Modulation of protein-DNA binding reveals mechanisms of spatiotemporal gene control in early Drosophila embryos
- Department of Chemical and Biomolecular Engineering, University of Pennsylvania, United States
- Master of Biotechnology Program, University of Pennsylvania, United States
Figures
Figure 1 with 1 supplement
Single TF binding site mutation greatly diminishes mRNA production from the distal sna enhancer.
(A) Schematic of the reporter construct containing the minimal sna distal enhancer, sna core promoter, MS2 stem loops, and the yellow reporter gene. The minimal enhancer contains binding sites for …
Figure 1—figure supplement 1
Mutations affect mRNA production and expression sharpness, not width of expression domain.
(A) Average mRNA production of all nuclei in wildtype embryos and Dl3, Twi2, and Dl3Twi2 mutant embryos across the snamin expression domain. (B) Plot showing the width of the expression domain …
Figure 2 with 2 supplements
Mutations cause lower mRNA production, mainly due to lower transcriptional amplitude.
(A) Schematic comparing wildtype and mutants. The mutant embryo may have fewer nuclei transcribing (shaded) and at a lower intensity. Each nucleus in mutant embryos may have late activation or a …
Figure 2—figure supplement 1
Mutations cause lower mRNA production, mainly due to lower transcriptional amplitude.
(A–C) Boxplots showing (A) the time to transcriptional activation, (B) the duration of active transcription, and (C) the transcriptional amplitude for all actively transcribing nuclei. Decreased …
Figure 2—figure supplement 2
Mutations in the full sna distal enhancer decrease duration of transcription in addition to lowering transcriptional amplitude.
(A–C) Boxplots showing (A) the time to transcriptional activation, (B) the duration of active transcription, and (C) the transcriptional amplitude for all actively transcribing nuclei. Decreased …
Figure 3 with 2 supplements
Thermodynamic equilibrium binding model reveals synergy among TF binding sites.
(A) Promoter states and statistical weights for each microstate. A bound activator will yield transcription. Cooperativity term ω is included when more than one TF is bound, which will result in …
Figure 3—figure supplement 1
Sensitivity analysis on thermodynamic model highlights the importance of cooperativity among all TFs.
(A–B) Sensitivity analysis for ω1 (A) and ω6 (B) for values of 1 (left), 10 (center), and 1e2 (right). The model is robust against changes in these parameters. (C) Sensitivity analysis for ω11 for …
Figure 3—figure supplement 2
Modulation of binding affinity and Dl concentration.
(A–B) Model results of low-affinity sites (A) and high affinity sites (B) with cooperativity. Higher cooperativity values are needed for the low-affinity model (see Table 2). (C–D) Modulation of Dl …
Figure 4 with 2 supplements
A two-state model reveals differences in koff rates and burst duration.
(A) Actively transcribing nuclei are false-colored for early and late NC14. Mutant embryos show more sporadic transcriptional activity in a given frame. Scale bar = 15 μm. (B) Schematic depicting a …
Figure 4—figure supplement 1
A two-state model reveals differences in koff rates and burst duration.
(A) Plot showing the number of actively transcribing nuclei at every timepoint for wildtype and all mutant embryos. In the wildtype, the number of nuclei steadily increases, while in the mutant, the …
Figure 4—figure supplement 2
Mutations in the full sna distal enhancer increase koff rates leading to reduced burst duration.
(A) Plot showing the number of actively transcribing nuclei at every timepoint for wildtype and all mutant embryos. In the wildtype, the number of nuclei steadily increases, while in the mutant, the …
Videos
Video 1
Live imaging of wildtype embryo expressing snamin > MS2-yellow during NC14.
MS2 signal is shown in yellow. Nuclei are marked with His2Av-mRFP. Histogram was adjusted for visualization purposes. Scale bar = 10 μm.
Video 2
Live imaging of Dl2 mutant embryo expressing snamin > MS2-yellow during NC14.
MS2 signal is shown in yellow. Nuclei are marked with His2Av-mRFP. Scale bar = 10 μm.
Video 3
False coloring of actively transcribing nuclei of a wildtype snamin> MS2-yellow embryo.
Scale bar = 10 μm.
Video 4
False coloring of actively transcribing nuclei of a Dl2 mutant snamin> MS2-yellow embryo.
Scale bar = 10 μm.
Tables
Table 1
Table of TF motifs and mutated sequences.
Table showing the sequences of the TF motifs, p-value, mutated sequences, and Patser score.
| Transcription factor | p-value | Patser score | Wildtype | Mutation |
|---|---|---|---|---|
| Dl1 | 1.43e-4 | 22.8 | AGGGATTTCCT | AGGGATCGCCT |
| Dl2 | 2.71e-05 | 19.8 | GGCGTTTTCCCA | GGCGATTGACCA |
| Dl3 | 8.52e-05 | 17.8 | TGGGAAATCGG | TGTTAAATCGG |
| Twi2 | 4.08e-05 | 7.8 | GTCCATGTGTTG | GTCCATGAATTG |
Table 2
Table of cooperativity values.
Table showing the cooperativity values for the optimized model and the cooperativity values when all binding sites are weakened or strengthened. Cooperativity values are much higher when the …
| Cooperativity term | Transcription factor | Optimized value | Weak binding value | Strong binding value |
|---|---|---|---|---|
| ω1 | Dl1, Dl2 | 28.00 | 69.90 | 2.03 |
| ω2 | Dl2, Twi2 | 1.00 | 1.00 | 1.00 |
| ω3 | Dl1, Twi2 | 1.00 | 1.00 | 1.00 |
| ω4 | Dl1, Dl3 | 1.00 | 1.00 | 1.00 |
| ω5 | Dl2, Dl3 | 1.01 | 1.00 | 1.00 |
| ω6 | Dl3, Twi2 | 55.30 | 139.00 | 6.79 |
| ω7 | Dl1, Dl2, Twi2 | 1.07 | 1.05 | 1.03 |
| ω8 | Dl1, Dl2, Dl3 | 1.03 | 1.02 | 1.01 |
| ω9 | Dl2, Twi2, Dl3 | 1.13 | 61.10 | 1.03 |
| ω10 | Dl1, Twi2, Dl3 | 1.03 | 1.03 | 1.02 |
| ω11 | Dl1, Dl2, Twi2, Dl3 | 1.59e5 | 5.00e5 | 3.10e4 |
Table 3
Table of promoter occupancies with and without cooperativity.
Table showing the promoter occupancies in the cases of cooperativity and no cooperativity for the three cooperativity terms that were greater than 1 in the optimized model. Cooperativity increases …
| Promoter state | Promoter occupancy with cooperativity | Promoter occupancy with no cooperativity |
|---|---|---|
| ω1 | 2.4378 | 0.0870 |
| ω6 | 2.1913 | 0.0397 |
| ω11 | 54.8073 | 3.45e-04 |
