A novel method using DNA binding fluorochromes as indirect probes to study linker histone-chromatin interactions in situ

Sammanfattning: Over the years, a variety of methods have been used to study chromatin structure. These techniques have provided valuable information about nuclear framework, chromatin organisation, and the proteins that are involved in the formation and maintenance of chromatin. However, neither ultrastructural nor biochemical methods are suitable for investigating the dynamics of chromatin structure in structurally preserved nuclei. DNA binding fluorochromes can be used as indirect probes of chromatin structure, because their accessibility to DNA is detennined by the chromatin structure, which enables cytochemical studies of relatively undamaged cells. The aim of the work presented in this thesis was to develop a cytochemical method using the fluorocbromes 7-aminoactinomycin D (7-AAMD) and 4', 6-diamidino-2- phenylindole (DAPI) to study protein-DNA interactions in essentially intact cell nuclei.Both fluorochromes proved to be useful as probes of chromatin structure, particularly to study Iinker histone-chromatin interactions. Suitable protocols for permeabilisation, salt extraction of linker histones, fixation, and staining were established. Permeabilisation with digitonin followed by linker histone extraction at various NaCl concentrations and subsequent fixation in paraformaldehyde was found to enable analysis of linker histone affinity for chromatin in situ. Although the cells became fragile during the salt extraction, this protocol provided individual cells that could be measured by image cytofluorometry.In such analyses, DAPI had several advantages over 7-AAMD. At a concentration of 50 nM DAPI, all of the high-affinity binding sites were occupied, with only minor contributions from binding sites with lower affinity. This approach was used to study linker histone affinity in different types of cells, in cells in different stages of the cell cycle, and during cell differentiation in vivo. Our results indicate differences between the investigated cell types in regard to average Iinker histone affinity. It is generally assumed that increased linker histone affinity is associated with chromatin condensation, but that is not supported by our findings. Human fibroblasts showed substantially lower linker histone affinity for highly condensed metaphase chromatin than for interphase chromatin. Moreover, we found a tendency toward decreasing linker histone affinities in amphibian erytbroblasts that were developing into tenninally differentiated erythrocytes. However, as expected, mature avian erythrocytes showed strongly bound linker histones, probably due to their high arginine content. In conclusion, our results suggest that linker histone affinity is not directly involved in chromatin condensation.