ROLE OF EDR3 IN NORMAL DEVELOPMENT AND TUMORIGENESIS

描述（由申请人提供）：我们在筛选人染色体3q 26区域中发现早期发育调节因子3（EDR 3），该区域以前显示在骨肉瘤中发生洛。我们发现EDR 3基因的基因内缺失与骨肉瘤原发肿瘤和肿瘤细胞系的肿瘤抑制病因学一致。EDR 3的表达模式的检查显示，mRNA和蛋白质都显示出在成人和胚胎组织中几乎无处不在的表达模式。EDR 3是果蝇Polyhomeotic基因的同源物，并且是Polycomb Group（PcG）蛋白质家族的成员。PcG蛋白家族通过促进异染色质的形成和稳定传递而充当长期阻遏物。通过免疫共沉淀法，我们发现EDR 3蛋白与E2 F6、Bmi 1、YY 1和M33结合，而不与pRB 1和CtBP结合。这表明EDR 3是先前描述的人Polycomb抑制复合物（hPRC-H）的一部分，其含有E2 F6并且在G 0中有活性。与这一观察结果一致，我们发现，随着细胞进入G 0，EDR 3在细胞核中的定位从弥漫性变为高度点状模式。这种转变与含EDR 3的复合物结合YY 1和E2 F DNA结合位点以及c-myc启动子的能力相一致。当我们测试EDR 3与E2 F6和Bmi 1的共定位时，我们发现当细胞进入G 0时，EDR 3和E2 F6而不是Bmi 1共享向点状定位模式的转变。我们发现EDR 3在>70%的骨肉瘤肿瘤中受到影响。对其他肿瘤的检查显示，EDR 3蛋白在其他肿瘤中不存在，这表明这可能是一个检查点，通常在肿瘤发生中丢失。与该模型一致，我们发现EDR 3在正常细胞中过表达时或在肿瘤细胞中诱导表达时可充当生长抑制因子。我们的假设是EDR 3蛋白作为阻遏物复合物的一部分，调节成骨细胞和间充质干细胞中G 0的长期维持。一个推论假设是，成骨祖细胞中EDR 3功能的丧失可能导致维持G 0的能力丧失，从而有利于肿瘤发生。为了验证我们的假设，我们提出了以下具体目标：1）检查EDR 3在骨肉瘤肿瘤发生和成骨细胞分化中的作用; 2）表征含有EDR 3和E2 F6的PcG复合物的组分; 3）确定EDR 3调节的靶点。

DESCRIPTION (provided by applicant): We identified Early Developmental Regulator 3 (EDR3), as a candidate tumor suppressor gene in a screen of a region of human chromosome 3q26 previously shown to undergo LoH in osteosarcoma. We found intragenic deletions in the EDR3 gene consistent with a tumor suppressor etiology in osteosarcoma primary tumors and tumor cell lines. Examination of the expression pattern of EDR3 revealed that both mRNA and protein showed a near ubiquitous pattern of expression in adult and embryonic tissues. EDR3 is a homolog of the Drosophila Polyhomeotic gene and a member of the Polycomb Group (PcG) family of proteins. The PcG family of proteins acts as long-term repressors by contributing to the formation and stable transmission of heterochromatin. By co-immunoprecipitation, we found that the EDR3 protein was bound to E2F6, Bmi1, YY1 and M33 but not pRB1 or CtBP. This suggested that EDR3 was part of a previously described human Polycomb repressive complex (hPRC-H), which contains E2F6 and is active in G0. Consistent with this observation, we found that as cells entered G0, EDR3 localization in the nucleus changed from a diffuse to a highly punctuate pattern. This shift coincided with the ability of the EDR3-containing complex to bind to YY1 and E2F DNA-binding sites and to the c-myc promoter. When we tested co-localization of EDR3 with E2F6 and Bmi1, we found that the shift to a punctuate pattern of localization as the cells entered G0, was shared by EDR3 and E2F6 but not Bmi1. We found that EDR3 was affected in >70% of osteosarcoma tumors. Examination of other tumors showed that EDR3 protein was absent in other tumors suggesting that this could be a checkpoint, which is commonly lost in tumorigenesis. Consistent with this model, we found that EDR3 could act as a growth suppressor when overexpressed in normal cells, or when inducibly expressed in tumor cells. Our hypothesis is that the EDR3 protein acts as part of a repressor complex to regulate long-term maintenance of G0 in developing osteoblasts and mesenchymal stem cells. A corollary hypothesis is that loss of EDR3 function in osteoblast progenitor cells could lead to a loss of the ability to maintain G0 and thus favor tumorigenesis. To test our hypotheses, we propose the following specific aims: 1) To examine the role of EDR3 in osteosarcoma tumorigenesis and osteoblastic differentiation; 2) To characterize the components of the PcG complex containing EDR3 and E2F6; and 3) To identify the targets of EDR3 regulation.