Gene expression in human lymphoblastoid cell-line GM12878 in response to doxorubicin treatment. Homo sapiens

To determine if induced p53 binding is associated with gene expression in genome-wide. We examined mRNA levels with the Affymetrix Human Exon 1.0 ST platform in human lymphoblastoid GM12878 cells treated with doxorubicin to activate p53. In response to various cellular stresses, the tumor suppressor gene p53 induces activation or repression of more than a thousand human genes. Selective binding and transactivation of a large potential pool of p53 response elements (REs) is believed to regulate the variation in stress response across stress types and between cell types. To elucidate how the human genome is targeted by p53 at the chromatin level, we mapped the genome-wide localization of p53 and H3K4me3 from Doxo-treated human lymphoblastoid cells, and examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 Hypersensitivity, DHS), ENCODE chromatin states, RE sequence specificity and evolutionary conservation. Overall design: Human lymphoblastoid (GM 12878) cells at a density of 900,000 cells/ml were prepared in triplicate for each time point and treated with 0.5 µM doxorubicin (Calbiochem) for 4, 18 hr or no treatment (at 0 time). Total RNA will be extracted from each culture (9 RNA samples) using the Qiagen RNeasy kit with DNase digestion. RNA was quantified using RiboGreen (Invitrogen), check for quality by OD and Bioanalyzer and stored at -80°C. Expression analysis was conducted at at NIEHS Microarray Core using Affymetrix Human Exon 1.0 ST arrays following the Affymetrix hybridization protocols. Exon expression data were analyzed through Affymetrix Expression Console using gene-level RMA summarization and sketch-quantile normalization methods.

为探究p53的诱导性结合是否与全基因组基因表达存在关联，我们采用Affymetrix Human Exon 1.0 ST 芯片平台（Affymetrix Human Exon 1.0 ST），对经阿霉素（doxorubicin）处理以激活p53的人类淋巴母细胞GM12878的mRNA水平进行了检测。在多种细胞应激刺激下，抑癌基因p53可诱导超过千条人类基因的激活或沉默。目前认为，对大量潜在p53应答元件（p53 response elements, REs）的选择性结合与反式激活，可调控不同应激类型及细胞类型间的应激应答差异。为阐明p53在染色质层面如何靶向人类基因组，我们对经阿霉素处理的人类淋巴母细胞进行了p53与组蛋白H3赖氨酸4三甲基化（H3K4me3）的全基因组定位分析，并探究了p53结合占据度、基因表达、H3K4me3、染色质可及性（DNase I超敏位点, DNase 1 Hypersensitivity, DHS）、ENCODE染色质状态、RE序列特异性与进化保守性之间的关联。

实验设计：将密度为9×10^5 cells/ml的人类淋巴母细胞GM12878按每个时间点设置三次生物学重复，分别用0.5 μM 阿霉素（Calbiochem品牌）处理4小时、18小时，同时设置未处理对照组（0小时）。我们使用Qiagen RNeasy试剂盒（Qiagen RNeasy kit）并辅以DNase消化，从每份培养物中提取总RNA，共获得9份RNA样本。采用RiboGreen试剂（RiboGreen，Invitrogen品牌）对RNA进行定量，通过光密度（OD）检测与生物分析仪（Bioanalyzer）评估RNA质量，并将合格样本保存于-80℃冰箱。表达分析在美国国立环境卫生科学研究所（National Institute of Environmental Health Sciences, NIEHS）微阵列核心实验室完成，实验严格遵循Affymetrix杂交实验流程，使用Affymetrix Human Exon 1.0 ST 芯片进行检测。外显子表达数据通过Affymetrix Expression Console软件进行分析，采用基因水平的稳健多阵列平均（Robust Multi-array Average, RMA）汇总算法与草图分位数归一化方法进行数据处理。