Thursday, April 3, 2025
4/3/2025
Characterization of TOX isoform-specific roles in T cell and ILC development - Project Summary RNA splicing, RNA modification, and protein post translational modification all play significant roles in diversification of the functional proteome. However, less explored in many contexts–including in lymphocyte development and function–is the role of translational mechanisms in regulating protein function. We have pioneered work on the TOX protein, an evolutionarily conserved HMG-box superfamily DNA- binding transcriptional regulator. Our previous studies demonstrated that TOX is required for CD4 T cell lineage development in the thymus and for early innate lymphoid cell (ILC) lineage specification in the bone marrow. Collaborative studies as well as independent work from others have defined an additional role for TOX in regulating the function of CD8 T cells in the context of autoimmunity, chronic infection, and cancer. What was not appreciated in these foundational studies, however, was that there are two evolutionarily conserved isoforms of TOX that differ in N-terminal sequence. Recently, we demonstrated that these proteoforms arise by the process of translational leaky scanning, in which the ribosome can bypass an upstream start codon in favor of a downstream start codon in a more favorable sequence context. Moreover, we found that TOX isoform production is a regulated process during thymic T cell development, and that TOX isoforms have differential effects on gene expression. In this exploratory grant application, we propose to investigate the function of these TOX isoforms in the context of T cell and ILC development. Not only will these studies open new avenues of investigation into this key transcriptional regulator, but also provide an entry into future study of translational regulation during lymphocyte development. To accomplish this work, we have developed a novel mouse strain (D37) that specifically lacks the TOX long (TOXL) isoform but otherwise maintains normal transcriptional and translational regulation. Preliminary data from this model strongly points to a role for TOXL in CD4 T cell development. Taking advantage of D37 mice, as well as a unique TOXL-specific antibody, we propose two specific aims. In Aim 1, we propose a comprehensive analysis of T cell and ILC development in these mutant mice. In Aim 2, we will investigate the molecular basis of TOXL activity. This includes comparison of subnuclear localization of TOX in wildtype and D37 mice and, using a proteoform-specific antibody, direct analysis of TOXL localization. We also propose single-cell multiome RNA-seq and ATAC-seq to compare the transcriptomic and epigenetic state of post-positive selection thymocytes from D37 and wildtype mice. This dataset will inform cell populations to then be tested by CUT&RUN methodology to determine the influence of loss of TOXL on TOX binding to genomic loci.
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