THE ROLE OF ZINC FINGER GENES IN HEMATOPOIESIS

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The objective of the proposed study is to understand the normal hematopoiesis and malignant transformation of hematopoietic cells by identifying biological functions of two genes, Ikaros and Helios. Ikaros encodes a zinc finger protein whose expression is restricted to hematopoietic cells. Ikaros protein isoforms are expressed in a constitutively phosphorylated form, most abundantly in lymphocytes. Helios encodes a zinc finger protein structurally similar to Ikaros, whose expression is restricted to T cells, Gene disruption experiments in mice showed that expression of Ikaros is necessary for the development of both B and T cells and suggested the role of Ikaros in malignant transformation, where it might act as a tumor suppressor. Subcellular localization of Ikaros and Helios showed that both proteins localize in heterochromatin and their distribution changes during different stages of cell cycle. The biological function of Helios and the role of Ikaros during myeloid development are unknown. Our preliminary data showed that: electrophoretic mobility of Ikaros protein isoforms changes at different stages of cell cycle in lymphoid cells; Ikaros is expressed at different levels during myeloid differentiation in human cell lines; expression of Helios is elevated at the early stage of T cell development comparing to mature T cells. The aims of this proposal are: Specific aim #1: Determine phosphorylation status of Ikaros and Helios in B and T cells during the different stages of cell cycle and study the significance of the phosphorylation of these proteins for normal progression of the cell cycle. We will perform phosphopeptide mapping of Ikaros and Helios in human B and T cell lines followed by immunoprecipitation experiments in order to identify the specific kinase(s) involved in their phosphorylation; the association of Ikaros and Helios with specific protein kinase(s) will be studied during the cell cycle; subcellular localization of the mutant forms of Ikaros and Helios, with mutations of the cell cycle-specific phosphorylation site(s) will be studied during the cell cycle. Specific aim #2: Determine the role of Ikaros in myeloid differentiation and the consequences of the altered expression of Ikaros during myeloid maturation. We will study the effect of Ikaros overexpression on differentiation of human promyelocytic cell line HL6O; Ikaros dominant-negative isoforms which contain the dimerization domain, but not the DNA binding domain, will be overexpressed to study the effect of the functional shut-down of Ikaros on differentiation of HL6O cells. Specific aim #3: Determine the role of Helios in the differentiation of lymphoid-committed cell precursors toward the T cell or B cell lineage. We will create transgenic mice with the Helios gene overexpressed in B and T cells (under control of the immunoglobulin enhancer) and analyze whether overexpression of Helios in B cells will impair their development and switch differentiation of lymphoid precursor toward T cell lineage and whether the upregulated expression of Helios in T and B cells will lead to their malignant transformation; we will create transgenic mice with Helios overexpressed at the early stage of T cell development - under the control of proximal 1ck promoter to study the role of Helios during the early stage of T cell development. These studies will provide new and important information on the mechanisms controlling development and proliferation of hematopoietic cells and will yield insights into the pathophysiology and treatment of leukemias.