The overall focus of the proposed research is to define, at the level of chromatin regulation, the molecular processes underlying expression of the IL-4, IL-5 and IL-13 genes in T cells and other cell types. These three genes are closely linked on mouse chromosome 11 and human chromosome 5. They are expressed by T helper type 2 (Th2) cells, mast cells, and certain other cell types; they are silenced in T helper type 1 (Thl) cells as well as most non-lymphoid cells. Within the conserved cytokine cluster, the IL-13 and IL-5 genes are separated by the RAD50 gene, which encodes an essential DNA repair protein that is expressed by all cells. During the last project period, DNase I hypersensitivity mapping and comparative genome sequence analyses revealed (with striking correspondence) a large number of potential regulatory regions in the IL-4/IL-13/RAD50/IL-5 locus. Deletion of three of these regions, CNS-1, DH sites V/VA and DH site IV, in the genomic context has confirmed their regulatory function; DH site IV is the first negative (silencer) element to be defined in the locus. The objective for this competing renewal application is to build on these data to understand gene regulation of the IL-4/ IL-13/ RAD50/ IL-5 locus at a more mechanistic level. Using a combination of biochemical and gene-targeting approaches, the early and late events which lead to activation or silencing at this locus will be delineated and the functional roles of selected transcription factors and regulatory elements will be explored. Specific objectives are to define the role of the known or presumed elements DH site IV andHSS3 (Aim 1); to define the role of the positive elements CNS-1 and CNS-2 (Aim 2); to explore the function of potential regulatory elements in the RAD50 gene (Aim 3); and to investigate the role of GATA3 (Aim 4). These experiments will elucidate the molecular basis for coordinate expression and silencing of the cytokine genes, and should provide clues to the mechanisms underlying irreversible lineage commitment to the Th2 or Th1 phenotype. The question is important given the striking pathologies associated with progressive Th1/Th2 polarisation in response to chronic antigen stimulation in vivo: chronic Th2 responses are associated with asthma and atopy, while Th1 cells are implicated in inflammation and autoimmune disease.
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