THE ROLE OF ZINC FINGER GENES IN HEMATOPOIESIS

这项研究的目的是通过鉴定两个基因Ikaros和Helios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蛋白亚型以一种结构性的磷酸化形式表达，在淋巴细胞中含量最高。Helios编码一种结构上类似于Ikaros的锌指蛋白，其表达仅限于T细胞；小鼠的基因干扰实验表明Ikaros的表达对B细胞和T细胞的发育都是必需的，并提示Ikaros在恶性转化中的作用，在那里它可能起到肿瘤抑制作用。Ikaros和Helios的亚细胞定位表明，这两种蛋白都定位在异染色质中，并且在细胞周期的不同阶段它们的分布发生了变化。Helios的生物学功能和Ikaros在髓系发育过程中的作用尚不清楚。我们的初步数据显示：在淋巴样细胞中，Ikaros蛋白亚型的凝胶迁移率在细胞周期的不同阶段发生变化；在人类细胞系中，Ikaros在髓系分化过程中有不同程度的表达；与成熟T细胞相比，Helios在T细胞发育早期的表达增强。这项建议的目的是：具体目标1：确定B细胞和T细胞在细胞周期的不同阶段中Ikaros和Helios的磷酸化状态，并研究这些蛋白的磷酸化对细胞周期正常进行的意义。我们将在人B和T细胞系中进行Ikaros和Helios的磷酸肽图谱，然后进行免疫沉淀实验，以确定参与其磷酸化的特定激酶(S)；将在细胞周期中研究Ikaros和Helios与特定蛋白激酶(S)的关联；将在细胞周期中研究Ikaros和Helios突变形式与细胞周期特异性磷酸化位点(S)突变的亚细胞定位。具体目标#2：确定Ikaros在髓系分化中的作用以及在髓系成熟过程中Ikaros表达改变的后果。我们将研究Ikaros过表达对人早幼粒细胞系HL6O分化的影响；通过过表达含有二聚化结构域而不含DNA结合结构域的Ikaros显性-阴性亚型来研究Ikaros功能关闭对HL6O细胞分化的影响。具体目标#3：确定Helios在淋巴前体细胞向T细胞或B细胞分化过程中的作用。我们将建立在B和T细胞中过表达Helios基因的转基因小鼠(在免疫球蛋白增强子的控制下)，并分析在B细胞中过表达Helios是否会损害它们的发育和淋巴前体向T细胞系的分化，以及在T和B细胞中Helios基因的上调表达是否会导致它们的恶性转化；我们将在T细胞发育的早期阶段建立Helios基因过表达的转基因小鼠-在近端1ck启动子的控制下，研究Helios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学和治疗提供新的见解。

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.