IFN-?, a cytokine produced primarily by NK cells and by Th1 CD4 and CD8 T cells, plays a central role in the immune response to infection with intracellular pathogens. Conversely, protection against extracellular metazoans is dependent on Th2 CD4 T cells and the cytokines they produce, which include IL-4, IL-13 and IL-5. Expression of IFN-? and of Th2 cytokines is primarily regulated at the level of transcription. While transcription factors that govern IFN-? expression have in recent years been elucidated in considerable detail, very little is known regarding where and how they act. By contrast to IFN-?, regulation of the Th2 cytokines IL-4, IL-13 and IL-5, which are clustered into a single ~150 kb locus in mammals, is now understood in considerable molecular detail. In this application, we propose to gain a similar level of knowledge regarding IFN-? by comprehensively identifying the regulatory elements governing IFN-? expression and the functional boundaries of the Ifng locus through 3 Aims: ? ? Aim 1: Identify regulatory elements governing the expression of IFN-? through comprehensive chromatin profiling of the murine Ifng locus. Hypothesis: DNase hypersensitive sites and regions enriched in transcriptionally favorable histone modifications extend ~50 kb upstream and downstream of the murine Ifng gene and identify regulatory elements governing IFN-? expression. ? ? Aim 2: Define the functional boundaries of the Ifng locus using BAG transgenes to complement the defect in IFN-? knockout (GKO) mice. Hypothesis: BAG transgenes containing the murine Ifng gene and extending >= 50 kb 5' and 3' will reconstitute proper IFN-? expression and thereby complement the immunological defect in GKO mice. ? ? Aim 3: Identify the mechanisms bv which the transcriptional regulatory elements within the Ifng locus influence IFN-? expression and the key transcription factors that act at these sites. Hypotheses: T-bet, STAT4, NFAT, NF-?B and AP-1 bind not only to the Ifng promoter but to additional upstream and downstream regulatory elements, thereby affecting IFN-? expression; Ifng5'CNS 2 and 3 and/or Ifng3'CNS3 help to insulate the Ifng locus from the influence of surrounding genes. ? ? ?
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