Shan Zha

Lymphocyte-specific compensation for XLF/cernunnos end-joining functions in V(D)J recombination.

Mutations in XLF/Cernunnos (XLF) cause lymphocytopenia in humans, and various studies suggest an XLF role in classical nonhomologous end joining (C-NHEJ). We now find that XLF-deficient mouse embryonic fibroblasts are ionizing radiation (IR) sensitive and severely impaired for ability to support V(D)J recombination. Yet mature lymphocyte numbers in XLF-deficient mice are only modestly decreased. Moreover, XLF-deficient pro-B lines, while IR-sensitive, perform V(D)J recombination at nearly wild-type levels. Correspondingly, XLF/p53-double-deficient mice are not markedly prone to the pro-B lymphomas that occur in previously characterized C-NHEJ/p53-deficient mice; however, like other C-NHEJ/p53-deficient mice, they still develop medulloblastomas. Despite nearly normal V(D)J recombination in developing B cells, XLF-deficient mature B cells are moderately defective for immunoglobulin heavy-chain class switch recombination. Together, our results implicate XLF as a C-NHEJ factor but also indicate that developing mouse lymphocytes harbor cell-type-specific factors/pathways that compensate for the absence of XLF function during V(D)J recombination.

Essential Role for DNA-PKcs in DNA Double-Strand Break Repair and Apoptosis in ATM-Deficient Lymphocytes

The DNA double-strand break (DSB) repair protein DNA-PKcs and the signal transducer ATM are both activated by DNA breaks and phosphorylate similar substrates in vitro, yet appear to have distinct functions in vivo. Here, we show that ATM and DNA-PKcs have overlapping functions in lymphocytes. Ablation of both kinase activities in cells undergoing immunoglobulin class switch recombination leads to a compound defect in switching and a synergistic increase in chromosomal fragmentation, DNA insertions, and translocations due to aberrant processing of DSBs. These abnormalities are attributed to a compound deficiency in phosphorylation of key proteins required for DNA repair, class switching, and cell death. Notably, both kinases are required for normal levels of p53 phosphorylation in B and T cells and p53-dependent apoptosis. Our experiments reveal a DNA-PKcs-dependent pathway that regulates DNA repair and activation of p53 in the absence of ATM.

The BCL11B tumor suppressor is mutated across the major molecular subtypes of T-cell acute lymphoblastic leukemia

The BCL11B transcription factor is required for normal T-cell development, and has recently been implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) induced by TLX overexpression or Atm deficiency. To comprehensively assess the contribution of BCL11B inactivation to human T-ALL, we performed DNA copy number and sequencing analyses of T-ALL diagnostic specimens, revealing monoallelic BCL11B deletions or missense mutations in 9% (n = 10 of 117) of cases. Structural homology modeling revealed that several of the BCL11B mutations disrupted the structure of zinc finger domains required for this transcription factor to bind DNA. BCL11B haploinsufficiency occurred across each of the major molecular subtypes of T-ALL, including early T-cell precursor, HOXA-positive, LEF1-inactivated, and TAL1-positive subtypes, which have differentiation arrest at diverse stages of thymocyte development. Our findings provide compelling evidence that BCL11B is a haploinsufficient tumor suppressor that collaborates with all major T-ALL oncogenic lesions in human thymocyte transformation.

Defective DNA repair and increased genomic instability in Cernunnos-XLF-deficient murine ES cells

Nonhomologous DNA end-joining (NHEJ) is a major pathway of DNA double-strand break (DSB) repair in mammalian cells, and it functions to join both specifically programmed DSBs that occur in the context of V(D)J recombination during early lymphocyte development as well as general DSBs that occur in all cells. Thus, defects in NHEJ impair V(D)J recombination and lead to general genomic instability. In human patients, mutations of Cernunnos-XLF (also called NHEJ1), a recently identified NHEJ factor, underlie certain severe combined immune deficiencies associated with defective V(D)J recombination and radiosensitivity. To characterize Cernunnos-XLF function in mouse cells, we used gene-targeted mutation to delete exons 4 and 5 from both copies of the Cernunnos-XLF gene in ES cell (referred to as CerΔ/Δ ES cells). Analyses of CerΔ/Δ ES cells showed that they produce no readily detectable Cernunnos-XLF protein. Based on transient V(D)J recombination assays, we find that CerΔ/Δ ES cells have dramatic impairments in ability to form both V(D)J coding joins and joins of their flanking recombination signal sequences (RS joins). CerΔ/Δ ES cells are highly sensitive to ionizing radiation and have intrinsic DNA DSB repair defects as measured by pulse field gel electrophoresis. Finally, the Cernunnos-XLF mutations led to increased spontaneous genomic instability, including translocations. We conclude that, in mice, Cernunnos-XLF is essential for normal NHEJ-mediated repair of DNA DSBs and that Cernunnos-XLF acts as a genomic caretaker to prevent genomic instability.

