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Transcriptional and Epigenetic Responses of Human Cells to Low Dose Ionizing Radiation Identified through High Throughput ChIP-Seq Analysis

Carl Anderson
Brookhaven National Laboratory

Abstract

The major consequence of human exposures to ionizing radiation (IR) is considered to be an increased incidence of cancer (Brenner et al., 2003). Exposure of cells to 1 Gy of IR produces approximately 40 double-stranded breaks, 1000 single-stranded breaks, and 1000 damaged bases per genome equivalent (Pandita and Richardson, 2009); however, most direct DNA damage is rapidly repaired. Exposure to IR also induces epigenetic changes including both increases and decreases in DNA methylation, and increasing evidence suggests that epigenetic changes can both initiate cancer and cause its progression (Feinberg et al., 2009) through at least four possible mechanisms: the transcriptional activation of oncogenes, the transcriptional suppression of tumor suppressor genes, the induction of genetic instability through methylation-mediated changes in chromatin structure and DNA replication timing, and genome instability caused by DNA demethylation-mediated activation of endogenous retroviruses. However, mechanisms of radiation-induced epigenetic changes are not well known. Epigenetic changes also could, in principle, prevent or reduce cancer incidence through the silencing of "survival" genes such as survivin (BIRC5). Others have shown that the exposure of human HCT-116 cells to doxorubicin, a commonly used anticancer drug that causes DNA damage, induced a p53-dependent methylation of the survivin gene promoter and silencing of survivin expression (Estève et al, 2005).

We are testing the hypothesis that low dose radiation-induced activation of transcription factors (and/or other DNA binding proteins) such as p53 induce changes in the epigenome that may affect cancer susceptibility through three approaches. First, using whole genome high throughput sequencing of chromatin immunoprecipitated DNA fragments (ChIP-Seq), we are identifying sites in the human genome to which p53 binds (p53REs) both before and after exposure to low dose IR. Second, we will determine if exposure to IR induces changes in the methylation state of the survivin promoter and at other genomic sites at which p53 binds. Third, we are developing novel tools for genome-wide DNA methylation surveys that can be used to efficiently identify radiation-induced changes in DNA methylation at both targeted loci and in whole genomes. An understanding of the molecular consequences of DNA methylation induced changes as a function of dose and dose rate will facilitate prediction of the long term consequences of exposure to IR. Furthermore, an understanding of the mechanisms that lead to stress-induced DNA methylation changes may lead to the development of countermeasures to protect radiation workers, astronauts, and others from such exposures.

To validate our approach, we have first identified p53REs in two human cell types, early passage normal fibroblasts (IMR90) and the colon cancer-derived human cell line HCT116, under stress conditions used by others (Wei et al., 2006). Immunoprecipitation of p53-selected chromatin fragments obtained 6 hr after treatment with 5-FU was followed by high-throughput Solexa/Illumina sequencing (ChIP-seq) for genome-wide mapping of p53 binding sites. Seven to eleven million sequencing reads were obtained per p53-specific library. The sequence reads were mapped to the human genome sequence (hg18), and the mapped reads were normalized to the chromatin background from input DNA libraries for each cell line to identify significant p53-specific binding peaks. More than 500 high-confidence binding sites were identified for each cell line, a subset of which was validated by qPCR. In addition to the identified p53 binding sites common to both cell types, cell type-specific p53 binding in response to 5-FU was observed. Thus, p53 binding to chromatin upon stress is partially cell type specific. We are analyzing the p53 binding pattern in IMR90 fibroblasts compared to the recently published, genome-wide, single-base-resolution map of methylated cytosines in the IMR90 genome (Lister et al. 2009) and will discuss p53 binding in the context of the genomic and chromatin landscape. Our data represent the largest and most comprehensive study of p53 binding sites in the human genome to date. Efforts currently are directed at extending this analysis to cells exposed to high and low dose ionizing radiation and to other human cell types.

This work was funded by the DOE's Office of Biological and Environmental Research Low Dose research Program. | PDF Version

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