. 2024 Jun 3;15(1):4716.

doi: 10.1038/s41467-024-48286-1.

Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency

Liana Goehring¹, Sarah Keegan^{1

2}, Sudipta Lahiri^{1

3}, Wenxin Xia¹, Michael Kong¹, Judit Jimenez-Sainz³, Dipika Gupta¹, Ronny Drapkin^{4

5}, Ryan B Jensen³, Duncan J Smith⁶, Eli Rothenberg¹, David Fenyö^{1

2}, Tony T Huang⁷

Affiliations

¹ Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, USA.
² Institute for Systems Genetics, New York University School of Medicine, New York University School of Medicine, New York, NY, USA.
³ Department of Therapeutic Radiology, Yale University, New Haven, CT, USA.
⁴ Penn Ovarian Cancer Research Center, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA.
⁵ Basser Center for BRCA, Abramson Cancer Center, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA.
⁶ Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA.
⁷ Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, USA. tony.huang@nyumc.org.

PMID: 38830843
PMCID: PMC11148086
DOI: 10.1038/s41467-024-48286-1

Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency

Liana Goehring et al. Nat Commun. 2024.

. 2024 Jun 3;15(1):4716.

doi: 10.1038/s41467-024-48286-1.

Authors

Affiliations

¹ Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, USA.
² Institute for Systems Genetics, New York University School of Medicine, New York University School of Medicine, New York, NY, USA.
³ Department of Therapeutic Radiology, Yale University, New Haven, CT, USA.
⁴ Penn Ovarian Cancer Research Center, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA.
⁵ Basser Center for BRCA, Abramson Cancer Center, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, USA.
⁶ Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA.
⁷ Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, USA. tony.huang@nyumc.org.

PMID: 38830843
PMCID: PMC11148086
DOI: 10.1038/s41467-024-48286-1

Abstract

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. Loss of BRCA2 causes dormant origin firing at TTSs of long, highly transcribed genes.**
a Identification of new origins at TTSs upon replication stress and schematic of Okazaki-fragment sequencing origin analysis. (i) 1 kb-binned Crick and Watson Ok-seq reads are Hann smoothed and replication origins calls are defined as regions of increasing Crick-strand derivative and decreasing Watson-strand derivative from smoothed reads (see “Methods” section). (ii) Relative distance calculation orients origins to genomic annotations (TSS, TTS). Replication origins are prevalent at active TSSs indicated by a high proportion of origins at low relative distances in active TSSs (left). If origins were randomly distributed with respect to active TSSs, there would be an equal proportion at all relative distances (right). b Relative distance origin analysis of previously published untreated and HU-treated RPE-1 cells (Chen et al.) relative to TSSs of transcriptionally high-volume genes (FPKM × length > median). N = # of origins per condition. Percent of origins at each relative distance were fit to a nonlinear regression curve (solid line) with a 90% confidence interval (shaded). p-values calculated using a one-sided, paired t-test between conditions for the first three relative distance bins (0, 0.01, 0.02) (see “Methods” section). c Relative distance origin analysis of untreated and HU-treated RPE-1 cells relative to TTSs of high-volume genes. The solid line represents origins at each relative distance fit to a nonlinear regression curve with a 90% confidence interval (shading). p-values calculated using a one-sided, paired t-test between conditions for the first three relative distance bins (0, 0.01, 0.02). d Whole cell lysates of immortalized fallopian tube epithelial cells (FTEs) with doxycycline (DOX)-inducible BRCA2-shRNA after 0, 3, and 6 days of treatment were analyzed by western blot for validation of knockdown (KD) and pRPA-related replication stress. Tubulin is a loading control for this blot and subsequent western blots. Results reproducible for at least 3 biological replicates. e (Right) Log₂ annotation enrichment of FTE Ok-seq origins at 0, 3, and 6 days of BRCA2-KD. N = 2 biological Ok-seq replicates each (left). Zoomed TTS annotation (right); lines connect biological replicates. f Relative distance origin analysis of untreated and BRCA2-KD FTEs relative to TSSs of high-volume genes. For all subsequent experiments, 3 days of dox treatment was used for BRCA2-KD. The solid line represents origins at each relative distance fit to a nonlinear regression curve with a 90% confidence interval (shading). p-values calculated using a one-sided, paired t-test between conditions for the first three relative distance bins (0, 0.01, 0.02). g Relative distance origin analysis of untreated and BRCA2-KD FTEs relative to TTSs of high-volume genes. The solid line represents origins at each relative distance fit to a nonlinear regression curve with a 90% confidence interval (shading). p-values calculated using a one-sided, paired t-test between conditions for the first three relative distance bins (0, 0.01, 0.02). h Log₁₀ normalized gene FPKM and normalized gene length of candidate genes with new origins at TTSs with BRCA2-KD (n = 418 genes). The dotted line represents the average FPKM and gene length of all genes (N = 18250) from RNA-seq analysis of FTEs. i Two representative candidate genes with new origins at TTSs with BRCA2-KD in two Ok-seq biological replicates each with 0, 3, and 6 days of knockdown: *MYO1B* and *TLE3*. Source data are provided as a Source Data file.

