The anti-tumor drug bleomycin preferentially cleaves at the transcription start sites of actively transcribed genes in human cells

Abstract

The genome-wide pattern of DNA cleavage at transcription start sites (TSSs) for the anti-tumor drug bleomycin was examined in human HeLa cells using next-generation DNA sequencing. It was found that actively transcribed genes were preferentially cleaved compared with non-transcribed genes. The 143,600 identified human TSSs were split into non-transcribed genes (82,596) and transcribed genes (61,004) for HeLa cells. These transcribed genes were further split into quintiles of 12,201 genes comprising the top 20, 20-40, 40-60, 60-80, and 80-100 % of expressed genes. The bleomycin cleavage pattern at highly transcribed gene TSSs was greatly enhanced compared with purified DNA and non-transcribed gene TSSs. The top 20 and 20-40 % quintiles had a very similar enhanced cleavage pattern, the 40-60 % quintile was intermediate, while the 60-80 and 80-100 % quintiles were close to the non-transcribed and purified DNA profiles. The pattern of bleomycin enhanced cleavage had peaks that were approximately 200 bp apart, and this indicated that bleomycin was identifying the presence of phased nucleosomes at TSSs. Hence bleomycin can be utilized to detect chromatin structures that are present at actively transcribed genes. In this study, for the first time, the pattern of DNA damage by a clinically utilized cancer chemotherapeutic agent was performed on a human genome-wide scale at the nucleotide level.

The Interaction of the Metallo-Glycopeptide Anti-Tumour Drug Bleomycin with DNA

“…For the globin and retinoblastoma DNA sequences, bleomycin was able to footprint transcription factors and positioned nucleosomes in human cells. Bleomycin is known to cleave in the linker region of nucleosomes [ 93 , 98 , 99 ] and hence bleomycin is also a useful agent to probe chromatin structure in human cells ( Figure 3 ).…”

“…In our laboratory, the sequence specificity of bleomycin-induced double-strand breaks in the entire human genome has been studied by use of massively parallel next-generation sequencing technology. Contrary to the approximately 100 sites investigated with the previous techniques, the next-generation sequencing technology has enabled more than 200 million bleomycin double-strand break sites in both cellular and purified DNA to be assessed in order to determine the genome-wide sequence specificity [ 80 , 99 , 100 , 101 ]. It should be noted that the genome-wide studies mainly detect double-strand breaks, whereas the experiments with purified end-labelled DNA sequences (discussed above) mainly detect single-strand breaks.…”

“…This implies that positioned nucleosomes are present at transcription start sites and that bleomycin preferentially cleaves in the linker region of the nucleosome and the nucleosome core protects DNA from bleomycin cleavage [ 109 , 110 , 111 ]. Hence, bleomycin can be used to detect chromatin structure at actively transcribed genes [ 99 ].…”

“…Bleomycin preferentially damaged actively transcribed genes ( Figure 4 ) and the degree of bleomycin cleavage correlated with the level of transcription [ 99 ]. This enhanced bleomycin damage occurred within 1000 bp of transcription start sites (TSSs).…”

The influence of p53 status on the cytotoxicity of fluorinated pyrimidine L-nucleosides

“…Genomic DNA was isolated from 10 6 cells using an SDSproteinase K-phenol extraction technique as previously described [30,31]. A three primer polymerase chain reaction (PCR) was used to confirm the p53 status of the primary mouse fibroblasts.…”