Modifications of DNA double stand break repair genes and risk of neurological and immune system disorders
As an initial part of the project, we generated several models to study impact of modifications of DNA repair genes on human health. In particular, we developed several new human and mouse cell lines, as well as new mouse models, when one or several genes are inactivated, and accumulated first results. During the first year of the project period, we largely developed aims 1-2 of the proposal and initiated aims 3-4. In particular, first in the world, we generated a collection of human cell lines (HAP1), where one or several DNA repair genes are inactivated. We used CRISPR/Cas9 technology to inactivate DNA repair genes outlined in the project, which include XLF, PAXX, DNA-PKcs, XRCC4 and DNA Ligase 4. In human, mutations in these genes correlate with immunodeficiency, neurological defects and cancer. We accumulated preliminary results on the ability of the cells to resist DNA damage, to maintain self-renewal potential and to maintain genomic stability. We goal to further study impact of the DNA repair genes in these cells to understand regulation of the cell cycle, as this parameter correlates with human syndromes such as immunodeficiency and cancer. Also, as a high priority, we plan to determine the mechanism leading to disease when one or several genes are not functioning properly. In addition, using CRISPR/Cas9 technology, we inactivated genes in mouse lymphocytes to study impact of the XLF and PAXX on immune system development. In particular, we generated PAXX- and XLF-deficient mature B lymphocyte cell lines, using mouse CH12F3 cell line as a starting point. We found that inactivation of PAXX gene has no effect on Class Switch Recombination and production of immunoglobulin A. We will also investigate the role of XLF and PAXX on the maturation of immunoglobuline and development of B and T lymphocytes. Finally, we generated a mouse model deficient for DNA repair protein PAXX. This model allows us to determine the role of PAXX protein in many systems, ranging from development of immune and nervous systems, cancer suppression, to development of behavior and memory. Using this new mouse model, we plan to determine effect of PAXX on mouse growth and development in general. To investigate impact of PAXX on development of immune system, we will estimate number and proportion of B lymphocytes and progenitors in bone marrow (pre-pro-B, pro-B, pre-B, immature B and mature B lymphocytes), spleen (mature B cells) and blood (mature B cells). We will also estimate number and proportion of developing and mature T lymphocytes in spleen, blood and thymus. We will investigate development of nervous system in PAXX-deficient mice using, at first, histopathology analyses to find proportion of live and dead neurons in brain. We will compare ability of PAXX-deficient mice and wild type mice to develop memory and standard behavior functions. A group of PAXX-deficient mice will be investigated in order to determine if PAXX suppresses cancer development, with particular focus on lymphoma and medulloblastoma, as related DNA repair factors are known to protect from these diseases.