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. 2016 Aug 23;5:e17548. doi: 10.7554/eLife.17548

Figure 2. 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 added as indicated. (B) Meta-gene analysis for indicated DRIP signal over indicated genomic features. Data are shown for DRIP-seq biological replicate # 3. An enrichment is seen in all data sets but relative read density at these sites varies between replicates. (C) DRIP-seq read counts normalized for total mapped reads from DRIP in 0 nM E2 conditions vs DRIP from cells treated with 100 nm E2 for 2 hr (top) or 24 hr (bottom). Graphs are from 3 biological experiments. Black dots indicate DRIP peaks and red dots indicate induced DRIP peaks relative to 0 nM E2. (D) Integrated Genome Viewer (IGV) display of DRIP-seq enrichment at XBP1. Scale = million mapped reads. RNase H was performed prior to DRIP-seq on one replicate each from 0 nM E2 and 100 nM E2 24 hr. Independent replicates are shown as 1–3. (E) IGV display of CCND1 (Cyclin D1), as described in (D). (F) DRIP-qPCR validation. Cells were treated with 0, 10, or 100 nM E2 for 24 hr and harvested for DRIP. MLKL and 83/84 are negative controls. Error bars represent S.E.M. of 2 biological experiments. RNase H treatment was performed for 24 hr prior to DRIP-qPCR where indicated. (G) Functional signatures by GREAT of E2-induced DRIP peaks found to be differentially induced in 100 nM E2, 2 hr (top) or 100 nM E2 24 hr (bottom) than in 0 nM E2 treated cells. The 7 highest enrichment scores are shown, with red highlighting E2-associated signatures. (H) Fold change in DRIP signal after 2 hr of 100 nM E2 relative to 0 nM E2 (x-axis) vs. fold change in GRO-seq signal after 160 min of 100 nM E2 relative to 0 nM E2 (y-axis). E2-induced DRIP peaks that show a positive (red) or negative (blue) fold change in GRO-seq upon E2 stimulation are highlighted. Negative changes in DRIP upon E2 that correspond to a positive (yellow) or negative (green) fold change in GRO-seq are also shown. GRO-seq data from (Hah et al., 2011).

DOI: http://dx.doi.org/10.7554/eLife.17548.010

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.
DOI: http://dx.doi.org/10.7554/eLife.17548.011
elife-17548-fig2-data1.bed (187KB, bed)
DOI: 10.7554/eLife.17548.011
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.
DOI: http://dx.doi.org/10.7554/eLife.17548.012
elife-17548-fig2-data2.bed (76.1KB, bed)
DOI: 10.7554/eLife.17548.012

Figure 2.

Figure 2—figure supplement 1. R-loops are induced with E2 prior to S phase and exhibit R-loop features.

Figure 2—figure supplement 1.

(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 above the diagonal represent DRIP peaks above input. Red dots correspond to E2-induced peaks. (B) RNase H treatment. Read per kilobase per million (RPKM) plots of DRIP peaks in 0 nM E2 versus DRIP peaks from 0 nM E2 sample treated with RNase H prior to IP (left) and of DRIP peaks from cells treated with 100 nM E2 for 24 hr versus DRIP peaks from cells treated with RNase H after 100 nM E2 for 24 hr (right). Red dots show identified E2-induced DRIP peaks and their sensitivity to RNase H. (C) GC skew density in DRIP peaks. The strongest 3000 DRIP peaks from each indicated samples are shown. GC skew obtained from Ginno et al., 2012. (D) G-quartet density in DRIP peaks. The strongest 3000 DRIP peaks from each indicated samples are shown. G-quartet data obtained from Chambers et al., 2015. (E) IGV display of DRIP-seq enrichment at SLC7A5. Scale = million mapped reads. An RNase H control was performed prior to DRIP-seq from 0 or 100 nM E2-treated cells for 24 hrs. Biological replicates are shown as 1–3. (F) FACS profiles of MCF7 cells treated with 0 nM E2 (left), or 100 nM E2 for 2 hr (middle) or 24 hrs (right). Cells were pulsed with 25 μM BrdU 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 the four cell-cycle quadrants is shown.
DOI: http://dx.doi.org/10.7554/eLife.17548.013
Figure 2—figure supplement 1—source data 1. Genomic coordinates for all identified DRIP peaks from MCF7 cells treated with 0 nM E2.
DOI: http://dx.doi.org/10.7554/eLife.17548.014
elife-17548-fig2-figsupp1-data1.bed (405.2KB, bed)
DOI: 10.7554/eLife.17548.014
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.
DOI: http://dx.doi.org/10.7554/eLife.17548.015
elife-17548-fig2-figsupp1-data2.bed (779KB, bed)
DOI: 10.7554/eLife.17548.015
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.
DOI: http://dx.doi.org/10.7554/eLife.17548.016
elife-17548-fig2-figsupp1-data3.bed (576.3KB, bed)
DOI: 10.7554/eLife.17548.016
Figure 2—figure supplement 2. Sequence features and expression analysis associated with DRIP-seq.

Figure 2—figure supplement 2.

(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 negative (blue) change in GRO-seq upon E2. Error bands for (A,B) represent the 1st and 3rd quartile from 1000x bootstrap. (C) DRIP peak strength compared to expression level for DRIP performed with 0 nM E2 (left) and 100 nM E2 for 2 hr (middle) or 100 nM E2 for 24 hr (right). Expression levels in non-E2 treated or E2-treated cells for similar time periods obtained from publically available RNA-seq (Honkela et al., 2015).
DOI: http://dx.doi.org/10.7554/eLife.17548.017