Myb protein specificity in human hematopoietic cells

描述（由申请人提供）：本修订申请拟比较正常细胞和白血病表达的c-Myb野生型和变体的活性，并确定其与人类疾病的相关性。c-myb原癌基因编码具有潜在转化活性的DNA结合转录因子。c-Myb蛋白质的相对较小的变化释放其在鸟类和啮齿动物中引起白血病的能力，并且微阵列测定已经表明，即使其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 mRNA，其编码具有改变的C-末端结构域的蛋白质，并且选择性剪接在白血病中比在正常细胞中更丰富和更多样，这表明c-Myb的变体可能参与白血病的发生。 在目的1中，将确定在正常造血细胞和白血病的代表性组中表达的最丰富的变体c-myb mRNA的结构。为了比较它们的活性，将在人单核细胞中表达c-Myb的正常形式和变体形式，并且将使用微阵列测定来鉴定基因表达中的指示性变化。目的2将测试变体c-Myb蛋白的生物学相关性。首先，在分化造血细胞中研究c-myb基因产物的选择性剪接，然后在正常造血祖细胞中表达野生型和变体c-Myb蛋白，以观察是否诱导分化或增殖的变化。在目标3中，将使用定量测定来跟踪确定的成人AML样本队列中正常和变异c-myb RNA的表达，然后统计分析将确定特定变异是否与AML患者样本中的临床参数、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，并有很大的相关性的RNA剪接，Myb蛋白的活性和人类白血病的研究。

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.