XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.
The DNA-end-joining reactions used for repair of double-strand breaks in DNA and for V(D)J recombination, the process by which immunoglobulin and T-cell antigen-receptor genes are assembled from multiple gene segments, use common factors. These factors include components of DNA-dependent protein kinase (DNA-PK), namely DNA-PKcs and the Ku heterodimer, Ku70-Ku80, and XRCC4. The precise function of XRCC4 is unknown, but it interacts with DNA ligase IV. Ligase IV is one of the three known mammalian DNA ligases; however, the in vivo functions of these ligases have not been determined unequivocally. Here we show that inactivation of the ligase IV gene in mice leads to late embryonic lethality. Lymphopoiesis in these mice is blocked and V(D)J joining does not occur. Ligase IV-deficient embryonic fibroblasts also show marked sensitivity to ionizing radiation, growth defects and premature senescence. All of these phenotypic characteristics, except embryonic lethality, resemble those associated with Ku70 and Ku80 deficiencies, indicating that they may result from an impaired end-joining process that involves both Ku subunits and ligase IV. However, Ku-deficient mice are viable, so ligase IV must also be required for processes and/or in cell types in which Ku is dispensable.
Ku70, Ku80, and DNA-PKcs are subunits of the DNA-dependent protein kinase (DNA-PK), an enzyme implicated in DNA double-stranded break repair and V(D)J recombination. Our Ku70-deficient mice were about 50% the size of control littermates, and their fibroblasts were ionizing radiation sensitive and displayed premature senescence associated with the accumulation of nondividing cells. Ku70-deficient mice lacked mature B cells or serum immunoglobulin but, unexpectedly, reproducibly developed small populations of thymic and peripheral alpha/beta T lineage cells and had a significant incidence of thymic lymphomas. In association with B and T cell developmental defects, Ku70-deficient cells were severely impaired for joining of V(D)J coding and recombination signal sequences. These unanticipated features of the Ku70-deficient phenotype with respect to lymphocyte development and V(D)J recombination may reflect differential functions of the three DNA-PK components.
We generated mice in which a functional RAG2:GFP fusion gene is knocked in to the endogenous RAG2 locus. In bone marrow and thymus, RAG2:GFP expression occurs in appropriate stages of developing B and T cells as well as in immature bone marrow IgM+ B cells. RAG2:GFP also is expressed in IgD+ B cells following cross-linking of IgM on immature IgM+ IgD+ B cells generated in vitro. RAG2:GFP expression is undetectable in most immature splenic B cells; however, in young RAG2:GFP mice, there are substantial numbers of splenic RAG2:GFP+ cells that mostly resemble pre-B cells. The latter population decreases in size with age but reappears following immunization of older RAG2:GFP mice. We discuss the implications of these findings for current models of receptor assembly and diversification.
The Ig heavy chain (IgH) constant region (C H ) genes are organized from 5 to 3 in the order C, C␦, C␥3, C␥1, C␥2b, C␥2a, C, and C␣. Expression of C H genes downstream of C␦ involves class-switch recombination (CSR), a process that is targeted by germ-line transcription (GT) of the corresponding C H gene. Previously, we demonstrated that insertion of a PGK-neo r cassette at two sites downstream of C␣ inhibits, in cultured B cells, GT of and CSR to a subset of C H genes (including C␥3, C␥2a, C␥2b, and C) that lie as far as 120 kb upstream. Here we show that insertion of the PGK-neo r cassette in place of sequences in the I␥2b locus inhibits GT of and CSR to the upstream C␥3 gene, but has no major effect on the downstream C␥2a and C genes. Moreover, replacement of the C exons with a PGK-neo r cassette in the opposite transcriptional orientation also inhibits, in culture, GT of and CSR to the upstream C␥3, C␥2b, and C␥2a genes. As with the PGK-neo r insertions 3 of C␣ studied previously, the C␥1 and C␣ genes were less affected by these mutations both in culture and in mice, whereas the C␥2b gene appeared less affected in vivo. Our findings support the existence of a long-range 3 IgH regulatory region required for GT of and CSR to multiple C H genes and suggest that PGK-neo r cassette insertion into the locus short circuits the ability of this region to facilitate GT of dependent C H genes upstream of the insertion.Ig variable regions are encoded by component gene segments that are assembled during early B cell differentiation by V(D)J recombination (reviewed in ref.
Thrombosis can lead to life-threatening conditions such as acute myocardial infarction, pulmonary embolism, and stroke. Although commonly used anti-coagulant drugs, such as low molecular weight heparin and warfarin, are effective, they carry a significant risk of inducing severe bleeding complications, and there is a need for safer drugs. Activated Factor XI (FXIa) is a key enzyme in the amplification phase of the coagulation cascade. Anti-human FXI antibody significantly reduces thrombus growth in a baboon thrombosis model without bleeding problems (Gruber, A., and Hanson, S. R. (2003) Blood 102, 953-955). Therefore, FXIa is a potential target for anti-thrombosis therapy. To determine the structure of FXIa, we derived a recombinant catalytic domain of FXI, consisting of residues 370 -607 (rhFXI 370 -607 ). Here we report the first crystal structure of rhFXI 370 -607 in complex with a substitution mutant of ecotin, a panserine protease protein inhibitor secreted by Escherichia coli, to 2.2 Å resolution. The presence of ecotin not only assisted in the crystallization of the enzyme but also revealed unique structural features in the active site of FXIa. Subsequently, the sequence from P5 to P2 in ecotin was mutated to the FXIa substrate sequence, and the structures of the rhFXI 370 -607 -ecotin mutant complexes were determined. These structures provide us with an understanding of substrate binding interactions of FXIa, the structural information essential for the structure-based design of FXIa-selective inhibitors.
Tumor necrosis factor ␣ (TNF␣) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNF␣, interleukin (IL)-1, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNF␣ expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS-and IL-1-induced TNF␣ production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E 2 , and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNF␣ and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.
Single-cell PCR analyses of expressed Ig H and L chain sequences presented here show that certain rearrangements occur repeatedly and account for a major segment of the well-studied repertoire of B-1 cell autoantibodies that mediate the lysis of bromelain-treated mouse erythrocytes, i.e. antibodies reactive with phosphatldyicholine (PtC). We repeatedly isolated at least 10 different types of VH region rearrangements, involving three distinct germline genes, among FACS-sorted PtC-binding B-1 cells from three strains of mice (C57BL/6J, BALB/c and C.B-17). The predominant rearrangement, VH11-DSP-JH1 (VH11 type 1), has been previously found in anti-PtC hybridomas in several studies. We show that within each of six mice from two strains (C57BL/6J and BALB/c), unique instances of IgH/IgL pairing arose either from different B cell progenitors prior to IgH rearrangement or from pre-B cells which expanded after IgH rearrangement but prior to IgL rearrangement. Together with other recurrent rearrangements described here, our findings demonstrate that clonal expansion of mature B cells cannot account for all repeated rearrangements. As suggested by initial studies of dominant idiotype expression, these findings confirm that clonal expansion is only one of the mechanisms contributing to the establishment of recurrent rearrangements.
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