Explore the vast range of titles available.

Sickle cell disease and β-thalassemia affect the production of the adult β-hemoglobin chain. The clinical severity is lessened by mutations that cause fetal γ-globin expression in adult life (i.e., the hereditary persistence of fetal hemoglobin). Mutations clustering ~200 nucleotides upstream of the HBG transcriptional start sites either reduce binding of the LRF repressor or recruit the KLF1 activator. Here, we use base editing to generate a variety of mutations in the −200 region of the HBG promoters, including potent combinations of four to eight γ-globin-inducing mutations. Editing of patient hematopoietic stem/progenitor cells is safe, leads to fetal hemoglobin reactivation and rescues the pathological phenotype. Creation of a KLF1 activator binding site is the most potent strategy – even in long-term repopulating hematopoietic stem/progenitor cells. Compared with a Cas9-nuclease approach, base editing avoids the generation of insertions, deletions and large genomic rearrangements and results in higher γ-globin levels. Our results demonstrate that base editing of HBG promoters is a safe, universal strategy for treating β-hemoglobinopathies. Antoniou and colleagues used base editing to generate a variety of mutations inducing γ-globin and rescue the β-hemoglobinopathy phenotype. This strategy was safe and effective in long-term repopulating hematopoietic stem/progenitor cells.

XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain ( Rag2 c/c mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2 c/c XLF −/− p53 −/− mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc / pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly. Antigen receptor diversity relies on careful DNA cleavage and repair. Here the authors identify a functional interplay between RAG2 and XLF during V(D)J recombination, revealing an important role for the RAG complex in repairing induced DNA double-strand breaks and maintaining genome integrity.

We developed an Xrcc4 M61R separation of function mouse line to overcome the embryonic lethality of Xrcc4-deficient mice. XRCC4 M61R protein does not interact with Xlf, thus obliterating XRCC4-Xlf filament formation while preserving the ability to stabilize DNA ligase IV. X4 M61R mice, which are DNA repair deficient, phenocopy the Nhej1-/- (known as Xlf -/-) setting with a minor impact on the development of the adaptive immune system. The core non-homologous end-joining (NHEJ) DNA repair factor XRCC4 is therefore not mandatory for V(D)J recombination aside from its role in stabilizing DNA ligase IV. In contrast, Xrcc4 M61R mice crossed on Paxx-/- , Nhej1-/- , or Atm -/- backgrounds are severely immunocompromised, owing to aborted V(D)J recombination as in Xlf-Paxx and Xlf-Atm double Knock Out (DKO) settings. Furthermore, massive apoptosis of post-mitotic neurons causes embryonic lethality of Xrcc4 M61R -Nhej1-/- double mutants. These in vivo results reveal new functional interplays between XRCC4 and PAXX, ATM and Xlf in mouse development and provide new insights into the understanding of the clinical manifestations of human XRCC4 -deficient condition, in particular its absence of immune deficiency.

The repair of DNA double-stranded breaks (DNAdsb) through non-homologous end joining (NHEJ) is a prerequisite for the proper development of the central nervous system and the adaptive immune system. Yet, mice with Xlf or PAXX loss of function are viable and present with very mild immune phenotypes, although their lymphoid cells are sensitive to ionizing radiation attesting for the role of these factors in NHEJ. In contrast, we show here that mice defective for both Xlf and PAXX are embryonically lethal owing to a massive apoptosis of post-mitotic neurons, a situation reminiscent to XRCC4 or DNA Ligase IV KO conditions. The development of the adaptive immune system in Xlf −/− PAXX −/− E18.5 embryos is severely affected with the block of B- and T-cell maturation at the stage of IgH and TCR β gene rearrangements, respectively. This damaging phenotype highlights the functional nexus between Xlf and PAXX, which is critical for the completion of NHEJ-dependent mechanisms during mouse development.

Biallelic NF2 gene inactivation is frequently found in human malignant mesothelioma. In order to assess whether NF2 hemizygosity may enhance susceptibility to asbestos fibres, we investigated the Nf2 status in mesothelioma developed in mice presenting a heterozygous mutation of the Nf2 gene ( Nf2 KO3/+ ), after intraperitoneal inoculation of crocidolite fibres. Asbestos-exposed Nf2 KO3/+ mice developed tumoural ascites and mesothelioma at a higher frequency than their wild-type (WT) counterparts ( P <0.05). Six out of seven mesothelioma cell lines established from neoplastic ascitic fluids of Nf2 KO3/+ mice exhibited loss of the WT Nf2 allele and no neurofibromatosis type 2 protein expression was found in these cells. The results show the importance of the NF2 gene in mesothelial oncogenesis, the potential association of asbestos exposure and tumour suppressor gene inactivation, and suggest that NF2 gene mutation may be a susceptibility factor to asbestos.

Previously, we have mimicked human neurofibromatosis type 2 (NF2) in conditional Nf2 mutant (P0 Cre ; Nf2 flox2/flox2 ) mice. Schwannomas, characteristic for NF2, were found at low frequency in older mice. Here, we report that these mice, upon additional hemizygosity for p53 , rapidly develop multiple tumours showing features consistent with malignant peripheral nerve sheath tumours. Thus, p53 hemizygosity promotes tumorigenesis of mutant Nf2 peripheral nerve cells. In contrast, young P0 Cre ; Nf2 flox2/+ ; p53 +/− cis mice mainly succumb to Nf2/p53 -related osteogenic tumours. Therefore, Cre-mediated early biallelic loss of Nf2 function in neural crest-derived cells hemizygous for p53 results in resistance to osteogenic tumours and increased susceptibility to peripheral nerve sheath tumours.