HEB MEDIATED PATHWAYS AND FUNCTION IN T CELL DEVELOPMENT

淋巴细胞的发育是一个动态的但受到严格调控的过程，产生T淋巴细胞和B淋巴细胞，这是我们免疫系统的主要细胞成分。关键调控基因的遗传改变可能而且确实经常导致免疫缺陷、自身免疫、淋巴瘤或白血病。我们的长期研究目标是了解淋巴细胞发育的分子机制，并为淋巴细胞疾病的诊断和治疗提供新的策略。在这个提案中，我们将具体地讨论HEB在t细胞发育中的功能。HEB编码一种bHLH型转录因子，这种转录因子在t淋巴细胞中高度表达（尽管不是完全表达）。我们在小鼠中使用基因靶向来证明HEB在T细胞发育的早期阶段起着至关重要的作用。有证据表明，HEB的这种功能需要与其他调控分子如E2A、TCF和CBF1alpha合作。为了更好地了解HEB的功能机制，深入了解HEB介导的调控途径，我们提出了以下实验：1)我们将进一步明确HEB的功能及其与E2A的关系，并评估HEB在细胞死亡途径中的潜在作用。这些将通过在过继转移试验和/或常规转基因拯救试验中使用基于逆转录病毒的cDNA载体来完成。2)我们将在小鼠中使用ENU诱变技术筛选HEB修饰剂。这种正向遗传方法将导致鉴定其功能与HEB相关的调节分子。3)我们将描述和绘制在早期筛选中发现的新突变和从该筛选中分离出的其他突变。此外，我们还将启动位置和功能克隆过程，以便这项研究最终将导致分离新的对t细胞发育重要的调控基因。虽然拟议的诱变实验代表了一个长期的承诺，但我们在资助期的直接目标是识别和表征几种淋巴细胞特异性突变，并将它们置于HEB途径中。最终目标是根据这些基因在t细胞发育中的预定功能分离出它们。

Lymphocyte development is a dynamic yet tightly regulated process to give rise to T- and B- lymphocytes, the major cellular components of our immune system. Genetic alterations in key regulatory genes and could and does often result in immune deficiency, autoimmunity, lymphoma, or leukemia. Our long term research goal is to understand molecular mechanisms underlying lymphocyte development and to provide novel strategies for diagnosis and treatment of lymphoid diseases. In this proposal, we will specifically address the function of HEB in T-cell development. HEB encodes a bHLH type of transcription factor which is highly (although not exclusively) expressed in T-lymphocytes. We have used gene targeting in mice to show that HEB plays an essential role in early stages of T cell development. Evidence indicates that this function of HEB requires collaboration with other regulatory molecules such as E2A, TCF, and CBF1alpha. To better understand the mechanism underlying HEB function and to gain insights into the regulatory pathways mediated by HEB, we propose the following experiments: 1) We will further define the function of HEB and its relationship with E2A and evaluate the potential role of HEB in the cell death pathway. These will be accomplished by using a retroviral-based cDNA vector in an adoptive transfer assay and/or conventional transgenic rescuing assay. 2) We will use ENU mutagenesis in mice to screen for modifiers of HEB. This forward genetic approach will lead to identification of regulatory molecules whose functions are linked with HEB. 3) We will characterize and map a novel mutations identified in an earlier screen and other mutations to be isolated from this screen. In addition, we will also initiate the positional and functional cloning process so that this research will eventually lead to isolating novel regulatory genes important for T-cell development. Although the proposed mutagenesis experiment represents a long term commitment, our immediate goal in the funding period is to identify and characterize several lymphoid specific mutations and place them into the HEB pathway. The ultimate goal is to isolate these genes based on their predetermined function in T-cell development.