APE1 downregulation promotes somatic hypermutation and predisposes lymphocytes toward apoptosis for selection - Summary Development of B and T lymphocytes with a diverse repertoire of antigen-binding receptors is essential to fight infection, but antigen receptor assembly and modification require programmed DNA breaks and hypermutation, which also generates genomic instability. To ensure that clones with residual DNA damage or self-reactive receptors do not survive, these processes are followed by multiple rounds of selection. Cells at these stages are “prone to apoptosis” and to survive, they must be positively selected. Selection occurs at several stages: following VDJ-recombination in pre-B cells in the bone marrow and CD4+CD8+ double-positive T cells in the thymus, as well as following AID-dependent somatic hypermutation (SHM) in mature B cells responding to antigen in germinal centers (GC). We found that an essential DNA repair enzyme, AP endonuclease 1 (APE1) that is normally highly expressed is dramatically downregulated specifically at these stages where lymphocytes undergo DNA damage followed by selection. In the GC, we showed that an error-prone homolog, APE2, is highly expressed and promotes error-prone repair of AID lesions, creating the mutations necessary for antibody diversification. It is likely that APE1- downregulation is necessary to suppress accurate repair, but APE1 is an essential gene, and, unlike other repair proteins that are downregulated to accommodate programmed DNA breaks, inhibition of APE1 is tightly linked to apoptosis. I propose that APE1 downregulation in the GC promotes somatic hypermutation, and that it also predisposes lymphocytes toward apoptosis, allowing for selection, not only in GCs, but in all stages where selection takes place. In the GC, more than 50% of the cells undergo apoptosis. This default pathway of cell death is essential, as inappropriate survival can lead to both lymphomagenesis and autoimmunity. We will test this hypothesis by enforcing APE1 expression in the GC, in progenitor B cells and in developing T cells. I propose to develop a transgenic mouse model to conditionally express APE1, regulated by a floxed stop codon. It will be crossed to mice expressing the recombinase Cre under cell stage-specific promoters (AID- Cre, mb-1-Cre, and CD4-Cre mice), to delete the stop codon and enforce APE1 expression in cell stages where selection takes place. We will validate APE1 expression in all lymphocyte stages. We will analyze mutations in GC cells to determine if downregulation of APE1 is necessary for SHM. We will analyze cells at all stages of selection (bone marrow, thymus and GC) for cell survival, proliferation, and apoptosis to determine if APE1 expression impacts selection and cell death. As time permits, we will follow these mice for propensity to develop autoimmunity and lymphoma. This one mouse model will allow us to test this novel hypothesis and support future studies that will lead to a mechanistic understanding of selection processes that are necessary to prevent autoimmunity and lymphomagenesis.
