Predictive modeling reveals that higher-order cooperativity drives transcriptional repression in a synthetic developmental enhancer
- Chan Zuckerberg Biohub, United States
- Department of Chemical Biology, University of California, Berkeley, United States
- Department of Physics, University of California, Berkeley, United States
- Department of Biology, Ecole Polytechnique, France
- Biophysics Graduate Group, University of California, Berkeley, United States
- Department of Biology, New York University, United States
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
- Institute for Quantitative Biosciences–QB3, University of California at Berkeley, United States
Figures
Figure 1
Building up predictive models of transcriptional repression.
(A) In the absence of repressor binding, gene expression can be characterized by a dissociation constant between RNAP and the promoter and the rate of transcription initiation when the promoter …
Figure 2 with 2 supplements
Thermodynamic model of transcriptional regulation by Bicoid activator and Runt repressor.
(A) States and statistical weights for the regulation of hunchback P2 with one Runt binding site in the limit of strong Bicoid-Bicoid cooperativity. Here, we use the dimensionless parameters , , …
Figure 2—figure supplement 1
General thermodynamic model for a hunchback P2 enhancer with six Bicoid binding sites.
(A) States, weights, and degeneracy considered for our thermodynamic model. (B) Simpler form of the thermodynamic model in the limit of .
Figure 2—figure supplement 2
General thermodynamic model for an enhancer with six-Bicoid binding sites and one Runt binding site.
(A) Statistical weights and degeneracy of each state the system can be found in. (B) Simpler form of the model from (A) in the limit of strong Bicoid-Bicoid cooperativity.
Figure 3 with 11 supplements
Measurement of input transcription factor concentrations and output rate of transcription to test model predictions.
(A) Snapshot of an embryo expressing eGFP-Bicoid spanning 20–60% of the embryo length. (For a full time-lapse movie, see Movie Figure 3—video 1) (B) Bicoid nuclear fluorescence dynamics taken at 40% …
Figure 3—figure supplement 1
Comparison of the predicted rate of transcription using dynamic and time-averaged transcription factor concentration profiles as inputs.
(A) Instantaneous predicted rate of transcription calculated using dynamic transcription factor concentration profiles at each time point (blue) and resulting averaged rate of transcription averaged …
Figure 3—figure supplement 2
Initial rate of RNAP loading in nuclear cycle 14 across the anterior-posterior axis for different constructs, with or without Runt protein.
(A) Schematic showing how the initial rate of RNAP loading is measured by extracting the slope resulting from a linear fit to the MS2 time traces at the beginning of nuclear cycle 14. (B) Initial …
Figure 3—figure supplement 3
Duration of transcription over nuclear cycle 14.
(A) An example MS2 time trace in nuclear cycle 14. The decay regime is defined from the peak of the signal to the end of the measurement. is defined by the x-intercept of the slope of the fitted …
Figure 3—figure supplement 4
Fraction of competent loci in nuclear cycle 14 along the anterior-posterior axis for each synthetic enhancer construct in the presence and absence of Runt protein.
(A) Heatmap showing the transcriptional signal from the hunchback P2 enhancer for individual nuclei (rows) demonstrating that there are two populations of loci: transcriptionally active and inactive …
Figure 3—figure supplement 5
Accumulated mRNA during nuclear cycle 14 along the anterior-posterior axis for each synthetic enhancer construct in the presence and absence of Runt protein.
(A) An illustrative MS2 time trace in nuclear cycle 14. The accumulated mRNA is calculated by integrating the MS2 time traces during nuclear cycle 14, indicated by the purple area under the MS2 …
Figure 3—figure supplement 6
Accumulated mRNA during nuclear cycle 14 versus Runt concentration for each synthetic enhancer construct in the presence and absence of Runt protein.
(A) An example MS2 time trace averaged over MS2 spots in a spatial window of 2.5% of the embryo length in nuclear cycle 14. The accumulated mRNA is calculated by integrating the MS2 time traces …
Figure 3—figure supplement 7
Snapshots of an embryo expressing eGFP:LlamaTag-Runt and the Histone-iRFP signal used for nuclear segmentation.
(A) Snapshots of an embryo for eGFP:LlamaTag-Runt (top) and Histone-iRFP (bottom) spanning 20–60% of the embryo length in nuclear cycle 14. The scale bars represent 50 μm.
Figure 3—figure supplement 8
Correlation between the initial RNAP loading rate and accumulated mRNA during nuclear cycle 14.
Correlation between the initial RNAP loading rates and accumulated mRNA levels at each position along the embryo length for all constructs for the wild-type and runt null backgrounds. A Pearson’s …
Figure 3—video 1
eGFP-Bicoid confocal movie.
Confocal microscopy movie taken on a developing fly embryo (eGFP-Bicoid; His2Av-mRFP; +) during nuclear cycle 13 and 14.
Figure 3—video 2
eGFP:LlamaTag-Runt confocal movie.
Confocal microscopy movie taken on a developing fly embryo (eGFP-Bicoid; His2Av-mRFP; +) during nuclear cycle 13 and 14.
