HEB MEDIATED PATHWAYS AND FUNCTION IN T CELL DEVELOPMENT

淋巴细胞发育是一个动态但受到严格调控的过程，以产生T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变化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，并为淋巴系统疾病的诊断和治疗提供新的策略。在这项提案中，我们将具体讨论Heb在T细胞发育中的作用。Heb编码一种bHLH型转录因子，它在T淋巴细胞中高度表达(尽管不是排他性的)。我们在小鼠中使用了基因打靶，以表明Heb在T细胞发育的早期阶段发挥着重要作用。有证据表明，Heb的这一功能需要与其他调节分子如E2A、TCF和CBF1pha协同工作。为了更好地理解Heb功能的机制，并深入了解HeB介导的调控途径，我们提出了以下实验：1)我们将进一步定义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。这将通过在过继转移试验和/或传统的转基因挽救试验中使用基于逆转录病毒的cDNA载体来实现。2)利用ENU诱变法筛选Heb的修饰物。这一正向遗传方法将导致识别其功能与Heb相关的调节分子。3)我们将表征和定位在早期筛查中发现的新突变和从该筛查中分离的其他突变。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对T细胞发育重要的新调控基因。虽然拟议的突变实验代表着一项长期的承诺，但我们在资助期的近期目标是识别和表征几种淋巴特异性突变，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来分离它们。

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.