Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage
- Stanford University School of Medicine, United States
- University of California, Davis, United States
Figures
Figure 1 with 6 supplements
Estrogen induces DNA damage and DSBs in a replication-dependent manner.
(A) Immunostaining for P-H2AX in MCF7 cells treated with 0, 1, 10, or 100 nM E2 for 24 hr. (B) Quantification of P-H2AX immunostaining for data shown in (A), ****p<0.0001. n = 2 biological …
Figure 1—figure supplement 1
Immunostaining for 53BP1 in MCF7 cells either treated with 0 or 100 nM E2 for 24 hr.
Nuclei stained with Hoechst.
Figure 1—figure supplement 2
FACS profiles of MCF7 cells treated with 0 nM E2, 1 nM E2, or 100 nM E2 for 24 hr.
Cells were pulsed with 25 μM BrdU for 30 min prior to fixation. DNA content is marked by propidium iodide (x-axis) and BrdU incorporation is shown on the y-axis. The percentage of cells in each of …
Figure 1—figure supplement 3
Effect of E2 on MCF10A cells.
(A) Quantification of P-H2AX immunostaining in MCF10A cells treated with 0 or 100 nM E2 for 24 hr. ns = not significant (non-parametric Mann-Whitney rank sum t-test). n = 3 biological replicates. >25…
Figure 1—figure supplement 4
Quantification of the percent of cells positive for EdU incorporation after treatment with 0 or 100 nM E2 for the indicated length of time and then pulsed with 10 μM EdU for 30 min prior to fixation.
h = hours. Errors bars represent SD of 2 biological replicates.
Figure 1—figure supplement 5
Effects of PHA 767491 on EdU incorporation and Ser2 phosphorylation of RNA Pol II in MCF7 cells.
(A) Quantification of the percent of cells positive for EdU incorporation for cells treated with 0 or 100 nM E2 concurrently with DMSO or 1 μM Cdc7 inhibitor PHA 767491 for 14 hrs. Cells were pulsed …
Figure 1—figure supplement 6
Quantification of the percent of cells positive for EdU incorporation for cells treated with 0 or 100 nM E2 for 12 hr prior to the addition of 0.8 μM flavopiridol or DMSO for 2 hr.
Cells were pulsed with 10 μm EdU for 30 min prior to harvesting. Error bars represent the SD of 3 biological replicates.
Figure 2 with 2 supplements
Estrogen induces robust R-loop formation at E2-responsive genes.
(A) Slot blot to detect global RNA-DNA hybrids with S9.6 antibody in MCF7 cells treated with 0 or 100 nM E2 for 24 hr. Total denatured DNA is stained with a single-strand DNA antibody. RNase H was …
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Figure 2—source data 1
Genomic coordinates for DRIP peaks identified as induced in MCF7 cells treated with 100 nM E2 for 2 hrs relative to MCF7 cells treated with 0 nM E2.
- https://doi.org/10.7554/eLife.17548.011
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Figure 2—source data 2
Genomic coordinates for DRIP peaks identified as induced in MCF7 cells treated with 100 nM E2 for 24 hrs relative to MCF7 cells treated with 0 nM E2.
- https://doi.org/10.7554/eLife.17548.012
Figure 2—figure supplement 1
R-loops are induced with E2 prior to S phase and exhibit R-loop features.
(A) Average read count in input (x-axis) versus S9.6 immunoprecipitation (y-axis) in MCF7 cells treated with 0 nM E2 (left), 100 nM E2 for 24 hr (middle) or 100 nM E2 for 2 hr (right). Black dots …
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Figure 2—figure supplement 1—source data 1
Genomic coordinates for all identified DRIP peaks from MCF7 cells treated with 0 nM E2.
- https://doi.org/10.7554/eLife.17548.014
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Figure 2—figure supplement 1—source data 2
Genomic coordinates for all identified DRIP peaks from MCF7 cells treated with 100 nM E2 for 2 hrs.
- https://doi.org/10.7554/eLife.17548.015
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Figure 2—figure supplement 1—source data 3
Genomic coordinates for all identified DRIP peaks from MCF7 cells treated with 100 nM E2 for 24 hr.
