Protein–DNA footprinting of the human ϵ-globin promoter in human intact cells using nitrogen mustard analogues and other DNA-damaging agents

Abstract

Nitrogen mustard analogues, bleomycin and dimethyl sulphate (DMS) have been used as probes of protein–DNA interactions in intact human cells. The sites of damage have been determined at base pair resolution in the single copy ϵ-globin gene promoter in erythroid K562 cells, non-erythroid HeLa cells and purified DNA. Exponential amplification of gene-specific damage fragments was achieved using the ligation-mediated polymerase chain reaction (LMPCR) technique and analysed on DNA sequencing gels. A comparison of the relative damage band intensities between purified DNA and intact cells revealed several significant differences – both protection (footprint) and enhancement. These differences occurred at putative transcription factor binding sites and hence are thought to be due to protein–DNA interactions. A major feature of the band intensity ratio plots was the footprint observed at the CCAAT box binding motif as revealed by nitrogen mustard analogues. Enhanced band intensity (hypersensitivity) was displayed at the 5′- and 3′-ends of the CCAAT box in K562 cells – this feature was absent in HeLa cells and in vitro reconstitutions. A footprint was found at the GATA-1 motif in K562 cells that was also absent in non-expressing HeLa cells. Footprints were also evident at the TATA box, CACC box and the ϵF1 DNA binding motif in K562 cells.