Abstract
BruUV-seq utilizes UV light to introduce transcription-blocking DNA lesions randomly in the genome prior to bromouridine-labeling and deep sequencing of nascent RNA. By inhibiting transcription elongation, but not initiation, pre-treatment with UV light leads to a redistribution of transcription reads resulting in the enhancement of nascent RNA signal towards the 5′-end of genes promoting the identification of transcription start sites (TSSs). Furthermore, transcripts associated with arrested RNA polymerases are protected from 3′–5′ degradation and thus, unstable transcripts such as putative enhancer RNA (eRNA) are dramatically increased. Validation of BruUV-seq against GRO-cap that identifies capped run-on transcripts showed that most BruUV-seq peaks overlapped with GRO-cap signal over both TSSs and enhancer elements. Finally, BruUV-seq identified putative enhancer elements induced by tumor necrosis factor (TNF) treatment concomitant with expression of nearby TNF-induced genes. Taken together, BruUV-seq is a powerful new approach for identifying TSSs and active enhancer elements genome-wide in intact cells.
Highlights
The principal steps of the transcriptional cycle are initiation, elongation and termination
To test what effect gene size has on the inactivation of nascent RNA synthesis on a genome-wide scale, we mock-irradiated or irradiated normal human fibroblasts (HF1 cells) with 20 J/m2 of UVC light (254 nm) and immediately labeled nascent RNA with 2 mM bromouridine (Bru) for 30 min
We found that BruUV-seq exhibits greater enrichment near the transcription start sites (TSSs) than GRO-seq, as the BruUV-seq signal substantially drops off ~5 kb into gene bodies, whereas GRO-seq captures signal throughout the gene body and in this way appears similar to bromouridine labeling and sequencing (Bru-seq) signal (Supplementary Fig. 9b)
Summary
The principal steps of the transcriptional cycle are initiation, elongation and termination. ChIP-seq on its own, does not conclusively demonstrate promoter or enhancer usage but rather indicates the potential of a chromatin region or transcription initiation complex to be used as a TSS or enhancer Another approach to identify TSS and enhancers genome-wide involves tagging of the 5′ -ends of transcripts, examples of which include “cap analysis gene expression” (CAGE)7,8, “5′ -end serial analysis of gene expression” (SAGE)[9] and “gene identification signature” analysis (GIS) with “pair end tags” (PET)[10]. UVC light (254 nm) introduces predominantly cyclobutane pyrimidine dimers and 6–4 photoproducts in DNA that are distributed more or less randomly in the genome[21] These lesions are strong transcriptional blocks, causing RNA polymerase II elongation complexes to stall[22,23]. BruUV-seq can readily identify TSSs and active enhancers as well as unstable transcripts genome-wide in intact cells, and assess changes in transcription levels both at promoter and enhancer regions following exposure to a stimulus or stress
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