**Fig. 2. BRCA2 knockdown causes HO-TRCs with elongating RNAP2.**
a Schematic of pSer5- and pSer2-RNAP2 phospho-form abundance distributed across gene bodies relative to intergenic replication origin firing. b Proximity-ligation assay (PLA) of pSer2-RNAP2 and PCNA in WT- and KD-BRCA2 FTEs including single and no antibody controls. The data presented shows 300 nuclei from 3 biological replicates. p-values calculated using unpaired two-tailed t-tests. Left: number of foci per nucleus. Right: average number of foci per nucleus per biological replicates. Error bars = mean, std. Scale bar = 2 µm in representative pictures. c PLA of pSer5-RNAP2 and PCNA in WT- and KD-BRCA2 FTEs including single antibody controls. The data presented shows 200 nuclei from 3 biological replicates. p-values calculated using unpaired two-tailed t-tests. Left: number of foci per nucleus. Right: average number of foci per nucleus per biological replicates. Error bars = mean, std. Scale bar = 2 µm in representative images. d PLA of RPB1-CTD and PCNA in WT- and KD-BRCA2 FTEs including single antibody controls. The data presented shows 200 nuclei from 3 biological replicates. p-values calculated using unpaired two-tailed t-tests. Left: number of foci per nucleus. Right: average number of foci per nucleus per biological replicates. Error bars = mean, std. Scale bar = 2 µm in representative images. e Representative SMLM image shows EdU (yellow) and pSer2-RNAP2 (cyan). Scale bar = 2 µm. Scatter plot quantification measuring pSer2-RNAP2 at EdU in WT- and KD-BRCA2 (see “Methods” section). p-values calculated using unpaired two-tailed t-test from at least two biological replicates (WT: N = 156, KD: N = 203, where N = # of S-phase nuclei). Error bars = mean, std. f Representative SMLM image shows EdU (yellow) and pSer5-RNAP2 (cyan). Scale bar = 2 µm. Scatter plot quantification measuring pSer5-RNAP2 at EdU in WT- and KD-BRCA2. p-values calculated using unpaired two-tailed t-test from at least two biological replicates (WT: N = 250, KD: N = 218, where N = # of S-phase nuclei). Error bars = mean, std. Source data are provided as a Source Data file.

