Myb protein specificity in human hematopoietic cells

描述(申请人提供)：这份修订后的申请建议比较正常细胞和白血病表达的c-Myb野生型和变异版本的活性，并确定它们与人类疾病的相关性。C-myb原癌基因编码一种具有潜在转化活性的DNA结合转录因子。C-Myb蛋白相对较小的变化释放了其在鸟类和啮齿动物中导致白血病的能力，微阵列分析表明，即使是对其C末端结构域的微小变化也会导致转录活性的显著变化。选择性RNA剪接产生多个c-myb mRNAs，编码具有改变C末端结构域的蛋白，并且选择性剪接在白血病中比在正常细胞中更加丰富和多样化，这表明c-Myb的变体可能参与了白血病的发生。 在目标1中，将确定在具有代表性的正常造血细胞和白血病中表达的最丰富的变异c-myb mRNAs的结构。为了比较它们的活性，c-Myb的正常形式和变异形式将在人类单核细胞中表达，微阵列分析将用于识别基因表达的明显变化。目的2将测试变异的c-Myb蛋白的生物学相关性。首先，将研究c-myb基因产物在分化造血细胞过程中的选择性剪接，然后在正常造血祖细胞中表达野生型和变异型c-Myb蛋白，以观察是否诱导分化或增殖的变化。在目标3中，将使用定量分析来跟踪正常和变异的c-myb RNA在确定的成人AML样本队列中的表达，然后统计分析将确定特定的变异是否与AML患者样本的临床参数、预后或基因表达模式相关。 拟议的实验建立在我们的专业知识和广泛的初步和已发表的数据的基础上，对替代RNA剪接、Myb蛋白的活性和人类白血病的研究具有很大的相关性。

DESCRIPTION (provided by applicant): This revised application proposes to compare the activities of the wild type and variant versions of c-Myb expressed by normal cells and leukemias, and to determine their relevance to human disease. The c-myb proto-oncogene encodes a DNA binding transcription factor with latent transforming activity. Relatively minor changes to the c-Myb protein unleash its ability to cause leukemias in birds and rodents and microarray assays have shown that even minor changes to its C-terminal domains result in dramatic changes in transcriptional activity. Alternative RNA splicing generates multiple c-myb mRNAs that encode proteins with altered C-terminal domains and the alternative splicing is more abundant and more varied in leukemias than in normal cells, suggesting that variants of c-Myb may participate in leukemogenesis. In Aim 1, the structures of the most abundant variant c-myb mRNAs expressed in a representative group of normal hematopoietic cells and leukemias will be determined. To compare their activities, the normal and variant forms of c-Myb will be expressed in human monocytes and microarray assays will be used to identify telltale changes in gene expression. Aim 2 will test the biological relevance of the variant c-Myb proteins. First, alternative splicing of c-myb gene products will be studied in differentiating hematopoietic cells, then wild type and variant c-Myb proteins will be expressed in normal hematopoietic progenitors, to see if changes in differentiation or proliferation are induced. In Aim 3, quantitative assays will be used to follow the expression of normal and variant c-myb RNAs in a defined cohort of adult AML samples, and then statistical analyses will determine whether specific variants correlate with clinical parameters, prognosis or gene expression patterns in AML patient samples. The proposed experiments build on our expertise and extensive preliminary and published data and have great relevance to the study of alternative RNA splicing, the activity of Myb proteins and human leukemia.