Mechanisms of Genotoxic Stress-regulated Gene Expression in Human Cells

描述（由申请人提供）：细胞暴露于电离辐射（IR）激活DNA损伤反应，通过调控转录和转录后事件导致基因表达的全面重编程。调控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拨款申请中使用新开发的BrU-Seq技术来详细研究这些机制。在Specific Aim #1中，将探讨IR对人类成纤维细胞中所有rna的合成和稳定性的整体影响。初步实验表明，应激激酶ATM对于人类成纤维细胞中某些mrna（如BTG2）的合成和稳定性的增加是必需的，需要测试的假设是，在暴露于IR后，可能有更多的基因以ATM依赖的方式在合成和/或稳定性水平上受到影响。在具体目标#2中，将探讨IR对选择性剪接的影响。初步结果表明，内含子保留在人成纤维细胞中加工的mrna中很常见，我们的假设是，由于大量剪接因子是激活ATM的底物，IR可能以ATM依赖的方式影响人细胞中的剪接代码签名。在特异性目标#3中，IR对转录起始位点（TSS）和增强子激活的影响

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. PUBLIC HEALTH RELEVANCE: Exposure of cells to ionizing radiation activates DNA damage responses leading to a comprehensive reprogramming of gene expression. The goal of this R21 proposal is to use the newly developed BrU-Seq technique to study the global effects of ionizing radiation on the regulation of both transcriptional and post-transcriptional events in normal human cells. A better understanding of these mechanisms will provide new insights about cell responses induced by human exposure to ionizing radiation and may lead to improved strategies of radiation therapy.