THE ROLE OF ZINC FINGER GENES IN HEMATOPOIESIS

本研究的目的是通过鉴定Ikaros和Helios两个基因的生物学功能来了解造血细胞的正常造血和恶性转化。Ikaros编码一种锌指蛋白，其表达仅限于造血细胞。Ikaros蛋白异构体以组成型磷酸化形式表达，在淋巴细胞中最丰富。Helios编码一种锌指蛋白，其结构与Ikaros相似，其表达仅限于T细胞。小鼠基因破坏实验表明，Ikaros的表达是B细胞和T细胞发育所必需的，并提示Ikaros在恶性转化中的作用，在那里它可能作为肿瘤抑制因子。Ikaros和Helios的亚细胞定位表明，这两种蛋白都定位于异染色质，并且它们的分布在细胞周期的不同阶段发生变化。Helios的生物学功能和Ikaros在髓细胞发育中的作用尚不清楚。我们的初步数据表明：Ikaros蛋白亚型的电泳迁移率在淋巴样细胞周期的不同阶段发生变化；Ikaros在人细胞系骨髓分化过程中以不同水平表达；与成熟的T细胞相比，Helios的表达在T细胞发育的早期阶段升高。具体目的1：确定Ikaros和Helios蛋白在细胞周期不同阶段在B细胞和T细胞中的磷酸化状态，并研究这些蛋白磷酸化对细胞周期正常进程的意义。我们将在人类B和T细胞系中对Ikaros和Helios进行磷酸肽定位，然后进行免疫沉淀实验，以确定参与其磷酸化的特定激酶；Ikaros和Helios与特定蛋白激酶的关联将在细胞周期中进行研究；Ikaros和Helios突变体的亚细胞定位，以及细胞周期特异性磷酸化位点的突变将在细胞周期中进行研究。具体目的#2：确定Ikaros在髓细胞分化中的作用，以及在髓细胞成熟过程中Ikaros表达改变的后果。我们将研究Ikaros过表达对人早幼粒细胞株hl60分化的影响；Ikaros的显性阴性亚型包含二聚化结构域，但不包含DNA结合结构域，将被过表达，以研究Ikaros功能关闭对hl60细胞分化的影响。具体目标#3：确定Helios在淋巴细胞前体向T细胞或B细胞谱系分化中的作用。我们将构建Helios基因在B细胞和T细胞中过表达的转基因小鼠（在免疫球蛋白增强子的控制下），分析Helios基因在B细胞中的过表达是否会影响B细胞的发育和淋巴样前体向T细胞谱系的分化，以及Helios基因在T细胞和B细胞中的上调表达是否会导致其恶性转化；我们将在近端1ck启动子的控制下，构建Helios基因在T细胞发育早期过表达的转基因小鼠，研究Helios基因在T细胞发育早期的作用。这些研究将为控制造血细胞发育和增殖的机制提供新的重要信息，并将为白血病的病理生理和治疗提供见解。

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.