supplementary file 1
Mathematical model parameters.
- https://cdn.elifesciences.org/articles/79743/elife-79743-supp1-v1.xlsx
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Supplementary file 2
Primers used in this study.
- https://cdn.elifesciences.org/articles/79743/elife-79743-supp2-v1.xlsx
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Supplementary file 3
smFISH probe sequences used to detect FLC Venus transcripts.
These probes were labeled with Quasar570.
- https://cdn.elifesciences.org/articles/79743/elife-79743-supp3-v1.xlsx
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Supplementary file 4
smFISH probe sequences from Duncan et al., 2016 (A) used to detect unspliced PP2A transcripts and (B) used to detect FLC sense spliced.
Both probe sets were labeled with Quasar670.
- https://cdn.elifesciences.org/articles/79743/elife-79743-supp4-v1.xlsx
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Supplementary file 5
Raw data from this study.
- https://cdn.elifesciences.org/articles/79743/elife-79743-supp5-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/79743/elife-79743-mdarchecklist1-v1.pdf
