E protein targets orchestrating γδ development and function

Research Inst Of Fox Chase Can Ctr, Philadelphia PA

Investigators

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Abstract

PROJECT SUMMARY/ABSTRACT - PROJECT 1 T lymphocytes comprise two major lineages,  and , which play critical, partially-distinct roles in host defense.  and  T cells arise from a common progenitor in the thymus; however, the molecular processes responsible for specification of these lineages during development in the thymus remain incompletely understood and this represents a major gap in knowledge. We seek to address this knowledge gap. In doing so, we have provided compelling evidence that specification of the  and  T cell fates is controlled by differences in T cell receptor (TCR) signal strength. These signaling differences regulate fate by proportional induction of Id3, which causes graded repression of the function of E box DNA binding proteins (E proteins; E2A and HEB). Nevertheless, the targets through which these E proteins regulate  T lineage commitment and effector fate specification remain unclear, and this presents a major gap in knowledge. To address this knowledge gap, we (all Projects) employed a comprehensive, genome-wide approach, which revealed a number of important insights into the mechanism by which strong TCR signals remodel E protein binding to specify fate. First, strong TCR signals have selective effects on E proteins, markedly repressing E2A binding to the genome, while preserving HEB binding (all Projects). The sparing of HEB binding is important because HEB function is required for development of interleukin-17 (IL-17) producing  T cells (Proj1/3/4). Second, we have identified Tcf7/TCF1 as both a novel E protein target and a critical cofactor that cooperates with E proteins in controlling  lineage commitment. Consequently, in the current proposal we seek to address two major questions relating to how E proteins control TCF1 expression and the targets through which they cooperate to control lineage fate. We will do so according to two aims: Aim1 seeks to interrogate the mechanism by which 3 critical E protein bound elements (EPE) near the Tcf7 locus regulate Tcf7/TCF1 expression and whether individual E protein family members (E2A, HEBCan, and HEBAlt) play distinct roles. In Aim 2, we will focus on the E/TCF targets through which the  lineage fate is facilitated. We have determined that many of the E/TCF co-bound targets regulate cell metabolism and  lineage commitment is coupled with changes in lipid composition. We will employ a series of gain and loss of function strategies to explore the importance of these targets in regulating  lineage commitment and peripheral effector fate. In addition, we will profile at a genome-wide scale how the reduction in TCF1 expression caused by strong TCR signals cooperates with E protein remodeling to specify the  lineage fate. We will do so using a novel method termed STARR-Seq (with Project 2 and Core B) that interrogates the regulatory element repertoire at a genome-wide scale in single cells. Consequently, in the current proposal, we integrate the expertise of all Project leaders and the Genomics Core to elucidate the mechanistic basis by which changes in E protein binding controls  lineage commitment and adoption of the IL-17 effector fate and determine how the molecular remodeling in mouse model systems aligns with that during development of human  T cells (Proj2).