- https://doi.org/10.7554/eLife.17548.016
Figure 2—figure supplement 2
Sequence features and expression analysis associated with DRIP-seq.
(A) GC skew density in E2-induced DRIP peaks that show a positive (red) or negative (blue) change in GRO-seq upon E2. (B) G-quartet density in E2-induced DRIP peaks that show a positive (red) or …
Figure 3 with 3 supplements
Breast cancer rearrangements are enriched in E2-responsive genes.
(A) Histogram of E2-induced expression changes (Z-score) for genes that overlap with breast cancer structural variants (red bars) compared to the distribution of E2-induced expression changes …
Figure 3—figure supplement 1
Comparison of (A) Replication timing based on RepliSeq signal across the gene body of regions containing structural variants in breast cancer (x-axis) relative to that of the matched regions (y-axis), and (B) Mean expression level based on RNA-seq of regions associated with structural variants (x-axis) relative to that of the matched regions (y-axis).
https://doi.org/10.7554/eLife.17548.019
Figure 3—figure supplement 2
Comparison of (A) Replication timing based on RepliSeq signal across the gene body of translocated regions (x-axis) relative to that of the matched regions (y-axis), and (B) Mean expression level based on RNA-seq of translocations (x-axis) relative to that of the matched regions (y-axis).
https://doi.org/10.7554/eLife.17548.020
Figure 3—figure supplement 3
Comparison of (A) Replication timing based on RepliSeq signal across the gene body of genes with simple somatic mutations (x-axis) relative to that of the matched regions (y-axis), and (B) Mean expression level based on RNA-seq of genes with simple somatic mutations (x-axis) relative to that of the matched regions (y-axis).
https://doi.org/10.7554/eLife.17548.021
Figure 4 with 4 supplements
E2-induced R-loops occur on chromatin marked by DNA damage and RNase H reduces E2-induced DSBs.
(A) Proximity ligation assay between S9.6 antibody and P-H2AX antibody in cells treated either with 0 or 100 nM E2 for 24 hr. (B) Quantification of the percentage of cells with ≥ 1 PLA focus per …
Figure 4—figure supplement 1
Quantification of the fold change in the percent of cells with >5 P-H2AX foci per cell in MCF7-tetON-RH cells treated with indicated concentrations of DOX. *p<0.05.
Error bars represent SD of 3 technical replicates.
Figure 4—figure supplement 2
Expression of RNase H prevents E2-induced DNA damage.
(A) Quantification of P-H2AX intensity per nucleus in MCF7 tetON-RH clone 6 (left) or MCF7 tetON-RH clone 8 (right) treated with 0 or 1000 ng/mL DOX for 24 hr prior to the addition of 0 nM or 100 nM …
Figure 4—figure supplement 3
FACS profiles of MCF7 tetON-RH cells treated with or without 1000 ng/mL DOX for 24 hr prior to the addition of 100 nM E2 for 24 hr.
Cells were pulsed with 25 μM BrdU for 30 min prior to fixation. DNA content is marked by propidium iodide (x-axis) and BrdU incorporation is shown on the y-axis. The percentage of cells in each of …
Figure 4—figure supplement 4
EU incorporation following RNase H expression in MCF7 cells.
(A) EU staining in MCF7 tetON-RH cells either treated with or without 1000 ng/mL DOX for 24 hr prior to the addition of 0 or 100 nM E2 for 24 hr (48 hr DOX total). 100 μM DRB was added for 2 hr. …
Figure 5 with 1 supplement
Knockdown of NER and R-loop processing factors reduces E2-induced DNA damage and DSBs.
(A) P-H2AX intensity based on immunostaining of MCF7 cells transfected with indicated siRNA 64 hrprior to the addition of 0 or 100 nM E2 for 24 hr. ****p<0.0001. Western blot shows the level of XPG. …
Figure 5—figure supplement 1
XPG knockdown does not alter E2-induced cell cycle progression.
(A) FACS profiles of MCF7 cells transfected with siXPG or siGL3 and grown for 64 hr prior to the addition of 0 or 100 nM E2 for 24 hr, with DNA content marked by propidium iodide (x-axis), and cell …
Additional files
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Supplementary file 1
DRIP-qPCR primers.
- https://doi.org/10.7554/eLife.17548.029
