1R01AI077574-01A2;WEAVER, Casey T. The goal of this proposal is to elucidate the molecular mechanisms that control lineage-specific expression or repression of the Ifng locus as a model for understanding factors regulating effector T cell lineage specification. Th1, Th2, and Th17 cells, though derived from common naIve CD4+ T cell precursors, differentiate under the control of distinct developmental programs that specify altemate adaptive immune functions based on unique gene expression phenotypes. Development of effector T cells is associated with epigenetic alterations that establish and maintain lineage-specific programs of gene expression that enable rapid and efficient production of cytokines in recall responses. While the Th2 cytokine locus, which includes the 114, 1113 and 115 genes, has become a premier model for understanding chromatin dynamics during T lineage development, only recently have insights into regulation of the Th1 cytokine gene, Ifng, begun to emerge. Although we are beginning to understand modifications of chromatin structure associated with the mutually exclusive expression of Ifng by Th1 cells, and 114, 115, and 1113 by Th2 cells, a detailed understanding of the interplay of multiple distal regulatory cis-elements in defining lineagespecific gene expression awaits further study. Recently, we have identified a distal conserved, noncoding sequence (CNS) element 22kb upstream of the Ifng gene (CNS-22) that enhances Ifng promoter expression in vitro, resides in an area of accessible chromatin in naIve T cells and in both Th1 and Th2 cells, and when conditionally deleted from a Ifng locus BAC reporter transgene, ablates Ifng expression in Th1 cells, CTLs, and NK cells. To our knowledge, this represents the first identification of a single distal element that is required for gene expression of an immune cell cytokine in multiple lineages, setting the stage for further investigations to more fully define mechanisms by which CNS-22 may act as a critical hub for coordination of chromatin remodeling of the Ifng locus and regulation of Ifng expression. Accordingly, dissection of the factors that interact with CNS-22 in different T cell and non-T-celilineages provides an attractive window to regulation of chromatin dynamics important for effector T lineage specification. Although CNS-22 appears essential for Ifng gene expression, it represents only one of at least eight highly conserved distal CNSs within the Ifng locus, and it is unclear how the different CNSs interact to control gene expression. We hypothesize that distal conserved non-coding sequences in the Ifng locus differentially contribute to Ifng expression or repression throughout effector T cell differentiation and development and that CNS- 22, in particular, is an essenb""""""""al regulatory element required for promob""""""""ng Ifng gene expression in IFNy-producing cel/s, or silencing Ifng in non-expressing lineages. This revised proposal will test two related, but independent, aspects of this hypothesis: one, that CNS-22 is a hub for the assembly of transcription factors required for lineage-specific expression and repression of the Ifng gene;and two, that CNS-22 directs accessibility and remodeling of the Ifng locus in effector T cell lineages.
Adaptive immunity is essential for protection against infectious disease and malignancy, and when dysregulated can lead to autoimmunity. This project seeks to define molecular mechanisms that regulate the development of distinct lineages of CD4 T cells, with a view towards defining new approaches to prevent or treat infectious disease, cancer and autoimmunity.
