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

Nitrogen mustard analogues, bleomycin and dimethyl sulphate (DMS) have been used as probes of protein-DNA interactions in intact human cells. The site s of damage have been determined at base pair resolution in the single copy E-globin gene promoter in erythroid K562 cells, non-erythroid HeLa cells a nd purified DNA. Exponential amplification of gene-specific damage fragment s was achieved using the ligation-mediated polymerase chain reaction (LMPCR ) technique and analysed on DNA sequencing gels. A comparison of the relati ve damage band intensities between purified DNA and intact cells revealed s everal significant differences - both protection (footprint) and enhancemen t. These differences occurred at putative transcription factor binding site s and hence are thought to be due to protein-DNA interactions. A major feat ure of the band intensity ratio plots was the footprint observed at the CCA AT box binding motif as revealed by nitrogen mustard analogues. Enhanced ba nd 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 vit ro 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 ev ident at the TATA box, CACC box and the epsilon F1 DNA binding motif in K56 2 cells. (C) 1999 Elsevier Science B.V. All rights reserved.