**Fig. 3. Replication stress modulators alter dormant origin firing and HO-TRCs in BRCA2-deficient cells.**
a Schematic showing the impact of replication stress modulators, CDC7i, PARPi, and ATRi, on fork speed and dormant origins. In unperturbed cells, passive replication prevents the firing of nearby licensed origins. Under-replication stress, failure of fork progression causes the nearby firing of dormant origins. CDC7i directly inhibits dormant origins under-replication stress. PARPi increases fork speed. ATRi causes unscheduled origin firing. b DNA fiber analysis of sequentially labeled nucleotide analogs (IdU and CldU, each 20′) followed by DNA spreading and fluorescent labeling to measure fork elongation as CldU track length in kilobases in FTEs. Representative images are shown of DNA fibers from WT- and KD-BRCA2 FTEs ± 5 µM CDC7i, 5 h. Scale bars = 10 µm. The scatter plot shows the quantification of elongating CldU track length. p-values calculated using unpaired two-tailed t-tests of 200 fibers per condition from three biological replicates. Error bars = mean, std. c DNA fiber analysis to measure inter-origin distance in kilobases. Representative images were shown of DNA fibers from WT- and KD-BRCA2 ± CDC7i. Scale bars = 10 µm. The scatter plot shows the quantification of inter-origin distance. p-values calculated using unpaired two-tailed t-tests from three biological replicates: WT-BRCA2(−)CDC7i, N = 48; WT-BRCA2(+)CDC7i, N = 27; KD-BRCA2(−)CDC7i, N = 65; KD-BRCA2(+)CDC7i, N = 30, where N = # of IOD fibers analyzed. Error bars = mean, std. d PLA of pSer2-RNAP2 and PCNA in WT- and KD-BRCA2 ± CDC7i. The data presented shows 200 nuclei from 3 biological replicates. Number of foci per nucleus, p-values calculated using unpaired two-tailed t-tests. Error bars = mean, std. e PLA of pSer5-RNAP2 and PCNA in WT- and KD-BRCA2 ± CDC7i. The data presented shows 200 nuclei from 3 biological replicates. Number of foci per nucleus, p-values calculated using unpaired two-tailed t-tests. Error bars = mean, std. f PLA of pSer2-RNAP2 and PCNA in WT- and KD-BRCA2 ± PARPi or ATRi. p-values calculated using unpaired two-tailed t-tests of 200 nuclei per condition from 3 biological replicates. Error bars = mean, std. g Representative SMLM image of EdU pulse-labeled FTE nucleus stained with pSer2-RNAP2 and γH2AX. Scale bar = 2 µm. h Scatter plot quantification measuring SMLM of gH2AX at pSer2-RNAP2 in WT- and KD-BRCA2. p-values calculated using unpaired two-tailed t-test of at least two biological replicates. WT = 108; KD, N = 165, where N = # of S-phase nuclei. Error bars = mean, std. i Scatter plot quantification measuring SMLM of gH2AX at pSer2-RNAP2 in KD-BRCA2 ± CDC7i. p-values calculated using unpaired two-tailed t-test of at least two biological replicates. (−)CDC7i, N = 80; (+)CDC7i, N = 73, where N = # of S-phase nuclei. Error bars = mean, std. j PLA of pSer2-RNAP2 and gH2AX in WT- and KD-BRCA2 ± CDC7i including single antibody controls. p-values calculated using unpaired two-tailed t-tests of 200 nuclei per condition from 3 biological replicates. Error bars = mean, std. k PLA of pSer2-RNAP2 and pRPA-Ser33 in WT- and KD-BRCA2 ± CDC7i including single antibody controls. p-values calculated using unpaired two-tailed t-tests of 200 nuclei per condition from 3 biological replicates. Error bars = mean, std. l PLA of pSer2-RNAP2 and S9.6 in WT- and KD-BRCA2 ± CDC7i including single antibody controls. p-values calculated using unpaired two-tailed t-tests of 200 nuclei per condition from 3 biological replicates. Error bars = mean, std. Source data are provided as a Source Data file.

**Fig. 4. BRCA2 and RNase H2 cooperate together to mitigate HO-TRCs.**
a Representative SMLM images of EdU pulse-labeled FTEs ± 0.2 mM HU treatment (4 h) stained with pSer2-RNAP2 and BRCA2. Scale bar = 2 µm. b Scatter plot quantification measuring pSer2-RNAP2 at EdU in untreated and HU-treated FTEs (left; untreated, N = 234; HU, N = 255, where N = # of S-phase nuclei), BRCA2 at EdU (middle; untreated, N = 125; HU, N = 139), and BRCA2 at pSer2-RNAP2 (right; untreated, N = 162; HU, N = 219). p-values calculated using unpaired two-tailed t-tests of at least two biological replicates. Error bars = mean, std. c PLA of BRCA2 and PCNA in FTEs treated with 0.2 mM HU for 4 h, and/or 100 µM DRB for 90 min, including single antibody controls. The data presented shows 200 nuclei from 3 biological replicates. p-values calculated using unpaired two-tailed t-tests. Left: number of foci per nucleus. Right: average number of foci per nucleus per biological replicates. Error bars = mean, std. Scale bar = 10 µm in representative images. d Representative SMLM image of EdU pulse-labeled FTE cell stained with pSer2-RNAP2 and RNASEH2A. Scale bar = 2 µm. e Scatter plot quantification measuring RNASEH2A at pSer2-RNAP2 in EdU-labeled WT- and KD-BRCA2. p-values calculated using unpaired two-tailed t-test of at least two biological replicates. Unt, WT = 160; KD, N = 169, where N = # of S-phase nuclei. Error bars = mean, std. f Scatter plot quantification measuring RNASEH2A at pSer2-RNAP2 in EdU-labeled untreated and HU-treated FTEs. p-values calculated using unpaired two-tailed t-test of at least two biological replicates. Unt, N = 92; HU, N = 101. Error bars = mean, std. g PLA of pSer2-RNAP2 and PCNA in siControl or siRNASEH2A FTEs ± CDC7i. The data presented shows 150 nuclei from 3 biological replicates. p-values calculated using unpaired two-tailed t-tests. Left: number of foci per nucleus. Right: average number of foci per nucleus per biological replicates. Error bars = mean, std. h PLA of BRCA2 and RNASEH2A or BRCA2 and RNase H1 in FTEs ± 0.2 mM HU for 4 h, including single antibody controls for each PLA. The data presented shows 200 nuclei from 3 biological replicates. p-values calculated using unpaired two-tailed t-tests between conditions. Left: number of foci per nucleus. Right: average number of foci per nucleus per biological replicates. Error bars = mean, std. Scale bar = 10 µm in representative pictures. i PLA of BRCA2 and RNaseH2A or BRCA2 in FTEs ± ATRi. The data presented shows 200 nuclei from 3 biological replicates. p-values calculated using unpaired two-tailed t-tests between conditions. Left: number of foci per nucleus. Right: average number of foci per nucleus per biological replicates. Error bars = mean, std. Source data are provided as a Source Data file.

