Interferon restores replication fork stability and cell viability in BRCA-defective cells via ISG15

DNA replication and repair defects or genotoxic treatments trigger interferon (IFN)-mediated inflammatory responses. However, whether and how IFN signaling in turn impacts the DNA replication process has remained elusive. Here we show that basal levels of the IFN-stimulated gene 15, ISG15, and its conjugation (ISGylation) are essential to protect nascent DNA from degradation. Moreover, IFNβ treatment restores replication fork stability in BRCA1/2-deficient cells, which strictly depends on topoisomerase-1, and rescues lethality of BRCA2-deficient mouse embryonic stem cells. Although IFNβ activates hundreds of genes, these effects are specifically mediated by ISG15 and ISGylation, as their inactivation suppresses the impact of IFNβ on DNA replication. ISG15 depletion significantly reduces cell proliferation rates in human BRCA1-mutated triple-negative, whereas its upregulation results in increased resistance to the chemotherapeutic drug cisplatin in mouse BRCA2-deficient breast cancer cells, respectively. Accordingly, cells carrying BRCA1/2 defects consistently show increased ISG15 levels, which we propose as an in-built mechanism of drug resistance linked to BRCAness

Interferon restores replication fork stability and cell viability in BRCA-defective cells via ISG15

DNA replication and repair defects or genotoxic treatments trigger interferon (IFN)-mediated inflammatory responses. However, whether and how IFN signaling in turn impacts the DNA replication process has remained elusive. Here we show that basal levels of the IFN-stimulated gene 15, ISG15, and its conjugation (ISGylation) are essential to protect nascent DNA from degradation. Moreover, IFNβ treatment restores replication fork stability in BRCA1/2-deficient cells, which strictly depends on topoisomerase-1, and rescues lethality of BRCA2-deficient mouse embryonic stem cells. Although IFNβ activates hundreds of genes, these effects are specifically mediated by ISG15 and ISGylation, as their inactivation suppresses the impact of IFNβ on DNA replication. ISG15 depletion significantly reduces cell proliferation rates in human BRCA1-mutated triple-negative, whereas its upregulation results in increased resistance to the chemotherapeutic drug cisplatin in mouse BRCA2-deficient breast cancer cells, respectively. Accordingly, cells carrying BRCA1/2 defects consistently show increased ISG15 levels, which we propose as an in-built mechanism of drug resistance linked to BRCAness.</p

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects

Distinct roles of meiosis-specific cohesin complexes in mammalian spermatogenesis

Mammalian meiocytes feature four meiosis-specific cohesin proteins in addition to ubiquitous ones, but the roles of the individual cohesin complexes are incompletely understood. To decipher the functions of the two meiosis-specific kleisins, REC8 or RAD21L, together with the only meiosis-specific SMC protein SMC1β, we generated Smc1βRec8 and Smc1βRad21L mouse mutants. Analysis of spermatocyte chromosomes revealed that besides SMC1β complexes, SMC1α/RAD21 and to a small extent SMC1α/REC8 contribute to chromosome axis length. Removal of SMC1β and RAD21L almost completely abolishes all chromosome axes. The sex chromosomes do not pair in single or double mutants, and autosomal synapsis is impaired in all mutants. Super resolution microscopy revealed synapsis-associated SYCP1 aberrantly deposited between sister chromatids and on single chromatids in Smc1βRad21L cells. All mutants show telomere length reduction and structural disruptions, while wild-type telomeres feature a circular TRF2 structure reminiscent of t-loops. There is no loss of centromeric cohesion in both double mutants at leptonema/early zygonema, indicating that, at least in the mutant backgrounds, an SMC1α/RAD21 complex provides centromeric cohesion at this early stage. Thus, in early prophase I the most prominent roles of the meiosis-specific cohesins are in axis-related features such as axis length, synapsis and telomere integrity rather than centromeric cohesion.This work was supported by grants fropm the Deutsche Forschungsgemeinschaft SPP1384 (RJ) and by the BFU (2014-59307-R) and JCyLe (AP).Peer Reviewe

Distinct Roles of Meiosis-Specific Cohesin Complexes in Mammalian Spermatogenesis

