Repression of new p53 targets revealed by ChIP on chip experiments
Repression of new p53 targets revealed by ChIP on chip experiments
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DOI：
10.4161/cc.5.10.2777
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发表时间：
2006-05-15
期刊：
影响因子：
4.3
通讯作者：
Mantovani, Roberto
中科院分区：
文献类型：
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作者：
Ceribelli, Michele;Alcalay, Myriam;Mantovani, Roberto
DNA 损伤后，肿瘤抑制因子 p53 激活 G(1)/S 阻断和凋亡基因，并抑制其他基因，包括参与 G(2)/M 转变的基因。在后一个系统中，它通过 CCAAT 结合组蛋白样 NF-Y 发挥作用。多个研究小组报告称，p53 在无应激条件下与启动子相关。我们开发了一种包含 179 个人类启动子的寡核苷酸阵列，富含参与 DNA 损伤和 ER 应激反应的基因。我们在正常生长条件下的细胞中使用 p53 和 NF-Y 进行了 ChIP 芯片实验。我们鉴定了 46 个新的 p53 靶标，并注意到 (i) ER 应激反应基因显着富集，包括 XBP1 和 C/EBP beta 等关键调节因子； (ii)其产物参与p53功能调节的基因。通过传统 ChIP 验证了多个基因。在大多数（但不是所有）启动子上观察到 DNA 损伤依赖性 PCAF 介导的乙酰化。通过 RT-PCR 和 HCT116 wt、E6 和 p53(-/-) 细胞中的转染检查 p53 激活的效果：大多数启动子在阿霉素处理后或 p53-/- 细胞中 p53 转染后受到积极抑制。特别是，一些基因（BRCA1、RAD23 和 RAD17）的行为与 p53 水平的反馈环路调节一致。最后，p53 和 NF-Y 靶标之间存在很大的重叠 (66%)。我们的数据重申了 p53 启动子识别和直接转录抑制作为应对 DNA 损伤机制的生理重要性。
Following DNA-damage, the tumor suppressor p53 activates G(1)/S blocking and apoptotic genes, and represses other genes, including those involved in G(2)/M transition. In this latter system, it acts through the CCAAT-binding histone-like NF-Y. Several groups have reported that p53 is associated to promoters in unstressed conditions. We developed an oligo-based array containing 179 human promoters, enriched in genes involved in the DNA-damage and ER-stress response. We performed ChIP on chip experiments with p53 and NF-Y in cells under normal growing conditions. We identified 46 new p53 targets and noted (i) a significant enrichment in genes of the ER-stress response, including crucial regulators such as XBP1 and C/EBP beta; (ii) genes whose products are involved in the regulation of p53 function. Several genes were validated by conventional ChIP. DNA-damage dependent PCAF-mediated acetylation was observed on most, but not all promoters. The effect of p53 activation was checked by RT-PCR and transfections in HCT116 wt, E6 and p53(-/-) cells: most promoters were actively repressed upon Adriamycin treatment or following p53 transfection in p53-/- cells. In particular, the behaviour of some of the genes (BRCA1, RAD23 and RAD17) is consistent with a feedback loop regulation on p53 levels. Finally, there is a large overlap (66%) between p53 and NF-Y targets. Our data reinstate the physiological importance of p53 promoter recognition and direct transcriptional repression as a mechanism to cope with DNA-damage.