FUNCTIONAL ANALYSIS OF THE T(17--19)-ALL CHIMERA E2A-HLF

异常转录调控在肿瘤发生中的重要作用 非随机疾病的频繁发生， 编码转录因子的基因中的特定突变。 一个这样 在ALL中观察到的t（17; 19）突变产生E2A-HLF融合， 具有嵌合转录因子特性的蛋白质。 功能类似的嵌合体经常由其他基因创造。 急性白血病和儿童肉瘤中的易位。 理解 异常的转录调控机制 恶性转化是发展的必要前提 新的治疗方法直接针对介导 肿瘤发生 拟议的研究将在两个平行 E2A-HLF具有生物活性相关的实验系统 在ALL中的作用：阻断生长因子诱导的细胞凋亡 在鼠IL 3依赖性pro-B细胞系FL5.12中的撤药， 转化NIH 3T3细胞。 具体目标1旨在 严格检查的假设，转录激活的 关键靶基因是E2 A-HLF生物学的主要机制 活动 明确定义的相关结构域的点突变 在转录调控中的作用将用于描绘E2A-HLF 生物活性所必需的功能。 功能 因此，足以进行生物活性的性质将被 定义为用异源结构域替换必需结构域， 完成同样的功能。 这项建议的第二个目的是 鉴定受E2A-HLF转录调控的靶基因， 评估它们在白血病发生中的作用。 的配体结合域 雌激素受体（ER）与E2A-HLF融合， 条件活性转录激活因子E2A-HLF-ER。 3t3和 FL5.12细胞将用E2A-HLF-ER和E2A-HLF-ER转染，并将其转染到细胞中。 转录失活突变体构建体和代表性的 差异分析将用于鉴别mRNA差异 在诱导E2A-HLF-ER活性后表达。 候选mRNA 其也在T（17; 19）+人白血病中表达， 其特征在于确定其蛋白质产物在以下方面的作用： 白血病发生

The prominent role of aberrant transcriptional regulation in oncogenesis is underscored by the frequent occurrence of non-random, disease specific mutations in genes encoding transcription factors. One such mutation, the t(17;19) observed in ALL, creates an E2A-HLF fusion protein with properties of a chimeric transcription factor. Functionally analogous chimeras are frequently created by other translocations in acute leukemias and childhood sarcomas. Understanding the mechanisms by which aberrant transcriptional regulation contributes to malignant transformation is an essential prerequisite to developing new therapies directly targeted at the molecular events mediating oncogenesis. The proposed studies will be performed in parallel in two experimental system in which E2A-HLF has biological activity relevant to its role in ALL: blocking apoptosis induced by growth factor withdrawal in the murine IL3-dependent pro-B cell line FL5.12 and transformation of NIH 3T3 cells. Specific Aim 1 is designed to rigorously examine the hypothesis that transcriptional activation of crucial target genes is the major mechanism for E2A-HLF biological activity. Point mutations that inactivate well-defined domains involved in transcriptional regulation will be used to delineate E2A-HLF functions that are necessary for biological activity. Functional properties which are sufficient for biological activity will then be defined by replacing necessary domains with heterologous domains capable of accomplishing the same function. The second aim of this proposal is to identify target genes transcriptionally regulated by E2A-HLF and evaluate their role in leukemogenesis. The ligand binding domain of the estrogen receptor (ER) has been fused to E2A-HLF to create a conditionally active transcriptional activator, E2A-HLF-ER. 3T3 and FL5.12 cells will be stably transfected with E2A-HLF-ER and a transcriptionally inactive mutant construct and representational difference analysis will be used to identify mRNAs differentially expressed following induction of E2A-HLF-ER activity. Candidate mRNAs which are also expressed in t(17;19)+ human leukemias will be further characterized to define the role of their protein products in leukemogenesis.