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

The DSIF Subunits Spt4 and Spt5 Have Distinct Roles at Various Phases of Immunoglobulin Class Switch Recombination

Class-switch recombination (CSR), induced by activation-induced cytidine deaminase (AID), can be divided into two phases: DNA cleavage of the switch (S) regions and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in a switch-proficient B cell line CH12F3-2A cells, and Spt4 and Spt5 carry out independent functions in CSR. While neither Spt4 nor Spt5 is required for transcription of S regions and AID, expression array analysis suggests that Spt4 and Spt5 regulate a distinct subset of transcripts in CH12F3-2A cells. Curiously, Spt4 is critically important in suppressing cryptic transcription initiating from the intronic Sμ region. Depletion of Spt5 reduced the H3K4me3 level and DNA cleavage at the Sα region, whereas Spt4 knockdown did not perturb the H3K4me3 status and S region cleavage. H3K4me3 modification level thus correlated well with the DNA breakage efficiency. Therefore we conclude that Spt5 plays a role similar to the histone chaperone FACT complex that regulates H3K4me3 modification and DNA cleavage in CSR. Since Spt4 is not involved in the DNA cleavage step, we suspected that Spt4 might be required for DNA repair in CSR. We examined whether Spt4 or Spt5 is essential in non-homologous end joining (NHEJ) and homologous recombination (HR) as CSR utilizes general repair pathways. Both Spt4 and Spt5 are required for NHEJ and HR as determined by assay systems using synthetic repair substrates that are actively transcribed even in the absence of Spt4 and Spt5. Taken together, Spt4 and Spt5 can function independently in multiple transcription-coupled steps of CSR

FACT複合体によって制御されるヒストンH3K4トリメチル化修飾はクラススイッチ組換えにおけるDNA鎖切断に必須である

京都大学0048新制・課程博士博士(医科学)甲第16746号医科博第31号新制||医科||3(附属図書館)29421京都大学大学院医学研究科医科学専攻(主査)教授 生田 宏一, 教授 篠原 隆司, 教授 武田 俊一学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Spt4 and Spt5 regulate small yet distinct sets of transcripts.

<p>(A) A Venn diagram showing the number of up- or down-regulated ranscripts by at least 2-fold in the absence of either Spt4 or Spt5, in CIT(+) treated CH12F3-2A cells. (B) Differential expressions of selected genes identified by microarray by either Spt4 or Spt5 knockdown stimulated for 24 hours with CIT; results are presented relative to control, which was set as 1. A complete list is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002675#pgen.1002675.s006" target="_blank">Table S1</a>. (C) Top: A schematic diagram of the positions of primers (triangles) used to quantify the cryptic S region transcripts. Bottom: after introducing the RNAi oligonucleotides indicated, the cryptic Sμ and Sα transcripts were quantified by RT-qPCR normalized to HPRT. SD values were determined from three independent experiments. The p-values were calculated using the unpaired two-tailed Student's <i>t</i> test (*, P<0.03; **, P<0.004; ***, P<0.0002).</p

The DSIF complex is required for efficient NHEJ.

<p>(A) ChIP assays of the various knockdown and control samples indicated, using anti-Ku80 antibody. Background values from controls with no antibody were subtracted. Values were normalized to the input DNA signals. The maximum value in each data set was set as 100%. SD values were derived from three independent experiments. The p-values were calculated using the unpaired two-tailed Student's <i>t</i> test (*, P<0.01). (B) Schematic diagram of the <i>I-Sce</i>I-induced NHEJ repair substrate. (C) Percentage of EGFP-positive cells, assessed by FACS analysis 48 hours after co-transfection of <i>I-Sce</i>I expression plasmids and the indicated RNAi oligonucleotides into H1299dA3-1 cells. (D) Relative EGFP-positive cells with respect to to <i>I-Sce</i>I-treated control cells. SD values were derived from three independent experiments. A.U: arbitrary unit. (E) Knockdown efficiency of the indicated genes was quantified by RT-qPCR. (F) <i>I-Sce</i>I(+)-treated control cells from (D) were divided and transfected with the indicated RNAi oligonucleotides (indicated by dotted line); the percent EGFP-positive population was quantified and compared to control samples. SD values were derived from three independent experiments. A.U: arbitrary unit. (G) PCR of genomic DNA products; uncut and repaired fragments derived from control and knockdown samples are indicated by arrowheads. (H) PCR products of repaired genomic DNA fragments derived from the indicated knockdown samples. Arrowheads represent insertions.</p

Spt4 and Spt5 differentially control S region DNA cleavage and H3K4me3 status.

<p>(A) DNA break assay by γH2AX ChIP using anti-γH2AX antibody were performed in Spt4- or Spt5-knockdown or control samples. Pulled-down DNA was subjected to Sμ- and Sα-specific detection by RT-PCR, normalized to the input DNA signals. The maximum value in each data set was set as 100%. SD values were derived from three independent experiments. (B) DNA break assay performed using biotin-dUTP end labeling method derived from Spt4- or Spt5-knockdown and control samples. Pulled-down DNA was subjected to Sμ- and Sα-specific detection by RT-PCR, normalized to the input DNA. SD values were derived from three independent experiments. (C) Top: schematic diagram of the position of the ChIP assay PCR products. Bottom: the knockdown and control samples indicated were assayed by ChIP, using anti-H3K4me3 and anti-H3 antibodies. Background values from controls with no antibody were subtracted. Values were normalized to the input DNA signals. The maximum value in each data set was set as 100%. SD values were derived from three independent experiments. The p-values were calculated using the unpaired two-tailed Student's <i>t</i> test (*, P = 0.05; **, P<0.03; ***, P<0.02; ****, P<0.005). (D) Immunoblotting of histone H3K4me3, H3, and tubulin derived from the indicated knockdown samples.</p

Spt4 and Spt5 are critical for CSR.

<p>(A) Flow cytometry (FACS) profile of the percent IgA-switching population, indicated by the number in the box, after introducing the indicated RNAi oligonucleotide under CIT(−) or (+) conditions. (B) Top: Summary of the percent IgA-switching population data derived from the indicated gene knockdown samples. Bottom: Percent of dead cells as determined by PI staining. SD values were determined from three independent experiments. Knockdown efficiency for each gene indicated was analyzed by (C) RT-qPCR and (D) immunoblotting. (E) Immunoblotting of Spt4 and Spt5 derived from the indicated knockdown samples. (F) Various transcripts quantified by RT-qPCR and normalized to HPRT, after introduction of the indicated RNAi oligonucleotide, under the CIT(+) condition. SD values were determined from three independent experiments. (G) After introducing Spt4 or Spt5 RNAi oligonucleotides, γ3GLT was quantified by RT-qPCR normalized to HPRT. SD values were determined from three independent experiments. (H) Summary of IgG3-switching population data derived from the indicated gene-knockdown samples. SD values were determined from three independent experiments. The p-values were calculated using the unpaired two-tailed. Student's <i>t</i> test (*, P<0.03; **, P<0.007; ***, P<0.001). (I) FACS profile of the IgG3-switching population after introducing the indicated RNAi oligonucleotide under CIT(−) or (+) conditions.</p

DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates