Linking TCR signal strength to Id3 transcriptional dynamics and nuclear positioning

PROJECT SUMMARY/ABSTRACT - PROJECT 4 T lymphocytes consist of two major lineages, αβ and γδ, that arise from common progenitors. The separation of these two lineages is instructed by TCR signaling, with weak and strong signals favoring development of the  and cell lineages, respectively. The signaling axis through which differences in signal strength are executed involves sequential activation of tyrosine and ERK MAPK kinases, that ultimately terminate with induction of the critical antagonist of E protein function, Id3. TCR-signals of differing strength elicit proportional induction of Id3, which antagonizes E2A and HEB occupancy of genomic sites, in a graded fashion. How strong versus weak TCR signals instruct  versus  T cell fate through graded reduction in E protein function remains a critical gap in knowledge to be addressed by this program. Here we hypothesize that gradients of TCR signal strength instruct differences in Id3 gene expression dynamics and propose that these differences underpin the mechanism that instructs differentiation into either the  or  lineage fate. To test this possibility, we engineered an approach that for the first time, enabled monitoring of mRNA synthesis in live immune cells that were derived from primary lymphoid organs. Using this approach, we were able to track mRNA synthesis in live immune cells that were derived from the bone marrow. We found that pre-TCR and -TCR signals activated distinct Id3 transcriptional bursting signatures at the nuclear envelope. Here we seek to determine how weak versus strong TCR-induced Id3 transcriptional bursting signatures are associated with the  versus  lineage fate decision and how TCR-induced Id3 gene expression dynamics is initiated at the nuclear envelope. Specifically, we would convert TCR-induced fluctuations in Id3 mRNA abundance from analog to digital signals. We would describe ligand-TCR interactions in terms of digital codes that define the spatiotemporal control of Id3 gene expression dynamics. We would examine where the Id3 locus is localized and whether and how the nuclear positioning of the Id3 locus is regulated by antigen receptor signaling. This project is heavily integrated with all other projects. In collaboration with Project 1, we would determine whether and how in response to pre-TCR and TCR signals ERK signal duration modulates transcriptional bursting frequencies, ON- and OFF-times and bursting amplitudes. In collaboration with Project 3, we would examine how feed-back circuitry involving Id3, E2A and HEB, instructs Id3 gene expression dynamics in response to pre-TCR and -TCR signals. In collaboration with Project 2, we would monitor Id3 gene expression dynamics in KN6- T cells in response to exposure of tetramers that interact with a gradient of affinities for the KN6-TCR.This aim will be done in close collaboration with Project 1. Ultimately, it is our goal, in collaboration with our P01 partners, to establish a new paradigm that defines TCR signals elicited in response to self- or non-self-antigens by their digital Id3 bursting codes.