Post-translational modifications affect c-Myb specificity.

描述（由申请方提供）：C-Myb是一种原癌基因，与病毒癌基因v-Myb具有序列相似性。V-Myb能够在鸡中引起急性髓性白血病（AMI），并且可以在体外转化造血细胞。虽然v-Myb已显示转化细胞，但c-Myb缺乏v-Myb转化能力。这两种转录因子都能够在体外结合靶基因的启动子，如mim-1基因，但v-Myb不能激活内源性mim-1。这沿着其他数据表明，这两个基因具有非常不同的特异性，这是受体内因素的影响。可能有助于体内转录因子特异性的一种机制是翻译后修饰。以下拟议的研究旨在确定特定的翻译后修饰如何影响原代人T细胞整个细胞周期的内源性基因表达。具体目标1涉及在细胞周期的特定时间点期间的翻译后修饰的表征。将用植物血凝素诱导静止T细胞，用抗修饰特异性抗体进行免疫沉淀和免疫印迹。作为目标1的后续，具体目标2将分析c-Myb的特异性修饰形式的启动子靶标。将在细胞周期的特定时间点采集样本，并进行染色质免疫沉淀。然后将那些富集的样品进行亲和层析镶嵌阵列以鉴定c-Myb靶基因。特异性目的1中的上述免疫沉淀的结果然后可以与特异性目的2中鉴定的启动子靶标相关联。这两个目的一起用于在细胞周期的整个特定时间点鉴定和表征c-Myb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。实验室已证明C-Myb与CDK 6结合，但这种相互作用的时间过程目前尚不清楚。具体目标3将使用细胞周期中的特定时间点来分析CDK 6在细胞周期的哪个阶段与c-Myb相互作用。将使用丙啶流式细胞术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修饰特异性基因在细胞周期中的表达可能会导致对造血和造血分化中许多未知过程的理解的突破。这项研究有助于全面了解c-Myb和v-Myb之间的表型差异，这可以增强我们对人类急性髓系白血病的理解。

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.