The Tox Gene Encodes Two Proteins with Distinct and Shared Roles in Gene Regulation

Here we report that the murine Tox gene encodes two proteins from a single mRNA, and we investigate the mechanism of production and function of these proteoforms. The annotated thymocyte selection-associated HMG-box protein (TOX) coding sequence is predicted to produce a 526-aa protein (TOXFL). However, Western blots reveal two bands. We found that the lower band consists of an N-terminally truncated variant of TOX (TOX Delta N), whereas the slower-migrating band is TOXFL. The TOX Delta N proteoform is alternatively translated via leaky ribosomal scanning from an evolutionarily conserved translation initiation site downstream of the annotated translation initiation site. When expressed exogenously from a cDNA in murine CD8 T cells or HEK cells, or endogenously from the murine Tox locus, both forms are translated, although the ratio of TOXFL/TOX Delta N significantly varies with cellular context. This includes regulation of proteoform production during development of murine CD4 T cells in the thymus, where the positive selection of CD4(+)CD8(+) cells and subsequent differentiation to CD4(+)CD8(lo) transitional and CD4SP cell subsets is associated with both an increase in total TOX protein and increased TOX Delta N production relative to TOXFL. Finally, we found that sole expression of TOXFL had a greater effect on gene regulation during chronic stimulation of murine CD8 T cells in culture mimicking exhaustion than did TOXDN, including uniquely regulated cell cycle and other genes.