Genetic interaction between the non-homologous end joining factors during B and T lymphocyte development: in vivo mouse models

Non-homologous end joining (NHEJ) is the main DNA repair mechanism for the repair of double strand breaks (DSBs) throughout the course of the cell cycle. DSBs are generated in developing B and T lymphocytes during V(D)J recombination to increase the repertoire of B and T cell receptors. DSBs are also generated during the class switch recombination (CSR) process in mature B lymphocytes, providing distinct effector functions of antibody heavy chain constant regions. Thus, NHEJ is important for both V(D)J recombination and CSR. NHEJ comprises core Ku70 and Ku80 subunits that form the Ku heterodimer, which binds DSBs and promotes the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, PAXX, Mri) and downstream core factors (XLF, Lig4 and XRCC4). In recent decades, new NHEJ proteins have been reported, increasing complexity of this molecular pathway. Numerous in vivo mouse models have been generated and characterized to identify the interplay of NHEJ factors and their role in development of adaptive immune system. This review summarizes the currently available mouse models lacking one or several NHEJ factors, with a particular focus on early B cell development. We also underline genetic interactions and redundancy in the NHEJ pathway in mice.


Introduction
DNA double strand breaks (DSBs) are generated both extrinsically, e.g., by chemotherapeutic agents; and physiologically, e.g., during V(D)J recombination in developing B and T lymphocytes, and class switch recombination (CSR) in activated mature B cells [1,2].
The DNA damage response (DDR) pathway is initiated upon the induction of DSBs. Ataxia telangiectasia mutated (ATM) is a DDR regulator protein kinase that phosphorylates multiple substrates in response to the DSBs, including histone H2AX, modulator of DNA damage checkpoint 1 (MDC1), and p53-binding protein 1 (53BP1). Phosphorylated H2AX facilitates the recruitment of MDC1, following the activation of really interesting protein (RING) finger 8 (RNF8) and RNF168, which are ubiquitin ligases. Phosphorylated and ubiquitinated H2AX facilitates recruitment of 53BP1, which in turn mediates recruitment of RIF1 and interacts with Rev7. The Shieldin complex also promotes DNA repair [1,3]. Accumulation of DSBs results in ATM-dependent activation of 3 of 12 nucleotides prior to ligation of DNA ends during V(D)J recombination [29,30]. Proteins such as nipped-B-like protein (NIPBL) and breast cancer 1 (BRCA1) have also been shown to play a role in NHEJ [31,32].

Non-Homologous End Joining-Deficient Mice
Before transgenic mice became popular, an inbred strain of immunodeficient mice with severe combined immunodeficiency (SCID) was identified [33]. First characterized by Bosma et al., SCID mice carry a recessive mutation in the Dna-pkcs gene, which results in impaired V(D)J recombination and a subsequent lack of mature B and T lymphocytes in homozygous mice [33]. Later, transgenic mouse models deficient for Dna-pkcs gene were generated by several groups [13][14][15][16]. DNA-PKcs-deficient mice (Dna-pkcs -/-) are live-born and possess a SCID phenotype due to inefficient coding-end (CE) joining during the V(D)J recombination [13] (Figure 1). Artemis -/-mice have also been observed to exhibit a SCID phenotype due to lack of CE joining [17]. Thus, both DNA-PKcs and Artemis are required for processing of RAG-mediated hairpin-sealed DNA ends (CEs) during V(D)J recombination, although repair of blunt signal ends (SEs) remains efficient in mice lacking Artemis [17] or DNA-PKcs [13][14][15]. Inactivation of Ku70 [11] or Ku80 [10] in mice results in reduced body weight and a SCID phenotype. Lack of B and T lymphocytes in Ku70 -/-and Ku80 -/-mice is explained by inefficient joining of both RAG-induced blunt SEs and hairpin-sealed CEs [10,11].
In contrast, inactivation of Lig4 [8] or Xrcc4 [9] results in embryonic lethality in mice, presenting challenges for in vivo studies. However, cell studies show that inactivation of Lig4 or Xrcc4 led to inefficient joining of both SEs and CEs, resembling Ku-deficient phenotypes in mice. This suggests that such in vivo models would yield an immunodeficient animal due to ablated V(D)J recombination and lack of B and T cells, if one could be generated [8,9]. Several single-deficient mouse models initially suggested that XLF, PAXX and Mri are dispensable for the V(D)J recombination. Particularly, mice lacking XLF/Cernunnos [34,35] possess both mature B and T cells, despite being characterized by modest lymphocytopenia, and reduced repertoires of B cell receptors (BCRs) and TCRs [34,35]. Xlf -/-lymphocytes support efficient V(D)J recombination in vitro, including both SE and CE repair [34,35] Genetic interaction No genetic interaction Single deficiency [18,19,21,36] or Mri [22,23] possess normal counts of mature B and T cells, efficiently supporting both SE and CE repair during the V(D)J recombination. However, more complex mouse models have revealed that XLF, PAXX and Mri are required for V(D)J recombination, although their functions are compensated by each other and additional proteins due to the extensive genetic interaction inside the NHEJ pathway, as well as interaction between the NHEJ and DDR pathways [1,37].
Furthermore, Xlf -/-Mri -/-double knockout mice are embryonic lethal [23], but can be rescued by inactivation of one or two alleles of Trp53 [42]. Mice lacking both XLF and Mri are characterized by leaky SCID with nearly no mature B and T lymphocytes due to the V(D)J recombination defect [23]. In particular, the lymphocytes lacking both XLF and Mri are unable to efficiently ligate both RAGmediated SEs and CEs in vitro [23]. Both the double-deficient mouse model lacking XLF and PAXX, as well as the model lacking XLF and Mri, are characterized by leaky SCID with low but detectable levels of mature B and T lymphocytes [42]. This phenotype is likely possible due to residual NHEJ activity in developing Xlf -/-Paxx -/-and Xlf -/-Mri -/-lymphocytes in vivo [39,42].