**Fig. 5. HO-TRCs are sites of genomic instability in BRCA2-deficient cells.**
a Whole cell lysates of FTEs were analyzed by western blot. Empty vector or V5-tagged RNase H1 (RH1) plasmids were transiently transfected for 24 h prior to collection in WT- or KD-BRCA2 FTEs. Results reproducible for at least 3 biological replicates. b PLA of pSer2-RNAP2 and PCNA of WT- and KD-BRCA2 ± RH1 overexpression. The data presented shows at least 150 nuclei from 3 biological replicates. p-values calculated using unpaired two-tailed t-tests between conditions. Left: number of foci per nucleus. Right: average number of foci per nucleus per biological replicates. Error bars = mean, std. c DRIP-qPCR analysis of 6 candidate gene TTSs in WT- and KD-BRCA2 FTEs ± RH1 overexpression. The bar graph shows % of input normalized to the control sample (0d + EV) of 3 biological replicates. p-values calculated using two-way ANOVA with Tukey’s multiple comparisons test. Error bars = mean, std. d Hallmark gene set analysis (GSEA) of genes length >50 kb and highly expressed (top 500 FPKM) from RNA-seq of FTEs. Top hallmark gene sets are shown as -log₁₀(p-value) p-values calculated using the hypergeometric distribution of overlapping genes over all genes in the gene universe. e Hallmark GSEA of candidate genes with new origins at TTSs in shBRCA2-KD based on Ok-seq origin analysis. p-values calculated using the hypergeometric distribution of overlapping genes over all genes in the gene universe. f Hallmark GSEA of genes with mutations enriched in mutBRCA2 samples vs all mutTP53 samples in ovarian serous cystadenocarcinoma patients (TCGA Firehose study). p-values calculated using the hypergeometric distribution of overlapping genes over all genes in the gene universe. g Characterization of genes with mutations enriched in mutBRCA2 samples vs all mutTP53 samples in ovarian serous cystadenocarcinoma patients (N = 376) (TCGA Firehose study): log normalized gene length (left); log normalized gene expression (RNA-seq V2 RSEM) (middle); mutation type (right). h Schematic model of HO-TRCs at TTSs in an early BRCA2-deficient HGSOC. Left: BRCA2 protects stalled replication forks to promote fork progression of canonical forks at TSSs. BRCA2 promotes transcription elongation and R-loop resolution through RNase H2 recruitment. Right: in BRCA2 deficiency, increased replication stress and transcriptional dysregulation. Dormant origin firing at TTSs of long, highly transcribed genes causes HO-TRCs with elongating RNAP2 and DNA damage. TRC-prone genes maintain fallopian tube epithelial (FTE) integrity, which is compromised in early BRCA2-deficient HGSOC lesions. Source data are provided as a Source Data file.

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Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency

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Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency

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