Mechanisms underlying the function of XLF in DNA double-strand break repair

Double strand break DNA repair pathway maintains the genomic stability of our cells, which is challenged by external and internal, physical and chemical factors. When DNA repair genes are mutated, correspondent factors and molecular pathways cannot function properly. It causes a variety of disorders, ranging from immunodeficiency with low count or lack of mature lymphocytes, abnormalities in the nervous system, and tumors. XLF is the DNA repair factor, and mutations in the XLF gene lead to immunodeficiency and brain abnormalities in humans. Mechanisms underlying the function of XLF are not clear yet, and we proposed the multidisciplinary approach to elucidate the role of XLF in DNA repair and in protection from disorders. Recently, we found that the function of XLF is partially compensated by other DNA repair factors, DNA-PKcs, Paxx, and Mri/Cyren. During 2016-2019 (this project), we developed and characterized two new mouse models lacking either PAXX or Mri. Combined deficiency for XLF and PAXX, or XLF and Mri, results in synthetic lethality in mice due to defect in neurodevelopment. Inactivation of p53 rescued synthetic lethality between XLF and PAXX. These data are partially published in international journals, DNA repair, FEBS open bio, and Biomolecules. Overall, our findings suggest several new candidate genes for the prognosis of immunodeficiency in human.

Primary immunodeficiency needs to be rapidly diagnosed and treated in children, otherwise, it can result in death. To determine the impact of new DNA repair factors on immune system development, we generated, characterized and published two mouse models, and multiple genetically modified cell lines. These models are available for researchers worldwide, e.g. mice lacking PAXX or MRI have been transferred to Oslo. Our results established roles for several DNA repair factors, including PAXX, Mri, MDC1, Gcn5, and PCAF in lymphocyte development. This knowledge can be translated to clinics for a more precise diagnosis of primary immunodeficiency. Moreover, one PhD candidate and ten MSc students were trained during this project. This positively influences society by providing experts in immunology and biomedical research.

In this project, I will use a multi-disciplinary approach combining cell biology, mouse genetics and proteomics to elucidate new aspects of the non-homologous DNA end joining (NHEJ) pathway. NHEJ repairs DNA double strand breaks (DSBs) to maintain genome stability and defects in the pathway cause immunodeficiency and neurological diseases. The NHEJ pathway includes Ku70/Ku80 (Ku), DNA-PKcs kinase, and XRCC4/Ligase4 complex that ligates two DSB ends together. XRCC4-like factor (XLF) is a protein that directly associates with XRCC4. Deficiency of XLF leads to genomic instability and results in microcephaly and immunodeficiency in humans. However, the exact function of XLF is unclear. Deficiencies of Ku, XRCC4, and Ligase4 abrogate NHEJ, while inactivation of XLF or DNA-PKcs leads to modest defects in DSB repair. Mice deficient in either XLF or DNA-PKcs are alive and show mild NHEJ defects; surprisingly, combined deficiency of XLF and DNA-PKcs leads to perinatal lethality associated with a complete loss of NHEJ. My preliminary data indicate that deletion of Ku completely rescues the lifespan of these double deficient mice, pointing to a toxic effect of Ku in the absence of both XLF and DNA-PKcs. To explain this remarkable genetic rescue, I propose that in the absence of DNA-PKcs, XLF associates with other proteins and facilitates the removal of Ku from DSBs during DNA repair. To test this hypothesis, I will investigate the role of XLF in recruitment of Ku70 and Ligase4 to DSB sites with live-cell imaging and perform quantitative proteomics to identify XLF-associated proteins. Finally, I will use mouse genetics to determine whether the perinatal lethality of XLF/DNA-PKcs mice is a consequence of p53-dependent apoptosis and perform histopathology analyses to elucidate previously underappreciated roles for DNA-PKcs and XLF in mouse development. The scientific understanding of XLF function can lead to new therapeutic strategies as well as prognostic markers for disease