Post-translational modifications affect c-Myb specificity.

翻译后修饰影响 c-Myb 特异性。

• 批准号: 7322280
• 负责人: Anita M Quintana
• 金额: $ 2.88万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-06 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7322280
• 关键词: Acute Myelocytic Leukemia; Affect; Antibodies; Binding; Binding Sites; Biological Assay; Biological Process; Cell Cycle; Cell Cycle Stage; Cell Differentiation process; Cells; Chickens; Chromatin; Co-Immunoprecipitations; DNA; DNA Binding; DNA Binding Domain; Data; Flow Cytometry; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Goals; Harvest; Hematopoiesis; Hematopoietic; Human; Immunoprecipitation; In Vitro; Knowledge; Laboratories; Lead; MYB gene; Mediating; Microarray Analysis; Modeling; Modification; Pattern; Phosphorylation; Phytohemagglutinins; Point Mutation; Post-Translational Protein Processing; Process; Propidium; Proteins; Proto-Oncogene Proteins c-myb; Proto-Oncogenes; Regulation; Research; Sampling; Specificity; Staging; T-Lymphocyte; Time; Viral Oncogene; cell transformation; chromatin immunoprecipitation; follow-up; in vivo; promoter; protein protein interaction; research study; tissue culture; transcription factor; v-myb Genes

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.

描述（由申请人提供）：C-Myb是一种原癌基因，序列与病毒癌基因v-Myb相似。V-Myb能引起鸡急性髓性白血病（AMI），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v-Myb的转化能力。这两种转录因子在体外均能结合靶基因（如mim-1基因）的启动子，但v-Myb不能激活内源性mim-1。这与其他数据一起表明，这两个基因具有非常不同的特异性，这些特异性受体内因素的影响。一个可能有助于体内转录因子特异性的机制是翻译后修饰。下面提出的研究旨在确定特异性翻译后修饰如何影响人类原代t细胞整个细胞周期的内源基因表达。特异性目标1涉及细胞周期中特定时间点翻译后修饰的表征。静止t细胞将被植物血凝素诱导，免疫沉淀和免疫印迹抗修饰特异性抗体。作为目标1的后续，特异性目标2将分析c- Myb特异性修饰版本的启动子靶标。样品将在细胞周期的特定时间点采集，并进行染色质免疫沉淀。这些富集后的样品将进行Affymetrix平铺阵列鉴定c- Myb靶基因。特异性靶1中上述免疫沉淀的结果可以与特异性靶2中确定的启动子目标相关联。这两个目标共同用于识别和表征细胞周期中特定时间点的c-Myb特异性。具体目标3与上述目标相关，并将导致进一步了解细胞周期。实验室已经证明C-Myb与CDK6结合，但这种相互作用的时间过程目前尚不清楚。Specific aim 3将使用细胞周期中的特定时间点来分析CDK6与c-Myb在细胞周期的哪个阶段相互作用。流式细胞术将用于测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。研究细胞周期中c-Myb修饰特异性基因的表达可能会为理解造血和许多造血分化的未知过程带来突破。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性白血病的认识。