Figure 3—video 3
[001]-MS2V5:MCP-GFP (+Runt) confocal movie.
Confocal microscopy movie taken on a developing fly embryo (yw; His2Av-mRFP; MCP-eGFP) for the [001] construct with MS2 reporter during nuclear cycle 13 and 14.
Figure 4 with 2 supplements
Enchancer-to-enhancer variability in the unrepressed transcription level stems from unique RNAP-dependent parameters.
(A) Measured initial rates of RNAP loading across the anterior-posterior axis of the embryo for all synthetic enhancer constructs in the absence of Runt protein. (The [111] synthetic enhancer …
Figure 4—figure supplement 1
Bioinformatically predicted architecture of major transcription factor binding sites in the hunchback P2 minimal enhancer with three Runt (Run) binding sites.
(A) PATSER scores for Bicoid and Zelda for hunchback P2 (blue) and hunchback P2 with three Runt sites (brown). The binding motifs with PATSER scores higher than three are shown. We concluded that …
Figure 4—figure supplement 2
Initial rate of RNAP loading in nuclear cycle 14 across the anterior-posterior axis for different constructs in individual embryos in the absence of Runt protein.
(A) Schematic showing how the initial rate of RNAP loading is measured for an individual embryo by extracting the slope resulting from a linear fit to the averaged MS2 time traces at the beginning …
Figure 5 with 3 supplements
Testing the direct repression model in the presence of one Runt binding site.
(A) Initial transcription rate as a function of position along the embryo for the three constructs containing one Runt binding site in the presence and absence of Runt repressor, together with their …
Figure 5—figure supplement 1
Thermodynamic models for different modes of repression.
States and statistical weights corresponding to the hunchback P2 enhancer with one Runt binding site for the (A) direct repression, (B) competition, and (C) quenching mechanisms.
Figure 5—figure supplement 2
MCMC fitting to the hunchback P2 with one Runt binding site constructs using different models of repression.
(A,B,C) MCMC fits for three modes of repression, (i) direct repression, (ii) competition, and (iii) quenching, for our three one-Runt site constructs, (A) [100], (B) [101], and (C) [001]. (D) Corner …
Figure 5—figure supplement 3
Assessment of alternative models for the one-Runt binding site case.
(A) A schematic of three synthetic enhancers with one Runt binding site at different positions in the enhancer (proximal, intermediate and distal) and their key parameters and . (B,C,D,E) Best …
Figure 6 with 7 supplements
Prediction for the transcription initiation rate of hunchback P2 with two-Runt binding sites under different models of cooperativity.
(A) Direct repression model for hunchback P2 with two Runt binding sites featuring Runt-RNAP interaction terms given by and, Runt-Runt cooperativity captured by , and Runt-Runt-RNAP …
Figure 6—figure supplement 1
Prediction for two-Runt binding sites constructs based on the inferred parameters from the one-Runt binding site cases for different modes of repression for the (A) [011], (B) [101], and (C) [110] constructs.
The model assumes no interactions between Runt molecules. (A,B, and C, error bars represent standard error of the mean over embryos.).
Figure 6—figure supplement 2
Prediction for hunchback P2 transcription initiation rate with two-Runt binding sites under the competition scenario for different combinations of cooperativities.
(A) Schematic of cooperativity terms considered: Runt-Runt cooperativity given by and Runt-Runt-Bicoid complex higher-order cooperativity captured by , in addition to the competition terms …
Figure 6—figure supplement 3
Prediction for hunchback P2 transcription initiation rate with two-Runt binding sites under the quenching mechanism for different combinations of cooperativities.
(A) Schematic of additional cooperativities considered: Runt-Runt cooperativity and Runt-Runt-Bicoid-RNAP complex higher-order cooperativity . (B) Zero-parameter prediction using the inferred …
Figure 6—figure supplement 4
Invoking Runt-Runt cooperativity in the thermodynamic model is not sufficient to explain the experimental data from hunchback P2 with two Runt binding sites.
(A) Model schematic where we add a new parameter representing Runt-Runt cooperativity. (B) Corresponding states and weights for hunchback P2 with two Runt binding sites in the presence of …
Figure 6—figure supplement 5
Statistical mechanics model incorporating Runt-Runt-RNAP higher-order cooperativity.
(A) Schematic of a model where we add Runt-Runt-RNAP higher-order cooperativity represented by . (B) Thermodynamic model states and weights for hunchback P2 with two Runt binding sites in the …
Figure 6—figure supplement 6
Invoking Runt-Runt cooperativity and higher-order cooperativity can explain the experimental data from hunchback P2 with two Runt binding sites.
(A) Schemati showing Runt-Runt cooperativity and higher-order cooperativity. (B) States and weights for hunchback P2 with two Runt binding sites with Runt-Runt cooperativity and higher-order …
Figure 6—figure supplement 7
Sensitivity test for by repeating the MCMC inference for different scenarios of cooperativities with different values of .
