Footprinting the ‘essential regulatory region’ of the retinoblastoma gene promoter in intact human cells

The retinoblastoma tumour suppressor protein is a key cell cycle regulator. Protein–DNA interactions at the retinoblastoma (RB1) promoter, including the ‘essential regulatory region’, were investigated using novel DNA-targeted nitrogen mustards in intact human cells. The footprinting experiments were carried out in two different environments: in intact HeLa and K562 cells where the access of DNA-targeted probes to chromatin is affected by cellular protein–DNA interactions associated with gene regulation; and in purified DNA where their access is unencumbered by protein–DNA interactions. Using the ligation-mediated PCR (LMPCR) technique, the sites of damage were determined at base pair resolution on DNA sequencing gels. Our results demonstrate that, in intact cells, footprints were observed at the E2F, ATF and RBF1/Sp1 DNA binding motifs in the RB1 promoter. In addition, a novel footprint was observed at a previously unidentified cycle homology region (CHR) and at four uncharacterised protein–DNA binding sites. In further experiments, nitrogen mustard-treated cells were FACS sorted into G 1, S and G 2 /M phases of the cell cycle prior to LMPCR analysis. Expression of the RB1 gene is cell cycle-regulated and footprinting studies of the promoter in FACS-sorted cells indicated that transcription factor binding at the GC box, CHR binding motif and the ‘essential regulatory region’ are cell cycle dependent.