Post-translational modifications affect c-Myb specificity.

描述(申请人提供)：C-Myb是一种原癌基因，与病毒癌基因v-Myb序列相似。V-Myb可引起鸡急性髓系白血病，并能在体外转化造血细胞。虽然v-Myb已被证明可以转化细胞，但c-Myb缺乏转化v-Myb的能力。这两种转录因子在体外都能与目的基因的启动子结合，如mim-1基因，但v-Myb不能激活内源性mim-1。这一点与其他数据一起表明，这两个基因具有非常明显的特异性，受到体内因素的影响。体内转录因子特异性的一个机制可能是翻译后修饰。这项拟议的研究旨在确定特定的翻译后修饰如何影响原代人类T细胞细胞周期中内源性基因的表达。具体目标1涉及细胞周期中特定时间点的翻译后修饰的特征。静止的T细胞将被植物血凝素诱导，免疫沉淀，并用抗修饰特异性抗体免疫印迹。作为目标1的后续，特定目标2将分析c-Myb的特定修饰版本的启动子靶标。样本将在细胞周期的特定时间点采集，并接受染色质免疫沉淀。然后，这些浓缩的样本将受到Affymetrix平铺阵列的影响，以确定c-Myb目标基因。然后，在特定目的1中的上述免疫沉淀的结果可以与特定目的2中确定的启动子靶相关联。这两个目的一起用于在细胞周期的特定时间点识别和表征c-Myb的特异性。具体目标3与上述目标相关，并将导致对细胞周期的进一步了解。实验室已经证明C-Myb与CDK6结合，但这种相互作用的时间进程目前尚不清楚。特定目标3将使用细胞周期中的特定时间点来分析细胞周期中CDK6在哪个阶段与c-Myb相互作用。丙二醛的流式细胞术将被用来确定细胞处于细胞周期的哪个阶段。这些实验将有助于确定翻译后修饰对c-Myb特异性的影响。检测c-Myb修饰基因在细胞周期中的表达可能会导致在理解造血和许多未知的造血分化过程中取得突破。本研究有助于全面了解c-Myb和v-Myb的表型差异，从而提高我们对人类急性髓系白血病的认识。

DESCRIPTION (provided by applicant): C-Myb is a proto-oncogene with sequence similarity to the viral oncogene v-Myb. V-Myb is capable of causing acute myeloid leukemia (AMI) in chickens and can transform hematopoietic cells in vitro. Although v-Myb has been shown to transform cells, c-Myb lacks v-Myb ability to transform. Both transcription factors are able to bind to promoters of target genes such as the mim-1 gene in vitro, but v-Myb cannot activate the endogenous mim-1. This along with other data suggests that the two genes have very distinct specificities, which are influenced by in vivo factors. One mechanism that may contribute to specificity of in vivo transcription factors is post-translational modifications. The following proposed research aims to determine how specific post-translational modifications influence endogenous gene expression throughout the cell cycle of primary human T-cells. Specific aim 1 involves the characterization of post-translational modifications during specific time points in the cell cycle. Quiescent T-cells will be induced with phytohemagglutinin, immunoprecipitated and immunobloted with anti-modification specific antibodies. As a follow up to aim 1, specific aim 2 will analyze the promoter targets for specifically modified versions of c- Myb. Samples will be taken at specific time points in the cell cycle and subjected to chromatin immunopreciptitation. Those enriched samples will then be subjected to Affymetrix tiling arrays to identify c- Myb target genes. The results from the above immunoprecipitations in specific aim 1 can then be correlated with the promoter targets identified in specific aim 2. Together these two aims serve to identify and characterized c-Myb specificity throughout specific time points in the cell cycle. Specific aim 3 is related to the above aims and will lead to further knowledge of the cell cycle. C-Myb has been shown by the laboratory to bind to CDK6, but the time course of this interaction is currently unknown. Specific aim 3 will use specific time points during the cell cycle to analyze at what stage in the cell cycle CDK6 interacts with c-Myb. Flow cytometry with propidium will be used to determine what stage of the cell cycle the cells are in. These experiments will help to determine the influence of post-translational modifications on c-Myb specificity. Examining c-Myb modification specific gene expression in the cell cycle could lead to break throughs in understanding hematopoiesis and many of the unknown processes in hematopoietic differention. This research stands to contribute to the overall understanding of the phenotypic differences between c-Myb and v-Myb, which can enhance our understanding of human acute myeloid leukemia.