Dysregulation of X-Ray Repair Cross Complementing 4 Expression in the Eutopic Endometrium of Women with Endometriosis

Recent data suggest that the DNA damage response (DDR) is altered in the eutopic endometrium (EE) of women with endometriosis and this probably ensues in response to higher DNA damage encountered by the EE in endometriosis. DDR operates in a tissue-specific manner and involves different pathways depending on the type of DNA lesions. Among these pathways, the non-homologous end joining (NHEJ) pathway plays a critical role in the repair of double-stranded DNA breaks. The present study was undertaken to explore whether NHEJ is affected in the EE of women with endometriosis. Towards this, we focused on the X-Ray Repair Cross-Complementing 4 (XRCC4) protein, one of the core components of the NHEJ pathway. Endometrial XRCC4 protein levels in the mid-proliferative phase were found significantly (p<0.05) downregulated in women with endometriosis, compared to control women. Investigation of a microarray-based largest dataset in the GEO database (GSE51981) revealed a similar trend at the transcript level in the EE of women with endometriosis, compared to control women. Further in-vitro studies were undertaken to explore the effects of H2O2-induced oxidative stress on DNA damage, as assessed by γ-H2AFX and 8-hydroxy-2’-deoxyguanosine (8-OHdG) immunolocalization, and XRCC4 protein levels in endometrial stromal (ThESCs) and epithelial (Ishikawa) cells. A significant decrease in XRCC4 protein levels and significantly higher localization of γ-H2AFX and 8-OHdG were evident in ThESCs and Ishikawa cells experiencing oxidative stress. Overall, the study demonstrates that the endometrial XRCC4 expression is dysregulated in women with endometriosis and this could be due to higher oxidative stress in endometriosis.

RIF1-ASF1-mediated high-order chromatin structure safeguards genome integrity

The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identified a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.

Associations of the Polymorphisms of the NHEJ Pathway Genes With HIV-1 Infection and Aids Progression Among Men Who Have Sex With Men in Northern China

Background: Men who have sex with men (MSM) are at high risk of HIV infection. Non-homologous end joining (NHEJ) pathway is the main way of double-stranded DNA break (DSB) repair in the higher eukaryotes, and can repair the DSB timely at any time in cell cycle. The objective of this study was to investigate the association of SNPs of the NHEJ pathway genes with susceptibility to HIV-1 infection and AIDS progression among MSM residing in northern China.Results: In the present study, a total of 481 HIV-1 seropositive men and 493 HIV-1 seronegative men were included. And genotyping of 22 SNPs in NHEJ pathway genes was performed using the SNPscanTM Kit. Our results disclosed significant associations of XRCC6 rs132770 and XRCC4 rs1056503 genotypes with susceptibility to HIV-1 infection. The generalized multifactor dimensionality reduction (GMDR) analysis found a significant SNP-SNP interaction between the XRCC6 and XRCC4 variants in the risk of HIV-1 infection. In stratified analysis, the positive effects of XRCC5 rs16855458 and LIG4 rs1805388 on the CD4+ T cell count and clinical phase of disease were validated.Conclusions: Our results confirmed that the NHEJ gene polymorphisms played an important role in HIV-1 infection and AIDS progression in the northern Chinese MSM population.

Inherently confinable split-drive systems in Drosophila

CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.

Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells

Classical nonhomologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but predominates in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 (“Ku”) and XRCC4/Ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Ligase IV are absolutely required for joining RAG1/2-endonucease (“RAG”)-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Ligase IV also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Ligase IV-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially, in association with G1-phase down-regulation of Ligase1. Finally, we suggest that differential impacts of Ku-deficiency versus Lig4-deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-defcient developing lymphocyte and neuronal cells.Significance StatementAlternative end-joining (A-EJ) is implicated in oncogenic translocations and mediating DNA double-strand break (DSB) repair in cycling cells when classical nonhomologous endjoining (C-NHEJ) factors of the C-NHEJ Ligase complex are absent. However, V(D)J recombination-associated DSBs that occur in G1 cell cycle-phase progenitor lymphocytes are joined exclusively by the C-NHEJ pathway. Until now, however, the overall mechanisms that join general DSBs in G1-phase progenitor B cells had not been fully elucidated. Here, we report that Ku, a core C-NHEJ double-strand break recognition complex, directs repair of a variety of different targeted DSBs towards C-NHEJ and suppresses A-EJ in G1-phase cells. We suggest this Ku activity explains how Ku-deficiency can rescue the neuronal development and embryonic lethality phenotype of Ligase 4-deficient mice.