<div><p>Mammalian meiocytes feature four meiosis-specific cohesin proteins in addition to ubiquitous ones, but the roles of the individual cohesin complexes are incompletely understood. To decipher the functions of the two meiosis-specific kleisins, REC8 or RAD21L, together with the only meiosis-specific SMC protein SMC1β, we generated <i>Smc1</i>β^<i>-/-</i><i>Rec8</i>^<i>-/-</i> and <i>Smc1β</i>^<i>-/-</i><i>Rad21L</i>^<i>-/-</i> mouse mutants. Analysis of spermatocyte chromosomes revealed that besides SMC1β complexes, SMC1α/RAD21 and to a small extent SMC1α/REC8 contribute to chromosome axis length. Removal of SMC1β and RAD21L almost completely abolishes all chromosome axes. The sex chromosomes do not pair in single or double mutants, and autosomal synapsis is impaired in all mutants. Super resolution microscopy revealed synapsis-associated SYCP1 aberrantly deposited between sister chromatids and on single chromatids in <i>Smc1β</i>^<i>-/-</i><i>Rad21L</i>^<i>-/-</i> cells. All mutants show telomere length reduction and structural disruptions, while wild-type telomeres feature a circular TRF2 structure reminiscent of t-loops. There is no loss of centromeric cohesion in both double mutants at leptonema/early zygonema, indicating that, at least in the mutant backgrounds, an SMC1α/RAD21 complex provides centromeric cohesion at this early stage. Thus, in early prophase I the most prominent roles of the meiosis-specific cohesins are in axis-related features such as axis length, synapsis and telomere integrity rather than centromeric cohesion.</p></div

List of secondary antibodies used.

<p>List of secondary antibodies used.</p

Telomere analysis.

<p>A. Staining of spermatocyte spreads by Telo FISH (red) to assess telomere length and anti-SYCP3 for AEs/LEs in WT, SKO and DKO mice (scale bar: 5 μm). Magnified images of individual chromosomes are shown below. Telomeres are seen as red dots at the ends of chromosomes or on disrupted chromosome structures. WT chromosomes show FISH signals of about equal intensities at both ends. Examples for aberrant telomeres are indicated as follows: SCs lacking telomeres—white arrow head; isolated telomeres/broken off SCs, yellow arrow; telomere fusions or close associations—white arrow. B. Immunofluorescence staining of spermatocyte chromosome spreads of WT, SKO and DKO mice, probed with anti-SYCP3 (red) for the AEs/LEs and anti-RAP1 (green) to stain telomeres (scale bar: 5 μm). Aberrant telomeres are marked as above. C. Immunofluorescence staining of spermatocyte chromosome spreads of WT, SKO and DKO mice, probed with anti-SYCP3 (green) for the AEs/LEs and anti-SUN1 (red) to stain telomere attachments (scale bar: 5 μm). D. Graph showing the average telomere intensity, maximum intensity and area of WT, SKO and DKO spermatocyte spreads as measured using the ImageJ software. The boxes show the median value, the upper 75th and the lower 25th percentile, along with maximal and minimal values. The p values for all comparisons of a mutant to wt are <0.0001. The p values for all other paired comparisons (e.g. <i>Smc1β</i>^<i>-/-</i> versus <i>Rec8</i>^<i>-/-</i> etc.) are also <0.0001 according to Dunn’s multiple comparison test.</p

Super resolution telomere analysis.

<p>SIM analysis of wild-type and mutant spermatocyte telomeres in chromosome spreads, stained with anti TRF2 and anti SYCP3. Sex chromosomes in wild-type are marked by a blue arrow. High magnification excerpts labeled with a grey asterisks are additional examples from separate images (scale bar: 5 μm).</p

Testis tubule analysis of wild-type and mutant mice.

<p>A. Immunofluorescence staining of testis sections of WT, SKO (single knockout) mice <i>Smc1β</i>^<i>-/-</i>, <i>Rec8</i>^<i>-/-</i>, <i>Rad21L</i>^<i>-/-</i>, and DKO (double knock out) mice: <i>Smc1β</i>^<i>-/-</i><i>Rec8-/-</i> and <i>Smc1β</i>^<i>-/-</i><i>Rad21L</i>^<i>-/-</i> mice, probed with anti-SYCP3 and anti-γH2AX; DNA was stained with DAPI (scale bar: 5 μm). Tubular stages are indicated by roman letters. B. Magnified images from Fig. 2A showing SYCP3-stained axes and γH2AX localization. The most advanced stages are shown, characterized by chromosome axes that are SYCP3-positive and γH2AX signals, which indicate the presence of DNA double-strand breaks und unsynapsed chromosomes. C. Immunofluorescence staining of testis sections of WT, SKO (single knockout) mice <i>Smc1β</i>^<i>-/-</i>, <i>Rec8</i>^<i>-/-</i>, <i>Rad21L</i>^<i>-/-</i>, and DKO (double-knock out) mice: <i>Smc1β</i>^<i>-/-</i><i>Rec8-/-</i> and <i>Smc1β</i>^<i>-/-</i><i>Rad21L</i>^<i>-/-</i> mice, probed with anti-SYCP1 and anti-SYCP3; DNA was stained with DAPI (scale bar: 5 μm). D. Magnified images from Fig. 2C showing SYCP1 (green)/SYCP3 (red)-stained axes indicated by yellow color.</p