Class swtich recombination
Class switch recombination (CSR) occurs in mature B cells following the efficient V(D)J recombination in vivo. However, several experimental models allow for the by-pass of V(D)J recombination to determine the impact of specific factors on CSR, even though these factors are required for earlier stages of B cell development (e.g., Artemis, DNA-PKcs). Knocking-in preassembled heavy and light chains of the immunoglobulin gene ("HL") allows for the development of mature B cells in mice that otherwise lack the capacity for V(D)J recombination [49]. For example, lack of Artemis or DNA-PKcs [49] moderately affects the CSR in mature B cells. Additionally, it was found that CSR levels were reduced 2-3 fold in cells lacking XLF [34,35], Ku70 [44], Ku80 [44], Lig4 [28], or XRCC4 [28]. PAXX seems to be dispensable for CSR in wild-type cells [18,19,21,36,50], while inactivation of Mri results in modest CSR defects [22,23,42].

Genetic interacion between Xlf and Rag
Xlf has also been shown to genetically interact with Rag2 [58]. Mutation in the Rag2 gene results in the truncated protein "core Rag 2", which continues to support DSB formation and DNA repair in developing B and T lymphocytes. However, in XLF-deficient cells, this "core Rag2" activity is lost, and V(D)J recombination does not proceed. This finding suggests a potential role for RAG in both the induction of DSBs and DNA repair, as the RAG complex supports tethering of DNA ends before ligation [58] (Figure 2).
Hence, deficiencies in NHEJ often result in neuronal apoptosis, likely due to accumulation of DSBs in post-mitotic neurons. Inactivation of p53 prevents neuronal apoptosis, for example, by allowing A-EJ to repair DSBs in NHEJ-deficient cells.

NHEJ in mouse and human
Mutations in several NHEJ genes have been identified in humans [61,62]. For instance, patients with mutations in XLF, DNA-PKCS/PRKDC and LIG4 genes display severe clinical features, characterized mainly by SCID, delayed growth and neurological abnormalities [61,62]. In mice, XLF deficiencies lead to a modest lymphocytopenia and defect in CSR [34,35]; DNA-PKcs-deficiencies lead to a SCID phenotype but no neural complications [59]; and Lig4-deficiencies are embryonic lethal [8]. In the same manner, ARTEMIS/DCLRE1C-deficient patients are characterized by SCID but not neurological defects [62]; similar to Artemis-deficient mice [17]. On the other hand, Xrcc4 -/-mice are embryonic lethal [9], unlike XRCC4-deficient patients who only display neurological problems [63]. Mutations in several NHEJ genes have not yet been found in human immunodeficient patients up to the present. These genes include Ku70, Ku80, PAXX and MRI. Ku70 and Ku80 might be essential in human cells, and therefore mutations in KU70 and KU80 genes might be identified only by analyzing embryonic samples. However, mutations in accessory factor genes PAXX and MRI might present without clinical features, based on the knowledge we have obtained from mouse models [18,19,[21][22][23]36]. In the latter case, XLF might compensate for deficiencies in PAXX and Mri in human cells. Sometimes, dramatic differences in phenotypic presentation between mice and humans lacking the same NHEJ factor can be explained, for example, by minor sequence changes between species, resulting in significant changes in protein-protein and protein-DNA interactions, and inability for other factors to compensate the protein loss.

