Genomic targets of oncoproteins and tumor suppressors

癌蛋白和肿瘤抑制因子的基因组靶点

• 批准号: 7409989
• 负责人: KEVIN STRUHL
• 金额: $ 78.65万
• 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 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7409989
• 关键词: Address; Affect; Binding; Binding Proteins; Binding Sites; Biological; Biological Process; Breast; Cancer Etiology; Cell Line; Cells; Chromosomes, Human, Pair 21; Collaborations; Condition; Development; Dimensions; Diploidy; Epithelial Cells; Estrogen Receptors; Family; Fibroblasts; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Goals; Growth; Human; Human Biology; Human Genome; Individual; Large T Antigen; Ligand Binding Domain; Link; Location; Malignant Neoplasms; Messenger RNA; Methods; Microarray Analysis; Molecular; Molecular Profiling; Nephroblastoma; Normal Cell; Numbers; Oncogene Proteins; Pathway interactions; Pattern; Physiological; Play; Process; Protein Binding; Protein p53; Proteins; Regulator Genes; Relative (related person); Repression; Research Personnel; Resolution; Retinoblastoma; Retinoic Acid Receptor; Role; Site; Specificity; System; TP53 gene; Tamoxifen; Telomerase; Time; Transcription Factor AP-1; Transcription Regulatory Protein; Tumor Suppressor Proteins; Viral Tumor Antigens; Work; base; cell transformation; cell type; chromatin immunoprecipitation; functional genomics; fusion gene; in vivo; insight; interest; member; metaplastic cell transformation; novel; programs; research study; sialosyl-T antigen; tool; transcription factor; v-src Oncogenes

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。尽管这一目标很重要，但对转录调控元件的无偏见全基因组鉴定从未以实验方式进行过描述。许多转录调节蛋白通过作为癌蛋白和肿瘤抑制蛋白在人类癌症中发挥因果作用。染色质免疫沉淀（ChIP）和微阵列分析的结合是在全基因组水平上识别体内靶点的有力工具，这种方法经常揭示转录调节蛋白的生物学功能的意想不到的和关键的见解。与Affymetrix的Tom Gingeras合作，我们使用了代表所有人类染色体21和22的平铺微阵列，对p53肿瘤抑制因子、Myc癌蛋白和Sp1进行了公正和全面的ChIP分析。这种分析已经确定了许多生理靶点，其中许多是在意想不到的基因组位置。我们打算继续这种合作，在全基因组规模上，确定许多直接参与人类癌症的转录调节蛋白的生理靶点。将采用两种实验系统。首先，我们将使用MCF-10A细胞，这是一种未转化的人乳腺上皮细胞系，在这种细胞系中，Src癌基因（融合到雌激素受体的配体结合域）的表达可以被快速诱导，从而可以比较“正常”细胞和不同状态的细胞在转化途径上的差异。其次，我们将分析h-TERT永生化成纤维细胞是否被T抗原+活化的Ras转化。最初的实验将在大约50个蛋白质（包括p53、Jun-Fos、Myc、Myb、Ets、Rb、E2F家族的肿瘤蛋白和肿瘤抑制因子的多个成员）上进行，这些蛋白质在ENCODE阵列上代表了1%的基因组。从这些ChIP结果和基因表达谱中，将在全基因组基础上使用该提案开始时可用的阵列确定大约35个蛋白质（平均1.5个条件/蛋白质）的最感兴趣的靶点。这些结果将提供大量公正和全面的信息，如1)已知和未知基因的生理靶点位置，2)致癌调节基因与特定因子之间的联系，3)在受调节基因上的因子占用是受调节还是不受调节，4)优先与致癌调节基因相关的因子的特定组合，5)多蛋白家族中蛋白结合的特异性和冗余性；6)调控回路的鉴定。这种正常细胞和转化细胞在两种状态之间的明确转变过程中的详细分子比较，对于研究癌症许多不同方面的众多研究人员来说，应该是非常有用的。更一般地说，这些结果应该代表了功能基因组学的一个新维度，它们应该催生各种实验和计算方法，以了解癌蛋白和肿瘤抑制蛋白是如何导致癌症的。