53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.We thank Anthony Tubbs for comments on the paper; Jennifer Mehalko and Dom Esposito (Protein Expression Laboratory, Frederick National Laboratory for Cancer Research) for transgenic constructs; Karim Baktiar, Diana Haines, and Elijah Edmonson (Pathology/Histotechnology Laboratory, Frederick National Laboratory for Cancer Research) for rodent necropsy, pathology analysis, and imaging; Joseph Kalen and Nimit Patel (Small Animal Imaging Program, Frederick National Laboratory for Cancer Research) for X-ray computed tomography (CT) scan imaging; Jennifer Wise and Kelly Smith for assistance with animal work; Davide Robbiani and Kai Ge for antibodies; Dan Durocher for shieldin constructs; David Goldstein and the CCR Genomics core for sequencing support; and Neil Johnson for discussions. Research in the J.M.S. laboratory is supported by NIH grant R01CA197506. Research in the N.M. laboratory is supported by NIH grant R01 227001. The A.N. laboratory is supported by the Intramural Research Program of the NIH, an Ellison Medical Foundation Senior Scholar in Aging Award (AG-SS-2633-11), the Department of Defense Idea Expansion (W81XWH-15-2-006) and Breakthrough (W81XWH-16-1-599) Awards, the Alex's Lemonade Stand Foundation Award, and an NIH Intramural FLEX Award.S

Bacteria Modulate the CD8+ T Cell Epitope Repertoire of Host Cytosol-Exposed Proteins to Manipulate the Host Immune Response

The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested

Bcl6 Is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells

<div><p>The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.</p></div

Super DIVAC does not potentiate mutation in the absence of Bcl6.

<p>A) Representative flow cytometry plots of GFP and Thy 1.1 levels after 28 days of culture of the indicated cell lines. B) Western blot analysis for AID expression in the sublcones analyzed for mutation in panels C, D, E, and F, with beta actin loading control shown below. * background band. Each WT subclone contains a unique Super DIVAC GFP2 reporter integration event while all Pax5^R AID^R SD#10 and Bcl6^R AID^R SD#10 subclones share an identical reporter integration site. C, D) Scatter plots showing the SHM/GCV event frequencies observed at the IgL V region (C) or <i>GFP</i> (D) for 2 independent subclones as indicated after 76 days of culture, presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149146#pone.0149146.g001" target="_blank">Fig 1B</a>. Horizontal bars indicate the mean frequency of mutations for each genotype. E, F) Pie charts showing the distribution of all SHM/GCV events observed at the IgL V region (E) or <i>GFP</i> (F) compiled from the 2 independent subclones in panels C, D after 76 days of culture, presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149146#pone.0149146.g001" target="_blank">Fig 1C</a>. Z test was used to assess significance differences among mutation frequencies. ***, p<0.001 and NS, not statistically significant.</p

Transcription, AID subcellular localization, and single stranded DNA in Bcl6-deficient DT40 cells.

<p>A, B) RT-PCR analysis of A) IgL V region or B) GFP RNA levels in the sequenced subclones from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149146#pone.0149146.g003" target="_blank">Fig 3C–3F</a>. Data are presented as the average of the signal obtained from two independent RNA preparations for each subclone after normalization to the signal obtained for Rab7a. Error bars represent the SEM. C) Western blot analysis for AID (lower panel) and Pax5 (upper panel) protein in cytoplasmic and nuclear fractions for the indicated cell lines. All fractions were quantitated and equivalent amounts of protein were loaded. *, unknown band. D) Pie charts showing the distribution of single stranded patches detected in the IgL V region after bisulfite treatment and sequence analysis for one subclone of each genotype. Bisulfite converts dCs on exposed single-stranded DNA to dUs and, after PCR amplification and sequencing, C to T transitions and G to A transitions reveal single-stranded dCs on the top and bottom strands, respectively. To be counted as a single-stranded DNA patch, at least two successive transition mutations were required.</p

Pax5 expression does not restore SHM/GCV in Bcl6-deficient DT40 cells.

<p>A) Western blot analysis for AID expression in the subclones analyzed for mutations in panels B and C with a beta actin loading control shown below. *, background bands. B) Scatter plot of IgL V region SHM/GCV event frequencies from individual subclones as indicated. Horizontal bars indicate the mean frequency of mutations for each genotype. Z test used to assess significant differences among mutation frequencies. ***, p<0.001 and NS, not statistically significant. C) Pie charts showing the distribution of all SHM/GCV events observed at the IgL V region from the independent subclones indicated in panel B after 28 days of culture.</p

ATM and PRDM9 regulate SPO11-bound recombination intermediates during meiosis

Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm–/– spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing

Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse

International audienceReplication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (\textgreater20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility