Nucleosome positioning in the human c-Fos promoter analyzed by in vivo footprinting with psoralen and ionizing radiation.

We performed detailed footprinting analysis of nucleosome positioning in the c-FOS promoter of living human fibroblasts. The translational position was determined by terminal transferase-dependent PCR with 4,5',8-trimethylpsoralen. The rotational position was determined by ligation-mediated PCR with ionizing radiation. In the middle of the c-FOS promoter, a nucleosome was positioned not only translationally but also rotationally. The comparison of the results of our in vivo footprinting with those of a previous report on the in vitro footprinting of reconstituted nucleosomes revealed that the major in vivo translational position was approximately 70 bp upstream of the in vitro position, whereas the rotational position was unchanged. The in vivo translational position appears to be strongly influenced by the presence of transcription factors, which may function as boundaries, while the rotational position appears to be determined predominantly by the DNA sequence. We also investigated the influence of the transcriptional activation of the c-FOS gene on the positioning of this nucleosome. Although it is well-known that there are rapid changes in general nuclease sensitivity and chemical modifications of histone in the c-FOS gene upon activation, we could not detect any change in the translational or rotational position of this nucleosome. The nucleoprotein complex in the c-FOS promoter containing the positioned nucleosome and several transcription factors seems to be structurally unaltered upon activation, despite the rapid chemical modifications of the nucleosome and some of the transcription factors.