Inherently confinable split-drive systems in Drosophila

CRISPR-based gene drive systems, which copy themselves based on gene conversion mediated by the homology directed repair (HDR) pathway, have potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway that are rendered resistant to Cas9 cleavage can interrupt the spread of genedrive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences to restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles, combined with recessive Mendelian processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that were inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multi-generational cage trials, sGD follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage or lethal/sterile mosaic phenotypes, leading to inherently confineable drive outcomes.

Inhibition of SETMAR–H3K36me2–NHEJ repair axis in residual disease cells prevents glioblastoma recurrence

Background Residual disease of glioblastoma (GBM) causes recurrence. However, targeting residual cells has failed, due to their inaccessibility and our lack of understanding of their survival mechanisms to radiation therapy. Here we deciphered a residual cell–specific survival mechanism essential for GBM relapse. Methods Therapy resistant residual (RR) cells were captured from primary patient samples and cell line models mimicking clinical scenario of radiation resistance. Molecular signaling of resistance in RR cells was identified using RNA sequencing, genetic and pharmacological perturbations, overexpression systems, and molecular and biochemical assays. Findings were validated in patient samples and an orthotopic mouse model. Results RR cells form more aggressive tumors than the parental cells in an orthotopic mouse model. Upon radiation-induced damage, RR cells preferentially activated a nonhomologous end joining (NHEJ) repair pathway, upregulating Ku80 and Artemis while downregulating meiotic recombination 11 (Mre11) at protein but not RNA levels. Mechanistically, RR cells upregulate the Su(var)3-9/enhancer-of-zeste/trithorax (SET) domain and mariner transposase fusion gene (SETMAR), mediating high levels of H3K36me2 and global euchromatization. High H3K36me2 leads to efficiently recruiting NHEJ proteins. Conditional knockdown of SETMAR in RR cells induced irreversible senescence partly mediated by reduced H3K36me2. RR cells expressing mutant H3K36A could not retain Ku80 at double-strand breaks, thus compromising NHEJ repair, leading to apoptosis and abrogation of tumorigenicity in vitro and in vivo. Pharmacological inhibition of the NHEJ pathway phenocopied H3K36 mutation effect, confirming dependency of RR cells on the NHEJ pathway for their survival. Conclusions We demonstrate that the SETMAR-NHEJ regulatory axis is essential for the survival of clinically relevant radiation RR cells, abrogation of which prevents recurrence in GBM.

NHJ-1 Is Required for Canonical Nonhomologous End Joining in Caenorhabditis elegans

DNA double-strand breaks (DSBs) are a particularly lethal form of DNA damage that must be repaired to restore genomic integrity. Canonical nonhomologous end joining (NHEJ), is a widely conserved pathway that detects and directly ligates the broken ends to repair the DSB. These events globally require the two proteins that form the Ku ring complex, Ku70 and Ku80, and the terminal ligase LIG4. While the NHEJ pathway in vertebrates is elaborated by more than a dozen factors of varying conservation and is similarly complex in other eukaryotes, the entire known NHEJ toolkit in Caenorhabditis elegans consists only of the core components CKU-70, CKU-80, and LIG-4. Here, we report the discovery of the first accessory NHEJ factor in C. elegans. Our analysis of the DNA damage response in young larvae revealed that the canonical wild-type N2 strain consisted of two lines that exhibited a differential phenotypic response to ionizing radiation (IR). Following the mapping of the causative locus to a candidate on chromosome V and clustered regularly interspaced short palindromic repeats-Cas9 mutagenesis, we show that disruption of the nhj-1 sequence induces IR sensitivity in the N2 line that previously exhibited IR resistance. Using genetic and cytological analyses, we demonstrate that nhj-1 functions in the NHEJ pathway to repair DSBs. Double mutants of nhj-1 and lig-4 or cku-80 do not exhibit additive IR sensitivity, and the post-IR somatic and fertility phenotypes of nhj-1 mimic those of the other NHEJ factors. Furthermore, in com-1 mutants that permit repair of meiotic DSBs by NHEJ instead of restricting their repair to the homologous recombination pathway, loss of nhj-1 mimics the consequences of loss of lig-4. Diakinesis-stage nuclei in nhj-1; com-1 and nhj-1; lig-4 mutant germlines exhibit increased numbers of DAPI-staining bodies, consistent with increased chromosome fragmentation in the absence of NHEJ-mediated meiotic DSB repair. Finally, we show that NHJ-1 and LIG-4 localize to somatic nuclei in larvae, but are excluded from the germline progenitor cells, consistent with NHEJ being the dominant DNA repair pathway in the soma. nhj-1 shares no sequence homology with other known eukaryotic NHEJ factors and is taxonomically restricted to the Rhabditid family, underscoring the evolutionary plasticity of even highly conserved pathways.