An understanding of lymphopoietic regulatory mechanisms is essential for the elucidation of aberrant events that result in leukemias, lymphomas, and immunodeficiencies. Ikaros family members, including Ikaros, Aiolos, and Helios, are DNA-binding proteins that are well- established as critical regulators of lymphopoiesis, but their intracellular functions appear to be atypical and remain unknown. Recent results from our laboratory, in collaboration with the laboratory of Amanda Fisher and Matthias Merkenschlager, have established the Ikaros and Helios often localize to foci of centromeric, heterochromatin. In addition, several cellular genes, including the terminal transferase (TdT) gene, become physically repositioned to these foci when heritably down-regulated. These results suggest that repositioning may be critical for the heritable inactivation of transcription, and that Ikaros may play an essential role in this process. The studies proposed here will use the TdT gene as a model system to examine these hypotheses in greater depth. The long-term objectives of the proposed research are to understand: 1. The order of events which occur when an active gene becomes inactivated and repositioned to ceteromeric foci, and 2. The intracellular functions of Ikaros and Helios, with special emphasis on functions relating to down- regulation and repositioning. To accomplish these objectives, the TdT locus will be examined in double-positive (CD4+CD8+) thymocytes and related cell lines as it comes inactivated, and repositioned. Among the properties that will be examined are its histone modification status, methylation status, and chromatin accessibility to nuclease cleavage. Experiments will also be performed to further explore the relevance of an Ikaros binding site within the TdT promoter for down-regulation and repositioning. To elucidate more fully the targeted to centromeric foci, its interaction with histone deacetylase complexes, and its direct interaction with genomic DNA targets. To facilitate the studies of repositioning and of the intracellular functions of Ikaros proteins, genetically altered mouse strains will be created. To determine if the T cell-specific Helios protein plays an important regulatory role, transgenic mice which express Helios ectopically in the B cell lineage will be analyzed To test the relevance of Ikaros for centromeric repositioning, transgenic mice have been created containing reporter genes downstream of multiple Ikaros binding sites. Finally, to examine the relevance of Ikaros for gene inactivation in lymphocytes, mouse strains allowing the conditional disruption of the Ikaros gene are being prepared.
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