Chromatin structure in living cells

The authors have used psoralen cross-linking to investigate the distribution of nucleosomes in living mouse L cells. The cells were studied in only one state, when their growth approached saturation. The most discrete pattern of cross-linking was seen when the cells were rapidly photoreacted at their normal growth temperature of 37 °C. The fraction of this DNA in nucleosome structure was estimated as 58-89%, based on the fraction of the DNA that showed me₃psoralen cross-links at 195-bp intervals. The remaining DNA appeared to be protected from me₃psoralen over lengths of 500 bp or more, but not necessarily protected by nucleosomes. The data provided strong evidence against the presence of long regions of protein-free DNA in the growing L cells, except perhaps at a low frequency (less than 2% of the DNA). When L cells were quickly cooled to 1° C and then rapidly photoreacted, the cross-linking pattern of the DNA again showed evidence for a nucleosome structure. However, several anomalous features appeared: The size distribution of the DNA protected from psoralen by nucleosomes was more heterogeneous (broader and less symmetric peaks); there was a higher ratio of dimer- to monomer-size protection units; and long regions of DNA (400-1000 bp) that appeared to be heavily cross-linked were observed at a 10% frequency, possibly representing regions of protein-free DNA or of structurally altered nucleosomes. The pattern of cross-linking in the DNA of isolated mouse-liver nuclei photoreacted at low temperature again showed strong evidence for nucleosomes, but shared many of the anomalous features seen when cells were photoreacted at low temperature. It appears that low temperature may produce some changes in chromatin structure, either changes in accessibility of nucleosome DNA to me₃psoralen or redistribution of nucleosomes on the DNA.