BRCA1 Recruitment to Transcriptional Pause Sites Is Required for R-Loop-Driven DNA Damage Repair

The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites

BRCA1 Recruitment to Transcriptional Pause Sites Is Required for R-Loop-Driven DNA Damage Repair

Summary The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites

Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx-/- revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome

Defective meiotic recombination and crossover formation in infertile <i>Setx^−/−</i> males.

<p>A. Initiation and repair of programmed DNA DSB as shown by γH2AX staining of spermatocytes spreads of <i>Setx^+/+</i> and <i>Setx^−/−</i> adult mice. At pachytene, γH2AX staining is restricted to the XY chromosomes (circle) in <i>Setx^+/+</i> spermatocytes, whereas some γH2AX foci remained on asynapsed autosomes indicating persistence of unrepaired DSB in <i>Setx^−/−</i>. Normal γH2AX staining of the XY chromosomes (circle) was observed in both <i>Setx^+/+</i> and <i>Setx^−/−</i> pachytene stage spermatocytes. Scale bar, 20 µm. XY, sex chromosomes. B. Persistence of Rad51 foci at pachytene stage in <i>Setx^−/−</i> spermatocytes indicating the presence of unrepaired DSBs (compare 1 and 2). Scale bar, 20 µm. C. Quantitation of Rad51 foci revealed a 6-fold increase in the number of Rad51 foci at pachytene stage in <i>Setx^−/−</i> as compared to <i>Setx^+/+</i> (Student's t-test, n = 50), * indicates p<0.05. D. Formation of chiasmata at pachytene stage in <i>Setx^+/+</i> spermatocytes as marked by Mlh1 staining. No Mlh1 foci were detected in <i>Setx^−/−</i> pachytene cells indicating that crossovers do not occur in <i>Setx^−/−</i>. 1 and 2 represent magnification of autosomes. Scale bar, 20 µm. SCP3 or SCP1 were used to identify the meiotic stages. E. Defect in senataxin leads to R-loop structures accumulation in germ cells. Staining with S9.6 antibody (R-loops) on adult spermatocytes revealed an increased formation of R-loops in <i>Setx^−/−</i> germ cells. Scale bar, 20 µm. F. Number of pachytene spermatocytes showing none-faint, medium, and strong R-loop staining intensities for <i>Setx^+/+</i> and <i>Setx^−/−</i>.</p

Targeted disruption of the mouse <i>Setx</i> gene.

<p>A. Diagram of the <i>Setx</i> wild type allele (WT), targeting vector, and mutant alleles (neo+ and KO). Primers used for PCR genotyping (In3F, In3R, LoxPR) and the length of the PCR fragments obtained for WT (In3F and In3R yielding a 600 bp product) and KO (In3F and LoxpR yielding a 339 bp product) are indicated. E, exon; I, intron. NeoR represents the neomycin cassette, and triangles the <i>loxP</i> sites. B. Representative image of PCR genotyping using In3F, In3R and LoxPR primers. Wild type (+/+), heterozygotes (+/−) and knockout (−/−) alleles generate PCR products of 600 bp, 600 bp and 339 bp, and 339 bp, respectively. A negative control for the PCR reaction (−ve) is also shown. M, 100 bp marker. C. RT-PCR of 35 day-old mice testes samples using primers specific to <i>Setx</i> cDNA indicates the absence of <i>Setx</i> expression in KO testes. GAPDH was used as an internal standard. D. Immunoprecipitation of senataxin using anti-human senataxin antibodies (Ab-1/Ab-3) from 35 day-old mouse testes extracts confirmed the absence of the protein in the <i>Setx^−/−</i>. Immunoprecipitation of senataxin from human lymphoblastoid cell extracts from normal (C3ABR) and an AOA2 patient (SETX2RM) confirmed the similar size of senataxin in both species. A species-matched non-specific serum (NSIg) was used as a negative control in for the IP experiments. As expected, no senataxin protein was brought down from <i>Setx</i>^+/+ testes following the IP with the non-specific serum (NSIg).</p

Defective localisation and diffusion of DNA damage response proteins in <i>Setx^−/−</i>.

<p>A. Absence of ATR diffusion over the XY chromatin domain in <i>Setx^−/−</i> compared to <i>Setx^+/+</i>. Scale bar, 5 µm. B. Incomplete diffusion of MDC1 over the XY chromatin domain in <i>Setx^−/−</i>, as indicated by the white arrow. Scale bar, 5 µm. C. Reduced intensity and diffusion of γH2AX staining on the XY chromosomes in <i>Setx^−/−</i> compared to <i>Setx^+/+</i>. D. Altered XY chromosomes structure and formation in <i>Setx^−/−</i> as shown by SCP3 staining. Scale bar 5 µm. E. Percentage of <i>Setx^+/+</i> and <i>Setx^−/−</i> pachytene spermatocytes at days 16, 20 and 22 with clearly distinguishable XY chromosomes. At every time point, a significant higher percentage of distinguishable XY chromosomes was observed in <i>Setx^+/+</i> (p<0.01 according to Student's t-test, n = 3600). *,**,*** indicates p<0.01.</p

Senataxin localises to the sex chromosomes during meiosis.

<p>A. Staining of spermatocytes spreads with an anti-senataxin antibody (Ab-1) revealed that senataxin localised in majority to the XY chromosomes (1) at pachytene stage in <i>Setx^+/+</i>. Some background staining was also observed on the autosomes. No senataxin was detected in <i>Setx^−/−</i> spermatocytes confirming the specificity of our senataxin antibody. (1) and (2) are magnification of the XY chromosomes. B. Double staining of <i>Setx^+/+</i> pachytene spermatocytes with senataxin (Ab-1) and SCP3 revealed a diffuse localisation of senataxin to the XY body. Scale bar, 20 µm. C. Partial co-localisation of senataxin with the XY chromosome marker Brca1. While Brca1 stains exclusively the unsynapsed axis of the XY chromosomes, senataxin staining is more diffuse. D. Brca1 staining of the XY chromosomes in <i>Setx^+/+</i> and <i>Setx^−/−</i> pachytene spermatocytes (day 20). In <i>Setx^+/+</i>, the unsynapsed axis of the XY chromosomes is entirely stained with Brca1 while an incomplete covering (white arrow) of the XY chromosomes is observed in <i>Setx^−/−</i>. Scale bar, 5 µm. E. Lack of senataxin recruitment to XY chromosomes in <i>Brca1^Δ11/Δ11 p53^+/−</i> as compared to <i>Brca1^+/+ p53^+/−</i>. F. Brca1 localised to only part of the unsynapsed axis of the XY chromosomes in <i>Brca1^Δ11/Δ11 p53^+/−</i> while Brca1 coated the entire unsynapsed axis of the XY chromosomes in <i>Brca1^+/+ p53^+/−</i>. G. Lack of evidence for an <i>in situ</i> direct endogenous interaction between senataxin and Brca1 on the XY chromosomes as revealed by negative Proximity Ligation Assay (PLA) results on pachytene spermatocyte spreads. Immunostaining of <i>Setx^+/+</i> pachytene spermatocytes with Brca1 and Setx individually with SCP3 is also shown. H. Endogenous interaction between ATR and Brca1 was confirmed by PLA. Here, we reveal for the first time a direct endogenous interaction between Brca1 and ATR <i>in situ</i> over the XY chromosomes.</p