Regulation of cytokine gene expression during T cell differentiation

This thesis examines the regulation of cytokine gene expression, with an emphasis on the role of chromatin structure, during the differentiation of peripheral CD4+ helper T lymphocytes. Helper T lymphocytes undergo two distinct phases of differentiation, resulting in the development of effector T cell subsets displaying mutually exclusive patterns of cytokine gene expression. T helper type-1 (Th1) cells characteristically transcribe the IFN-γ gene, whereas Th2 cells transcribe the IL-4, IL-5, and IL-13 genes.; The transcriptional activity of genetic loci is regulated by structural changes in chromatin, which activate or silence gene transcription. By deoxyribonuclease (DNAse) I hypersensitivity analysis of the endogenous murine IL-4, IL-13 and IFN-γ loci, we found that the initial response of naive T cells to antigen was accompanied by long-range changes in the chromatin structure of effector cytokine genes. Differentiation of naive T cells into effector Th2 cells was associated with chromatin remodeling throughout a 43 kb interval spanning the linked IL-4/IL-13 loci, suggesting coordinate regulation of these genes at the level of chromatin. In contrast, differentiation of the same precursor population into Th1 cells evoked chromatin remodeling of the IFN-γ but not of the IL-4/IL-13 locus. IL-4 locus remodeling was accompanied by demethylation and required both antigen stimulation and STAT6 activation. Antigenic stimulation of Th2 cells resulted in the induction of a novel DNAse I hypersensitive site in the IL-4 locus, which contained transcriptional enhancer activity in vivo. The enhancer contained consensus binding sites for the antigen-induced transcription factor NFAT, which was bound to the endogenous enhancer only in stimulated Th2 cells.; Together, our results support a model whereby signaling pathways initiated in naive T cells by primary contact with antigen and cytokines recruit nuclear factors and chromatin remodeling enzymes to novel regulatory regions in cytokine genetic loci. Upon subsequent antigenic stimulation, transcription factors such as NFAT gain access to cytokine regulatory regions only in the appropriate subset of differentiated T cells in vivo, enabling antigen-dependent and subset-specific transcription of cytokine genes. Further analysis of mice carrying targeted deletions of DNAse I hypersensitive sites will permit a functional analysis of these potential regulatory elements in the endogenous cytokine genetic loci.