Potential reasons for genetic interactions between the NHEJ factors
There are several types of genetic interaction between the DNA repair factors and several potential explanations for them, although detailed mechanisms have not been elucidated yet.
Why does inactivation of Ku70 [11] or Ku80 [10] result in viable mice, while inactivation of Lig4 [8] or Xrcc4 [9] results in embryonic lethality? This cannot simply be due to lack of NHEJ activity, because cells lacking any of these factors are characterized by similar genomic instability [38,40,57]. The Ku70/Ku80 complex seems to be toxic for the cells when the NHEJ pathway is blocked due to defects in downstream factors. It is possible that Ku may block access to DSB sites from other DNA repair pathway proteins, preventing DNA ligation and eventually resulting in the accumulation of DSBs, activation of p53 and apoptosis. Interestingly, Ku-deficient cells rely on other DNA repair pathways, such as homologous recombination and alternative end-joining. This could also explain why inactivation of Ku70 or Ku80 rescues embryonic lethality of Lig4 -/-mice [43,46]. Similarly, lack of Ku rescues embryonic lethality in mice lacking XLF/DNA-PKcs [38,40], as well as in mice with an inactivating DNA-PKcs point mutation [60]. Following the same logic, one can predict that inactivation of Ku70 or Ku80 would also have the ability to rescue synthetic lethality between Xlf and Paxx, and between Xlf and Mri. We can also predict that inactivation of all NHEJ genes in a mouse would result in a phenotype similar to those of Ku70 -/-or Ku80 -/-mice [22], and suggest that all NHEJ genes function in a purely Ku-dependent manner.
Synthetic lethality between Xlf and Dna-pkcs [38][39][40], Xlf and Paxx [18,19,36,39,42] and Xlf and Mri [23,42] results in phenotypes similar to Lig4 -/-and XRCC4 -/-. In all these cases, it is likely that the DNA ligation step is impaired, while Ku70/Ku80 remains functional. There are several potential explanations for the functional redundancy observed between XLF and other factors in NHEJ and DDR [1,38,54,56]. First, XLF and the second factor could have identical functions, such as having a role in stabilizing the DNA repair complex. A second explanation could be that XLF and the second factor could have purely complementary functions; for example, one protein stimulates DNA ligation while another one is required for DNA end tethering. However, the question of XLF's functionally redundancy with so many other factors [1,18,19,23,36,[38][39][40]42,54] is still an enigma in the field of DNA repair.
ATM and DNA-PKcs are both protein kinases. Synthetic lethality between Atm and Dna-pkcs [46] is reasonable to predict because these two proteins can partially compensate for each other's activity when one is inhibited, but no other protein can compensate combined ATM/DNA-PKcs deficiency [52,53,64]. DNA-PKcs is part of the DNA-PK holoenzyme, which includes Ku70 and Ku80. Both Ku70 and Ku80 are synthetic lethal with Atm [46], meaning that ATM is functionally redundant with the Ku70/Ku80/DNA-PKcs complex, and DNA-PKcs will likely be inactive in cells lacking Ku70 or Ku80.

Conclusion
Overall, there are complex genetic interactions between the genes of the NHEJ pathway, and between NHEJ and DDR factors. Genetically modified mouse models and murine cell lines have helped to uncover specific functions of DNA repair factors previously hidden due to the functional redundancy. Further studies will uncover additional genetic interactions between the DNA repair factors and pathways. Only a portion of genetic interaction is analyzed today, and empty cells represent potential future studies (Figures 1 and 2).
Author Contributions: SCZ and VO wrote the first draft of the paper. All the authors contributed to the final manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.