Genomic targets of oncoproteins and tumor suppressors

癌蛋白和肿瘤抑制因子的基因组靶标

• 批准号: 7093400
• 负责人: KEVIN STRUHL
• 金额: $ 78.22万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-18 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093400
• 关键词: RNA interference; binding sites; biotechnology; cell line; chromatin immunoprecipitation; fibroblasts; functional /structural genomics; gene expression profiling; genetic regulation; microarray technology; neoplasm /cancer genetics; neoplastic transformation; oncoproteins; p53 gene /protein; protein binding; retinoblastoma protein; telomerase; transcription factor

DESCRIPTION (provided by applicant): A key goal of functional genomics is to identify transcriptional regulatory elements throughout the entire human genome that are of physiological significance. Despite the importance of this goal, unbiased, genome-wide identification of transcriptional regulatory elements has never been described in an experimental fashion. Many transcriptional regulatory proteins play causal roles in human cancer by acting as oncoproteins and tumor suppressor proteins. The combination of chromatin immunoprecipitation (ChIP) and microarray analysis is a powerful tool for identifying in vivo target sites on a genome-wide level, and this approach has often revealed unexpected and crucial insights into the biological functions of transcriptional regulatory proteins. In collaboration with Tom Gingeras at Affymetrix, we have used tiled microarrays representing all of human chromosomes 21 and 22 to perform an unbiased and comprehensive ChIP analysis on the p53 tumor suppressor, the Myc oncoprotein, and Sp1. This analysis has identified numerous physiological targets, many of which are in unexpected genomic locations. We intend to continue this collaboration to define, on a whole-genome scale, physiological targets of many transcriptional regulatory proteins directly involved in human cancer. Two experimental systems will be employed. First, we will use MCF-10A cells, a non-transformed human breast epithelial cell line, in which expression of the Src oncogene (fused to the ligand-binding domain of estrogen receptor) can be rapidly induced, thereby permitting a comparison of "normal" cells with cells at different states along the transformation pathway. Second, we will analyze h-TERT immortalized fibroblasts that are or are not transformed with T antigen + activated Ras. The initial experiments will be performed on approximately 50 proteins (including multiple members of the p53, Jun-Fos, Myc, Myb, Ets, Rb, E2F families of oncoproteins and tumor suppressors) on the ENCODE array representing 1 % of the genome. From these ChIP results and gene expression profiles, target sites of approximately 35 proteins (average 1.5 conditions/protein) of highest interest will be determined on a whole-genome basis using arrays that should be available when this proposal would begin. The results will provide a massive amount oi unbiased and comprehensive information such as 1) the location of physiological target sites with respect to known and unknown genes, 2) links between oncogenically-regulated genes and specific factors, 3) whether factor occupancy at regulated genes is regulated or unregulated, 4) specific combinations of factors that preferentially associate with oncogenically-regulated genes, 5) the specificity and redundancy of binding by proteins in multiprotein families, 6) identification of regulatory circuits. This detailed molecular comparison of normal versus transformed cells during a defined transition between the two states should be extremely useful both to numerous investigators who work on many different aspects of cancer. More generally, the results should represent a new dimension in functional genomics, and they should spawn a variety of experimental and computational approaches to understand how oncoproteins and tumor suppressor proteins cause cancer.

描述（由申请人提供）：功能基因组学的一个关键目标是鉴定整个人类基因组中具有生理意义的转录调控元件。尽管这一目标的重要性，公正的，全基因组的转录调控元件的鉴定从来没有在实验中描述。许多转录调控蛋白作为癌蛋白和抑癌蛋白在人类癌症中发挥着重要作用。染色质免疫沉淀（ChIP）和微阵列分析的组合是一个强大的工具，用于在全基因组水平上识别体内靶位点，这种方法往往揭示了意想不到的和关键的见解转录调控蛋白的生物学功能。在与Affytown的Tom Gingeras的合作中，我们使用了代表所有人类21号和22号染色体的平铺微阵列对p53肿瘤抑制因子、Myc癌蛋白和Sp1进行了无偏倚和全面的ChIP分析。该分析已经确定了许多生理靶点，其中许多位于意想不到的基因组位置。我们打算继续这种合作，以确定，在全基因组规模，生理目标的许多转录调控蛋白直接参与人类癌症。将采用两个实验系统。首先，我们将使用MCF-10A细胞，一种非转化的人乳腺上皮细胞系，其中Src癌基因（融合到雌激素受体的配体结合结构域）的表达可以被快速诱导，从而允许比较“正常”细胞与转化途径上沿着不同状态的细胞。其次，我们将分析用T抗原+活化的Ras转化或未转化的h-TERT永生化成纤维细胞。将在代表1%基因组的ENCODE阵列上对约50种蛋白质（包括癌蛋白和肿瘤抑制因子的p53、Jun-Fos、Myc、Myb、Ets、Rb、E2 F家族的多个成员）进行初始实验。根据这些ChIP结果和基因表达谱，将在全基因组基础上使用阵列确定最感兴趣的约35种蛋白质（平均1.5种条件/蛋白质）的靶位点，该阵列应在本提案开始时可用。结果将提供大量无偏见和全面的信息，例如1）关于已知和未知基因的生理靶位点的位置，2）致癌调节基因和特定因子之间的联系，3）在调节基因处的因子占据是受调节的还是不受调节的，4）优先与致癌调节基因相关的因子的特定组合，5）多蛋白质家族中蛋白质结合的特异性和冗余性; 6）调控回路的鉴定。这种在两种状态之间定义的过渡期间对正常细胞与转化细胞的详细分子比较对于研究癌症许多不同方面的许多研究人员都非常有用。更一般地说，这些结果应该代表功能基因组学的一个新维度，它们应该产生各种实验和计算方法来理解癌蛋白和肿瘤抑制蛋白如何导致癌症。