The role of Ikaros in regulation of Notch target gene expression

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DESCRIPTION (provided by applicant): Ikaros is a nuclear protein and key regulator of T lymphopoiesis. Ikaros deficiency in mice results in abnormal T cell development and, with 100% penetrance, T cell leukemogenesis. However, the mechanistic role of Ikaros in these events is not yet known. Another important protein in T cell development is Notch. Deregulation of Notch signaling, like deregulation of Ikaros function, leads not only to defects in T cell development but to leukemogenesis as well. Deregulation of Ikaros and Notch are hypothesized to cooperate in leukemogenesis due to a correlation between loss of Ikaros and constitutive activation of Notch in leukemia cells in mouse and man. Therefore, it is likely that these two pathways also function cooperatively during T cell development. We hypothesize that deregulation of Notch signaling pathways, which is observed in the absence of Ikaros, is a major contributing factor, not only to the leukemogenesis, but also to the T cell developmental defects observed in Ikaros null mice. Notch is a transmembrane protein that is cleaved upon recognition of its ligand, freeing the "intracellular" domain (ICN), which travels to the nucleus. Here, ICN regulates transcription of Notch target genes through binding to and activation of the CSL transcription factor. CSL is believed to function as an active repressor of Notch target gene expression in the absence of ICN. We have demonstrated that in the absence of Ikaros, Notch target genes are de-repressed, suggesting that Ikaros is an obligate repressor of these genes. But how does Ikaros function in gene repression relative to CSL? And to what degree does de-repression of Notch target gene expression contribute to the T cell defects observed in Ikaros null mice? The studies described here will begin to address these questions using both ex vivo and in vivo approaches. In the first specific aim, we propose to utilize a combination of shRNA knockdown and biochemical approaches to define the role of Ikaros in regulation of the Notch target gene Hes1, with the ultimate goal of understanding the mechanism of CSL/Ikaros-mediated repression of Notch target genes in T cells. In the second specific aim, we propose to utilize conditional CSL knockout mice to determine the extent to which de-repression of Notch target genes underlies T cell developmental defects observed in Ikaros null mice. Taken together, these studies will provide a functional dissection of the interplay between CSL and Ikaros in gene repression, which, in turn, will illuminate a centrally important pathway in T cell development. PUBLIC HEALTH RELEVANCE: Genetic abnormalities occurring within an immune cell during its development can lead to catastrophic disease, such as leukemia. Identifying genes that regulate development is one approach to understanding the disease process, which is the first step to designing more effective therapies. In the studies proposed here, we will investigate the interaction between two important developmental genes, Ikaros and Notch, both of which are mutated in human leukemia.