(A) Direct repression model for hunchback P2 with two-Runt binding sites featuring Runt-RNAP interaction terms given by and , Runt-Runt cooperativity captured by , and Runt-Runt-RNAP …
Figure 7
Prediction for hunchback P2 with three-Runt binding sites and multiple sources of cooperativity.
(A) Prediction using previously inferred Runt-RNAP, Runt-Runt, and Runt-Runt-RNAP cooperativity parameters. (B) Best MCMC fit obtained by incorporating an additional Runt-Runt-Runt-RNAP higher-order …
Author response image 1
Inferred values of Kr and [RNAP]/Kp across all synthetic enhancer constructs.
Kr is inferred from one-Runt binding site constructs, and [RNAP]/Kp is inferred from all constructs in the absence of Runt protein.
Author response image 2
Comparison of accumulated mRNA profiles measured by FISH and MS2 for a reporter construct driven by the hunchback P2 enhancer.
Normalized profiles of accumulated mRNA averaged over embryos from FISH (blue, Park et al., eLife 8:e41266, 2019) and MS2 (red, Eck and Liu et al., eLife 9:e56429). The blue and red curves show the …
Tables
Table 1
Interaction energies for the Runt-related cooperativity parameters from one-, two-, and three-Runt sites constructs.
Note that we used the Boltzmann relation of , where the is the interaction energy, is the Boltzmann constant, and is the temperature.
| Interaction energies for the Runt-related cooperativity parameters | ||
|---|---|---|
| model parameter | construct | interaction energy (KBT) |
| Runt-RNAP interaction, | [001] | 2.34 ± 0.63 |
| [010] | 1.36 ± 0.36 | |
| [100] | 0.18 ± 0.24 | |
| Runt-Runt interaction, | [011] | 0 (manually set) |
| [110] | -0.95 ± 0.12 | |
| [101] | 0 (manually set) | |
| Runt-Runt-RNAP interaction, | [011] | -2.09 ± 0.27 |
| [110] | 4.15 ± 1.14 | |
| [101] | 1.12 ± 0.51 | |
| Runt-Runt-Runt-RNAP interaction, | [111] | -2.12 ± 0.14 |
Table 2
List of plasmids used to create the transgenic fly lines used in this study.
| Plasmids | |
|---|---|
| Name (hyperlinked to Benchling) | Function |
| pIB-hbP2-evePr-MS2v5-LacZ-Tub3UTR | [000]-MS2v5 reporter construct |
| pIB-hbP2+r1-far-evePr-MS2v5-LacZ-Tub3UTR | [100]-MS2v5 reporter construct |
| pIB-hbP2+r1-mid-evePr-MS2v5-LacZ-Tub3UTR | [010]-MS2v5 reporter construct |
| pIB-hbP2+r1-close-evePr-MS2v5-LacZ-Tub3UTR | [001]-MS2v5 reporter construct |
| pIB-hbP2+r2-2+3-evePr-MS2v5-LacZ-Tub3UTR | [011]-MS2v5 reporter construct |
| pIB-hbP2+r2-1+3-evePr-MS2v5-LacZ-Tub3UTR | [101]-MS2v5 reporter construct |
| pIB-hbP2+r2-1+2-evePr-MS2v5-LacZ-Tub3UTR | [110]-MS2v5 reporter construct |
| pIB-hbP2+r3-evePr-MS2v5-LacZ-Tub3UTR | [111]-MS2v5 reporter construct |
| pHD-scarless-LlamaTag-Runt | Donor plasmid for LlamaTag-Runt CRISPR knock-in fusion for the N-terminal |
| pU6:3-gRNA(Runt-N-2) | gRNA plasmid for LlamaTag-Runt CRISPR knock-in fusion for the N-terminal |
| pCasper-vasa-eGFP | vasa maternal driver for ubiquitous eGFP expression in the early embryo |
Table 3
List of fly lines used in this study and their experimental usage.
| Fly lines | |
|---|---|
| Genotype | Use |
| LlamaTag-Runt; +; vasa-eGFP, His2Av-iRFP | Visualize LlamaTagged Runt protein and label nuclei |
| LlamaTag-Runt; +; MCP-eGFP(4F), His2Av-iRFP | Visualize LlamaTagged Runt protein, nascent transcripts and label nuclei |
| run3/FM6; +; + | Visualize LlamaTagged Runt protein, nascent transcripts and label nuclei |
| yw; His2Av-mRFP; MCP-eGFP | Females to label nascent RNA and nuclei |
| yw; [000]-MS2v5; + | Males carrying the MS2 reporter transgene |
| yw; [100]-MS2v5; + | Males carrying the MS2 reporter transgene |
| yw; [010]-MS2v5; + | Males carrying the MS2 reporter transgene |
| yw; [001]-MS2v5; + | Males carrying the MS2 reporter transgene |
| yw; [011]-MS2v5; + | Males carrying the MS2 reporter transgene |
| yw; [101]-MS2v5; + | Males carrying the MS2 reporter transgene |
| yw; [110]-MS2v5; + | Males carrying the MS2 reporter transgene |
| yw; [111]-MS2v5; + | Males carrying the MS2 reporter transgene |
