Dissecting the sharp response of a canonical developmental enhancer reveals multiple sources of cooperativity
- Harvard Medical School, United States
Figures
Figure 1
The WTHbP2 enhancer directs a sharp expression pattern in the Drosophila blastoderm embryo.
(A) Schematics of the hunchback (hb) gene locus showing the three enhancers active in the blastoderm (top) and our reporter construct designed to express lacZ mRNA from the proximal enhancer (HbP2) …
Figure 2 with 1 supplement
Sequence variants of HbP2 contradict the classical Hill function model.
(A) (left) Schematics of hbP2-reporter constructs with Bcd binding sites sequentially removed by mutation to preserve the length of the construct. (center) mRNA expression profiles obtained from …
Figure 2—figure supplement 1
Residual expression remains even after removing all six canonical Bcd TFBS, and also two additional predicted TFBSs.
The WTHbP2 sequence contains six canonical predicted Bcd binding sites and one additional predicted site that overlaps with X3. The native hb promoter also contains an additional predicted Bcd …
Figure 3 with 1 supplement
Synthetic HbP2 enhancer reveals the contribution of Bicoid and other transcription factors to the shape and location of expression.
(A) Schematics of WTHbP2 illustrating the location and affinity of predicted binding sites for Bicoid (Bcd, dark blue), Hunchback (Hb, green), Zelda (Zld, red), Tailless (tll, grey) and Krüppel (Kr, …
Figure 3—figure supplement 1
Removing Bcd TFBS from SynHbP2 reporter construct.
We deleted binding sites from a version of SynHbP2 that contained 12 bp of endogenous sequence flanking each Bcd binding site. (A) Average expression profile from SynP2 deletion constructs color …
Figure 4
Chromatin remodelers and histone modifiers influence the WTHbP2 GRF.
We knocked down the maternal contribution of multiple trans factors using RNAi in embryos harboring the WTHbP2 reporter. We stained for LacZ expression and present the average expression profile …
Figure 5
Mediator subunits affect the shape of the WTHbP2 GRF.
Mediator subunits were knocked down using RNAi (see Materials and Methods). The number of embryos imaged is listed in Table 2. (Left column) Average gene expression profiles from hbP2-reporter …
Figure 6 with 4 supplements
Deleting binding sites from WTHbP2 and SynHbP2 reveals discrepancies between experimentally measured GRFs and a model at thermodynamic equilibrium.
(A) Using an updated version of the mathematical model in Estrada et al., we calculated the boundaries of the steepness and position for GRFs that can be generated by six transcription factor …
Figure 6—figure supplement 1
Deleting binding sites and comparing against model with extra sites.
(Top) Estimated position and steepness for wild-type and synthetic Hb enhancer with subsequent TF binding site deletions (indicated in the title of each panel), plotted against the boundaries of the …
Figure 6—figure supplement 2
Procedure for obtaining normalized position (P) and normalized steepness (S) to compare the experimental data to the boundaries of mathematical GRFs calculated using equilibrium modeling.
(A) (top left) Estimated non-normalized GRFs for WTHbP2 deletion series as shown in Figure 2. Average expression profile from each deletion construct. (bottom left) Non-normalized ‘raw’ position and …
Figure 6—figure supplement 3
Graph for gene regulation model.
(Top) Graph for the gene regulation model with two sites for a transcription factor, L, and a single site for RNA polymerase, P, giving a 2 + 1 model. (Bottom) The set-theoretic notation used for …
Figure 6—figure supplement 4
Boundary stabilization.
Position-steepness boundaries are shown for GRFs generated by six transcription factor binding sites, as in Figure 6, for varying parametric ranges. The boundaries stabilise rapidly as the …
Tables
Table 1
List of shRNA lines
https://doi.org/10.7554/eLife.41266.008| Target gene name | TRiP ID | # of embryos |
|---|---|---|
| Nejire (CBP) | HMS01570 | 8 |
| Bin1/SAP18 | GL00127 | 8 |
| Sin3A | HMS00359 | 13 |
| HDAC6 | HMS00077 | 2 |
| Mediator complex subunit 1 | HMS01139 | 9 |
| Mediator complex subunit 11 | HMS01094 | 7 |
| Mediator complex subunit 14 | HMS01049 | 5 |
| Mediator complex subunit 20 | HMS01051 | 12 |
| Mediator complex subunit 22 | HMS01047 | 8 |
| Mediator complex subunit 27 | HMS01050 | 4 |
| Mediator complex subunit 28 | HMS00458 | 5 |
| Mediator complex subunit 7 | HMS01140 | 5 |
| Vielfaltig (Zld) | HMS02441 | 7 |
| hunchback | GL01321 | 11 |
Table 2
Number of Embryos Imaged
https://doi.org/10.7554/eLife.41266.016| Reporter contructs | # of embryos |
|---|---|
| Endogeous hb | 6 |
| WThbP2 | 6 |
| WThbP2 (5TFBS) | 10 |
| WThbP2 (4TFBS) | 10 |
| WThbP2 (3TFBS) | 7 |
| WThbP2 (2TFBS) | 12 |
| WThbP2 (1TFBS) | 10 |
| WThbP2 (Δ1b) | 11 |
| WThbP2 (Δ2b) | 13 |
| WThbP2 (Δ3b) | 13 |
| SynHbP2 (2 bp) | 4 |
| SynHbP2 (7 bp) | 15 |
| SynHbP2 (12 bp) | 10 |
| SynHbP2 (12 bp, 4TFBS) | 15 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Drosophila melanogaster) | Nejire (CBP) RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 36682, RRID:BDSC_36682 | |
| Strain, strain background (D. melanogaster) | Bin1/SAP18 RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 36781, RRID:BDSC_36781 | |
| Strain, strain background (D. melanogaster) | Sin3A RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 32368, RRID:BDSC_32368 | |
| Strain, strain background (D. melanogaster) | HDAC6 RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 34072, RRID:BDSC_34072 | |
| Strain, strain background (D. melanogaster) | MED1 RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 34662, RRID:BDSC_34662 | |
| Strain, strain background (D. melanogaster) | MED11 RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 34083, RRID:BDSC_34083 | |
| Strain, strain background (D. melanogaster) | MED14 RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 34575, RRID:BDSC_34575 | |
| Strain, strain background (D. melanogaster) | MED20 RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 34577, RRID:BDSC_34577 | |
| Strain, strain background (D. melanogaster) | MED22 RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 34573, RRID:BDSC_34573 | |
| Strain, strain background (D. melanogaster) | MED27 RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 34576, RRID:BDSC_34576 | |
| Strain, strain background (D. melanogaster) | MED28 RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 32459, RRID:BDSC_32459 | |
| Strain, strain background (D. melanogaster) | MED7 RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 34663, RRID:BDSC_34663 | |
| Strain, strain background (D. melanogaster) | Vielfaltig (Zld) RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 42016, RRID:BDSC_42016 | |
| Strain, strain background (D. melanogaster) | hunchback (hb) RNAi | Bloomington Drosophila Stock Center | BDSC Cat# 54478, RRID:BDSC_54478 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.41266.017