Complementary functions of ATM and H2AX in development and suppression of genomic instability

Upon DNA damage, histone H2AX is phosphorylated by ataxia-telangiectasia mutated (ATM) and other phosphoinositide 3-kinase-related protein kinases. To elucidate further the potential overlapping and unique functions of ATM and H2AX, we asked whether they have synergistic functions in the development and maintenance of genomic stability by inactivating both genes in mouse germ line. Combined ATM/H2AX deficiency caused embryonic lethality and dramatic cellular genomic instability. Mechanistically, severe genomic instability in the double-deficient cells is associated with a requirement for H2AX to repair oxidative DNA damage resulting from ATM deficiency. We discuss these findings in the context of synergies between ATM and other repair factors.

Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway

Non-homologous end joining (NHEJ) is a major pathway to repair DNA double-strand breaks (DSBs), which can display different types of broken ends. However, it is unclear how NHEJ factors organize to repair diverse types of DNA breaks. Here, through systematic analysis of the human NHEJ factor interactome, we identify PAXX as a direct interactor of Ku. The crystal structure of PAXX is similar to those of XRCC4 and XLF. Importantly, PAXX-deficient cells are sensitive to DSB-causing agents. Moreover, epistasis analysis demonstrates that PAXX functions together with XLF in response to ionizing radiation-induced complex DSBs, whereas they function redundantly in response to Topo2 inhibitor-induced simple DSBs. Consistently, PAXX and XLF coordinately promote the ligation of complex but not simple DNA ends in vitro. Altogether, our data identify PAXX as a new NHEJ factor and provide insight regarding the organization of NHEJ factors responding to diverse types of DSB ends.

Robust chromosomal DNA repair via alternative end-joining in the absence of X-ray repair cross-complementing protein 1 (XRCC1)

Classical nonhomologous DNA end-joining (C-NHEJ), which is a major DNA double-strand break (DSB) repair pathway in mammalian cells, plays a dominant role in joining DSBs during Ig heavy chain (IgH) class switch recombination (CSR) in activated B lymphocytes. However, in B cells deficient for one or more requisite C-NHEJ factors, such as DNA ligase 4 (Lig4) or XRCC4, end-joining during CSR occurs by a distinct alternative end-joining (A-EJ) pathway. A-EJ also has been implicated in joining DSBs found in oncogenic chromosomal translocations. DNA ligase 3 (Lig3) and its cofactor XRCC1 are widely considered to be requisite A-EJ factors, based on biochemical studies or extrachromosomal substrate end-joining studies. However, potential roles for these factors in A-EJ of endogenous chromosomal DSBs have not been tested. Here, we report that Xrcc1 inactivation via conditional gene-targeted deletion in WT or XRCC4-deficient primary B cells does not have an impact on either CSR or IgH/c-myc translocations in activated B lymphocytes. Indeed, homozygous deletion of Xrcc1 does not impair A-EJ of I-SceI–induced DSBs in XRCC4-deficient pro–B-cell lines. Correspondingly, substantial depletion of Lig3 in Lig4-deficient primary B cells or B-cell lines does not impair A-EJ of CSR-mediated DSBs or formation of IgH/c-myc translocations. Our findings firmly demonstrate that XRCC1 is not a requisite factor for A-EJ of chromosomal DSBs and raise the possibility that DNA ligase 1 (Lig1) may contribute more to A-EJ than previously considered.

ATM-deficient thymic lymphoma is associated with aberrant tcrd rearrangement and gene amplification

Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor α/δ (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Eα) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5′ boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination–initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Eα. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells.

Mre11: roles in DNA repair beyond homologous recombination.

The Mre11 protein has well-documented functions in the repair of DNA double-strand breaks via homologous recombination. Now, several new studies reveal that Mre11 also has a role in mammalian DNA double-strand break repair by nonhomologous end joining.

Kinase-dead ATM protein causes genomic instability and early embryonic lethality in mice

Expression of a kinase-deficient ATM protein leads to severe genomic instability and embryonic lethality.