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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21ES020946-02
Application #
8462978
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Mcallister, Kimberly A
Project Start
2012-05-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
2
Fiscal Year
2013
Total Cost
$190,488
Indirect Cost
$67,988
Name
University of Michigan Ann Arbor
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Veloso, Artur; Kirkconnell, Killeen S; Magnuson, Brian et al. (2014) Rate of elongation by RNA polymerase II is associated with specific gene features and epigenetic modifications. Genome Res 24:896-905
Veloso, Artur; Biewen, Benjamin; Paulsen, Michelle T et al. (2013) Genome-wide transcriptional effects of the anti-cancer agent camptothecin. PLoS One 8:e78190
Paulsen, Michelle T; Veloso, Artur; Prasad, Jayendra et al. (2013) Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response. Proc Natl Acad Sci U S A 110:2240-5