Fig 2: BRD2 depletion results in loss of Pol II at BET-bound enhancers. (A) Venn diagram showing the overlap between each BET protein and the UCSC annotated hg19 coding gene regions. Note that BRD4 has the lowest percentage of inter-genic peaks, while BRD2 has the highest percentage of inter-genic peaks (Fig. 1D). (B) Clustering of H3K27ac, H3K4me1, and H3K4me3 ChIP-seq at the 8421 inter-genic peaks called for one or more of the BRDs using the mAID antibody. (C) Heat map of mAID ChIP-seq ± auxin for BET proteins and sorted by H3K27ac occupancy at the enhancer regions defined by clusters 2 and 3. (D) Genome browser views of Pol II ChIP-seq at two different enhancer regions upon auxin treatment in BET degron cells and dBET6 treatment in parental DLD-1 cells for 2 h. H3K27ac ChIP-seq is shown for comparison. (E) Heat map of Pol II ChIP-seq at enhancer regions before and after auxin treatment in BET degron cells or dBET6 treatment in parental DLD-1 cells for 2 h. (F) Box plot showing the log2RPKM of Pol II ChIP-seq corresponding to the conditions in E. Auxin treatment in BRD2-AID cells and dBET6 treatment in parental cells exhibit significant reductions in Pol II occupancy at enhancer regions. (****) P < 2.210 × -16, (ns) not significant. Mann–Whitney U-test.

Fig 3: ASXL3 is a direct target of BET inhibitors and predicts drug sensitivity. a The log2 fold change of nearby gene expression in ASXL3-depleted cells, and cells treated with either DMSO, dBET6 (300 nM), JQ1 (1 µM), or IBET-151 (1 µM). b The Venn-diagram shows the overlap of downregulated genes in cells treated with dBET6, JQ1, or IBET-151 from (a). c The bar plot shows ASXL3 expression (RPM) in NCI-H1963 cells treated with different BET inhibitors, n = 2, two-tailed unpaired Student’s t test. **P < 0.01; *P < 0.05. d Real-time PCR was performed to determine the relative gene expression of EIF4E and ASXL3 in NCI-H1963 cells treated with dBET6, JQ1, or IBET-151 for 4 h or 8 h. DMSO was used as negative control, n = 3, two-tailed unpaired Student’s t test. **P < 0.01; *P < 0.05. e BRD4 ChIP-seq was performed with NCI-H1963 SCLC cells treated either DMSO or dBET6 for 2 h. The representative track shows the occupancy of BRD4 at ASXL3 locus. f Whole-cell lysates were used for western blot with BRD2, BRD3, BRD4, Pol II, CDK9, and CCNT1 antibodies in human SCLC cell line NCI-H1963 treated with dBET6 for 0, 2, 8, and 24 h, n = 3. g Whole-cell lysates were used for western blot with ASXL3, JMJD6, NSD3, and CHD4 antibodies in human SCLC cell line NCI-H1963 treated with dBET6 for 0, 2, 8, and 24 h. HSP90 was used as an internal control, n = 3. h Whole-cell lysates were used for western blot with BRD2, BRD3, BRD4, ASXL3, JMJD6, NSD3, and CHD4 antibodies in human SCLC cell line NCI-H1963 treated with different concentrations of dBET6 for 8 h; HSP90 was used as an internal control, n = 3. i 8 different human SCLC cell lines NCI-H748, NCI-H1882, NCI-H1963, NCI-H209, NCI-H1436, NCI-H1105, NCI-H889, and NCI-H2171 were treated with different concentrations of dBET6 for 72 h. The cell viability was determined by CellTiter-Glo Luminescent Cell Viability Assay, n = 6. j The protein levels of ASXL3 and BRD4 from the previously cell lines mentioned (i) were determined by western blot, n = 3. k The representative track shows H3K27Ac and H3K4me1 levels at ASXL3 locus in ASXL3-high (NCI-H1963) and ASXL3-low (NCI-H2171) SCLC cells. l The Venn-diagram shows SE-associated genes in ASXL3-high and ASXL3-low cells from (k)

Fig 4: BRDs protein degradation is progeasome-dependent. 6 T-CEM, Molt4, Jurkat and CCRF cells were treated with 1 µM ARV-825 and different concentration of MG132. After treatment with 24 h, BRD2, BRD3 and BRD4 protein were presented by Western blot

Fig 5: Specific loss of BRD4 leads to R-loop-induced DNA damage.a WB of BRD2, BRD3, and BRD4 following transfection of HeLa cells with control siRNA, or siRNAs to BRD2, BRD3, and BRD4 for 48 h. b Left: WB of ?H2AX, total PARP, and total H2AX in cells following siRNA knockdown of BRD2, BRD3, or BRD4 in HeLa cells. Right: Quantification of ?H2AX and cleaved PARP product (cPARP; lower band on PARP western blot) signal following knockdown of BRD2, BRD3, or BRD4. Data presented as mean ± SEM (n = 3 independent experiments). Significance assessed using ANOVA followed by Sidak’s test (?H2AXCtl si vs BRD4 si **Adjusted P = 0.0080; cPARPCtl si vs BRD4 si *Adjusted P = 0.0254). c Quantification and representative IF images of ?H2AX and nuclear S9.6 intensities in cells following siRNA knockdown of BRD2, BRD3, or BRD4. Data presented as mean ± SEM (n = 3 separate experiments). Box-whisker plots are drawn as in Fig. 1b. Significance was assessed using ANOVA followed by Dunnett’s test (?H2AXCtl si vs BRD4 si ***Adjusted P = 0.0004; S9.6Ctl si vs BRD4 si ****Adjusted P < 0.0001). Scale bar = 2.5 µm. d HeLa cells expressing doxycycline-inducible FLAG-tagged RNase H1 were treated with doxycycline for 24 h then were transfected with control or BRD4 siRNA for 48 h. Lysates were probed for BRD4 and FLAG-tagged RNase H1 by WB. Tubulin serves as loading control. e Fluorescence microscopy images of single cell gel electrophoresis showing tail moments following knockdown of BRD4 (BRD4 si) in the absence or presence of RNase H1 induction (n = 3). Box-whisker plots are drawn as in Fig. 1b. Significance was assessed using ANOVA followed by Tukey’s test ****Adjusted P < 0.0001, **Adjusted P = 0.0069. d, e Are from the same experiment. f Representative WB of BRD4, FLAG-tagged RNase H1, and ?H2AX following knockdown of BRD4 and doxycycline-induced expression of FLAG-tagged RNase H1. Quantification of ?H2AX intensity presented as mean (n = 2 independent experiments). g Cell counts of RNase H-inducible HeLa cells following siRNA knockdown of BRD2, BRD3, or BRD4 for 72 h, in the presence (+RNase H, red line) or absence (-RNase H, blue line) of induced RNase H expression (n = 2 independent experiments). Source data are provided as a Source data file.