Mechanisms of Genotoxic Stress-regulated Gene Expression in Human Cells

描述（由申请方提供）：细胞暴露于电离辐射（IR）激活DNA损伤反应，导致通过调节转录和转录后事件对基因表达进行全面重编程。调节转录和转录后事件的机制尚未完全阐明，更好地了解这些机制有助于改善临床放射治疗。我们将在修订后的R21拨款提案中使用新开发的BrU-Seq技术来详细研究这些机制。在具体目标#1中，将探索IR对人成纤维细胞中所有RNA合成和稳定性的整体影响。初步实验表明，应激激酶ATM是增加某些mRNA（如BTG 2）在人成纤维细胞中的合成和稳定性所必需的，待检验的假设是，在暴露于IR后，可能有更多的基因以ATM依赖性方式在合成和/或稳定性水平受到影响。在具体目标#2中，将询问IR对可变剪接的影响。测序结果表明，内含子保留是常见的人成纤维细胞中加工的mRNA和我们的假设是，因为大量的剪接因子的底物激活ATM，IR可能会影响剪接代码签名在人类细胞中的ATM依赖性的方式。在具体目标#3中，研究了IR对转录起始位点（TSS）和增强子的激活的影响。 基因组中的元件将使用涉及在BrU脉冲标记之前进行UV照射的技术进行研究，以在基因组中引入随机转录阻断损伤。初步结果表明，利用这种技术，所有的转录起始位点和潜在的增强子元件可以在基因组中定位。有待检验的假设是，IR可能会影响TSS和增强子元素的选择，ATM可能参与调节这些改变。这项R21提案的主要创新是使用新的BrU-Seq技术来全面探索IR如何影响“转录组”、“RNA稳定组”、剪接密码以及TSS和增强子元件的选择。分析不仅包括mRNA，还包括非编码RNA，如microRNA。这些研究可能不仅对我们理解暴露于IR后改变的基因和microRNA调控机制产生重大影响，而且这种新技术将在其他环境中研究基因表达机制方面具有普遍应用。

DESCRIPTION (provided by applicant): Exposure of cells to ionizing radiation (IR) activates DNA damage responses leading to a comprehensive reprogramming of gene expression by regulation of both transcriptional and post- transcriptional events. The mechanisms regulating transcriptional and post-transcriptional events are not fully elucidated and a better knowledge about these mechanisms could help improve radiation therapy in the clinic. We will in this revised R21 grant proposal use the newly developed BrU-Seq technique to study these mechanisms in detail. In Specific Aim #1, the global effects of IR on the synthesis and stability of all RNAs in human fibroblasts will be explored. Preliminary experiments show that the stress kinase ATM is required for increased synthesis and stability of certain mRNAs such as BTG2 in human fibroblasts and the hypothesis to be tested is that there may be many more genes affected at the level of synthesis and/or stability in an ATM-dependent way following exposure to IR. In Specific Aim #2, the effects of IR on alternative splicing will be interrogated. Preliminry results show that intron retention is common among processed mRNAs in human fibroblasts and our hypothesis is that since a large number of splicing factors are substrates for activated ATM, IR may affect the splicing code signature in human cells in an ATM-dependent manner. In Specific Aim #3 the effects of IR on the activation of transcription start sites (TSS) and enhancer elements in the genome will be studied using a technique involving UV-irradiation prior to the BrU pulse-labeling to introduce random transcription-blocking lesions in the genome. Preliminary results show that with this technique, all transcription start sites and potentially enhancer elements can be mapped in the genome. The hypothesis to be tested is that IR may affect the selection of TSS and enhancer elements and that ATM may be involved in regulating these alterations. The major innovation of this R21 proposal is the use of the novel BrU-Seq technique for global exploration of how IR affects the "transcriptome", the "RNA stabilome", the splicing code and selection of TSS and enhancer elements. The analysis will not only include mRNAs but also non-coding RNAs such as microRNAs. These studies may have a great impact on not only our understanding of the mechanisms of altered gene and microRNA regulation following exposure to IR but also, this new technique will have general applications for studying mechanisms of gene expression in other settings.