Different promoter affinities account for specificity in MYC-dependent gene regulation
- Biocenter, University of Würzburg, Germany
- Max-Delbrück-Center for Molecular Medicine, Germany
- University of Würzburg, Germany
Figures
Figure 1 with 1 supplement
MYC saturates certain binding sites.
(A) Immunoblot of MYC and Vinculin in U2OSTet-On cells treated with EtOH or with 1 µg/ml of doxycycline. (B) Immunoblot of MYC and Vinculin in several transformed (U2OS, HeLa, HCT116) and …
Figure 1—figure supplement 1
Effect of different peak calling programs and parameters on peak numbers.
(A) Number of peaks for endogenous MYC according to different peak calling programs at default parameters. (B) Percentage of overlapping MYC peaks called by different peak calling programs. Shown is …
Figure 2 with 2 supplements
MYC binds with a wide range of affinity (EC50 values) to target genes.
(A) Immunoblot of MYC in U2OSTet-On cells treated with 1 µg/ml doxycycline and a recombinant MYC protein fragment, which was used for absolute quantification of cellular MYC levels (M: marker). …
Figure 2—figure supplement 1
Quantification of MYC molecules per U2OS cell.
(A) Coomassie staining of the recombinant MYC fragment used to quantify cellular MYC levels documenting the purity of the protein. (B) Coomassie staining of a polyacrylamide gel after the transfer …
Figure 2—figure supplement 2
Variation of MYC levels within the cell population demonstrates validity of the model conclusions for the majority of cells.
(A) Confocal fluorescence showing the variation of MYC expression in U2OSTet-On cells treated with 1 µg/ml doxycycline and ethanol control. Images were taken under the same imaging conditions. The …
Figure 3
Binding behavior of MYC in U2OS cells analyzed by mathematical modeling.
(A) Schematic representation of model 1. For details see Appendix 1. (B, C) Plot illustrating regions of occupancy in the parameter space of dissociation constants KEbox and KNNNNNN as well as total …
Figure 4 with 2 supplements
E-box occurrence, expression level and chromatin status of target genes influence MYC binding.
(A) Binned plot for the number of genes in each bin with a canonical E-box (CACGTG) in the MYC peak versus MYC occupancy in U2OSTet-On treated with EtOH. Genes were sorted according to MYC occupancy …
Figure 4—figure supplement 1
E-box occurrence and level of expression correlate with MYC binding and EC50 values.
(A, B) Gaussian kernel density estimate of endogenous MYC occupancy stratified by the number of consensus (A) or non-consensus (B) E-boxes in the promoter. p-Values were calculated with a two-sided …
Figure 4—figure supplement 2
Promoters that are bound by WDR5 and contain E-boxes are most strongly occupied by MYC.
(A) Gaussian kernel density estimate of endogenous MYC occupancy stratified by the number of consensus E-boxes in concert with WDR5 peaks in the promoter. p-values were calculated with a two-sided …
Figure 5 with 1 supplement
Promoter affinity for MYC stratifies functionally different gene sets.
(A) Immunoblot of MYC upon transfection of U2OSTet-On cells with the indicated siRNA. Vinculin was used as loading control. (B) Plot for regulation of genes upon siRNA-mediated depletion of MYC vs …
Figure 5—figure supplement 1
Changes in chromatin binding upon MYC depletion correlate with transcriptional responses.
(A) Immunoblot of MYC in U2OSTet-On cells tranfected with siRNA targeting MYC and control siRNA. (B) Summary of MYC ChIP-sequencing results of MYC-depleted U2OSTet-On cells and control cells. (C) …
Figure 6
MYC levels determine the regulation of different transcriptional response programs.
(A) Bar plot indicating MYC levels in U2OSTet-On cells treated with EtOH or with different concentrations of doxycycline. Data are shown as mean ± standard deviation of three independent biological …
Figure 7
Schematic models.
(A) High-affinity binding sites, for instance characterized by E-boxes and WDR5 binding, are already highly occupied at physiological MYC levels (medium MYC) in dividing cells. At oncogenic …
Appendix 1—figure 1
Schemes of models 1 and 2.
https://doi.org/10.7554/eLife.15161.019
Appendix 2–figure 1
Plot shows surfaces of equal occupancy as function of dissociation constants and as well as total amount of MYC.
Different regions of occupancy of E-boxes (A) and NNNNNN sequences (B) are shown in this parameter space. The experimentally measured range of the total amount of MYC is marked in grey. Total MYC …
Appendix 2–figure 2
Plot shows surface of of Appendix 2–figure 1A in two different orientations: rotated into the - -plane and into the - -plane.
https://doi.org/10.7554/eLife.15161.024
Appendix 2–figure 3
Ratio of the dissociation constants and determines how much occupancy of E-boxes differs from occupancy of NNNNNN sequences.
The panels show the dependency of the occupancies of E-boxes (red curve) and NNNNNN sequences (blue curves) on total number of MYC molecules. For each panel a different combination of dissociation …
Appendix 2–figure 4
Impact of number of E-boxes and NNNNNN sequences.
Different combinations of number of E-boxes and NNNNNN sequences are assumed in the simulations: and represent the reference values and the range of 50% to 200% of these reference values is …
Tables
Appendix 1—table 1
Parameters generally applicable to models 1 and 2.
| Parameter | Value | Unit | Comment, Reference |
|---|---|---|---|
| Vol | 7.7·10–13 | L | 1) |
| Genometotal | 9.5·109 | base pairs | 2) |
Appendix 1—table 2
Parameters of model 1.
| Parameter | Value | Unit | Comment, Reference |
|---|---|---|---|
| Eboxtotal | 9.3·105 | molecules | 3) |
| NNNNNNtotal | 9.5·109 | molecules | 4) |
| KEbox | 4.6·101 | molecules | 5); 0.1 nM (Guo et al., 2014) |
| KNNNNNN | 9.3·103 | molecules | 5); 20 nM (Guo et al., 2014) |
Appendix 1—table 3
Parameters of model 2.
| Parameter | Value | Unit | Comment, Reference |
|---|---|---|---|
| Eboxtotal | 9.2·105 | molecules | 6) |
| NNNNNNtotal | 9.5·109 | molecules | 6) |
| EboxWDR5total | 1.2·104 | molecules | 6) |
| NNNNNNWDR5total | 4.7·104 | molecules | 6) |
| KEbox | 4.6·101 | molecules | 5); 0.1 nM (Guo et al., 2014) |
| KNNNNNN | 9.3·103 | molecules | 5); 20 nM (Guo et al., 2014) |
| KEboxWDR5 | 4.3·10–4 | molecules | 7) |
| KNNNNNNWDR5 | 8.6·10–2 | molecules | 7) |
Additional files
-
Supplementary file 1
This file provides the raw data and calculation for absolute protein quantification.
- https://doi.org/10.7554/eLife.15161.016
-
Supplementary file 2
This file lists gene sets used in this study.
- https://doi.org/10.7554/eLife.15161.017
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Supplementary file 3
This file lists primer sequences used in this study.
- https://doi.org/10.7554/eLife.15161.018
