oa Mechanisms of Transcriptional Inhibition and Induction by Environmental Mutagens

Exposure of cells to environmental mutagens activates cellular responses, leading to a reprogramming of gene expression by mechanisms that are not fully elucidated. To investigate the mechanisms of how environmental mutagens affect global gene expression in human cells both at the level of RNA synthesis and transcript stability, we developed a new technique, called BruChase-Seq, based on bromouridine (BrU) pulse-chase labeling coupled to deep sequencing. Using BruChase-Seq we found that ionizing radiation, cadmium or the pro-inflammatory cytokine TNF rapidly altered both the synthesis and the transcript stability of specific sets of genes in human fibroblasts. To directly assess the effect of UV light on nascent RNA transcription, we developed BrUV-Seq, where cells are irradiated with UV-light immediately before labeling nascent RNA with BrU. We found that UV-induced DNA lesions inhibited transcription elongation but not initiation, leading to a dramatic enhancement of sequencing signal at transcription start sites. Unexpectedly, we found that UV-irradiation also caused an enrichment of non-coding RNA and enhancer sequences, potentially making it possible to use BrUV-Seq to map poorly annotated non-coding RNAs and active enhancer elements genome-wide. We believe that the BruChase-Seq and BrUV-Seq approaches will be useful in elucidating the mechanisms of gene regulation in many biological settings.