Elevated inflammatory responses and targeted therapeutic intervention in a preclinical mouse model of ataxia-telangiectasia lung disease.

Ataxia-telangiectasia (A-T) is an autosomal recessive, multisystem disorder characterized by cerebellar degeneration, cancer predisposition, and immune system defects. A major cause of mortality in A-T patients is severe pulmonary disease; however, the underlying causes of the lung complications are poorly understood, and there are currently no curative therapeutic interventions. In this study, we examined the lung phenotypes caused by ATM-deficient immune cells using a mouse model of A-T pulmonary disease. In response to acute lung injury, ATM-deficiency causes decreased survival, reduced blood oxygen saturation, elevated neutrophil recruitment, exaggerated and prolonged inflammatory responses and excessive lung injury compared to controls. We found that ATM null bone marrow adoptively transferred to WT recipients induces similar phenotypes that culminate in impaired lung function. Moreover, we demonstrated that activated ATM-deficient macrophages exhibit significantly elevated production of harmful reactive oxygen and nitrogen species and pro-inflammatory cytokines. These findings indicate that ATM-deficient immune cells play major roles in causing the lung pathologies in A-T. Based on these results, we examined the impact of inhibiting the aberrant inflammatory responses caused by ATM-deficiency with reparixin, a CXCR1/CXCR2 chemokine receptor antagonist. We demonstrated that reparixin treatment reduces neutrophil recruitment, edema and tissue damage in ATM mutant lungs. Thus, our findings indicate that targeted inhibition of CXCR1/CXCR2 attenuates pulmonary phenotypes caused by ATM-deficiency and suggest that this treatment approach represents a viable therapeutic strategy for A-T lung disease.

Reversible mislocalization of a disease-associated MRE11 splice variant product.

Ataxia-telangiectasia (AT) and related disorders feature cancer predisposition, neurodegeneration, and immunodeficiency resulting from failure to respond to DNA damage. Hypomorphic mutations in MRE11 cause an AT-like disorder (ATLD) with variable clinical presentation. We have sought to understand how diverse MRE11 mutations may provide unique therapeutic opportunities, and potentially correlate with clinical variability. Here we have undertaken studies of an MRE11 splice site mutation that was found in two ATLD siblings that died of pulmonary adenocarcinoma at the young ages of 9 and 16. The mutation, termed MRE11 alternative splice mutation (MRE11ASM), causes skipping of a highly conserved exon while preserving the protein's open reading frame. A new mouse model expressing Mre11ASM from the endogenous locus demonstrates that the protein is present at very low levels, a feature in common with the MRE11ATLD1 mutant found in other patients. However, the mechanisms causing low protein levels are distinct. MRE11ASM is mislocalized to the cytoplasm, in contrast to MRE11ATLD1, which remains nuclear. Strikingly, MRE11ASM mislocalization is corrected by inhibition of the proteasome, implying that the protein undergoes strict protein quality control in the nucleus. These findings raise the prospect that inhibition of poorly understood nuclear protein quality control mechanisms might have therapeutic benefit in genetic disorders causing cytoplasmic mislocalization.

Correction: Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells.

[This corrects the article DOI: 10.1371/journal.pgen.1006410.].

Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells.

A proportion of homologous recombination (HR) events in mammalian cells resolve by "long tract" gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer.

Recruitment and activation of the ATM kinase in the absence of DNA-damage sensors.

Two kinases, ATM and DNA-PKcs, control rapid responses to DNA double-strand breaks (DSBs). The paradigm for ATM control is recruitment and activation by the Mre11-Rad50-NBS1 (MRN) sensor complex, whereas DNA-PKcs requires the sensor Ku (Ku70-Ku80). Using mouse cells containing targeted mutant alleles of Mre11 (Mre11a) and/or Ku70 (Xrcc6), together with pharmacologic kinase inhibition, we demonstrate that ATM can be activated by DSBs in the absence of MRN. When MRN is deficient, DNA-PKcs efficiently substitutes for ATM in facilitating local chromatin responses. In the absence of both MRN and Ku, ATM is recruited to chromatin, where it phosphorylates H2AX and triggers the G2-M cell-cycle checkpoint, but the DNA-repair functions of MRN are not restored. These results suggest that, in contrast to straightforward recruitment and activation by MRN, a complex interplay between sensors has a substantial role in ATM control.

Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks.

Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.

Cell cycle-dependent induction of homologous recombination by a tightly regulated I-SceI fusion protein.

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase.

Mechanisms of double-strand break repair in somatic mammalian cells.

DNA chromosomal DSBs (double-strand breaks) are potentially hazardous DNA lesions, and their accurate repair is essential for the successful maintenance and propagation of genetic information. Two major pathways have evolved to repair DSBs: HR (homologous recombination) and NHEJ (non-homologous end-joining). Depending on the context in which the break is encountered, HR and NHEJ may either compete or co-operate to fix DSBs in eukaryotic cells. Defects in either pathway are strongly associated with human disease, including immunodeficiency and cancer predisposition. Here we review the current knowledge of how NHEJ and HR are controlled in somatic mammalian cells, and discuss the role of the chromatin context in regulating each pathway. We also review evidence for both co-operation and competition between the two pathways.

XRCC2 and XRCC3 regulate the balance between short- and long-tract gene conversions between sister chromatids.

Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DNA lesions associated with replication and is thought to be important for suppressing genomic instability. The mechanisms regulating the initiation and termination of SCR in mammalian cells are poorly understood. Previous work has implicated all the Rad51 paralogs in the initiation of gene conversion and the Rad51C/XRCC3 complex in its termination. Here, we show that hamster cells deficient in the Rad51 paralog XRCC2, a component of the Rad51B/Rad51C/Rad51D/XRCC2 complex, reveal a bias in favor of long-tract gene conversion (LTGC) during SCR. This defect is corrected by expression of wild-type XRCC2 and also by XRCC2 mutants defective in ATP binding and hydrolysis. In contrast, XRCC3-mediated homologous recombination and suppression of LTGC are dependent on ATP binding and hydrolysis. These results reveal an unexpectedly general role for Rad51 paralogs in the control of the termination of gene conversion between sister chromatids.

SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging.

Genomic instability and alterations in gene expression are hallmarks of eukaryotic aging. The yeast histone deacetylase Sir2 silences transcription and stabilizes repetitive DNA, but during aging or in response to a DNA break, the Sir complex relocalizes to sites of genomic instability, resulting in the desilencing of genes that cause sterility, a characteristic of yeast aging. Using embryonic stem cells, we show that mammalian Sir2, SIRT1, represses repetitive DNA and a functionally diverse set of genes across the mouse genome. In response to DNA damage, SIRT1 dissociates from these loci and relocalizes to DNA breaks to promote repair, resulting in transcriptional changes that parallel those in the aging mouse brain. Increased SIRT1 expression promotes survival in a mouse model of genomic instability and suppresses age-dependent transcriptional changes. Thus, DNA damage-induced redistribution of SIRT1 and other chromatin-modifying proteins may be a conserved mechanism of aging in eukaryotes.

Distinct roles of chromatin-associated proteins MDC1 and 53BP1 in mammalian double-strand break repair.

Phosphorylated histone H2AX ("gamma-H2AX") recruits MDC1, 53BP1, and BRCA1 to chromatin near a double-strand break (DSB) and facilitates efficient repair of the break. It is unclear to what extent gamma-H2AX-associated proteins act in concert and to what extent their functions within gamma-H2AX chromatin are distinct. We addressed this question by comparing the mechanisms of action of MDC1 and 53BP1 in DSB repair (DSBR). We find that MDC1 functions primarily in homologous recombination/sister chromatid recombination, in a manner strictly dependent upon its ability to interact with gamma-H2AX but, unexpectedly, not requiring recruitment of 53BP1 or BRCA1 to gamma-H2AX chromatin. In contrast, 53BP1 functions in XRCC4-dependent nonhomologous end-joining, likely mediated by its interaction with dimethylated lysine 20 of histone H4 but, surprisingly, independent of H2AX. These results suggest a specialized adaptation of the "histone code" in which distinct histone tail-protein interactions promote engagement of distinct DSBR pathways.