VISUALIZATION AND ANALYSIS OF CHROMATIN BY SCANNING FORCE MICROSCOPY

The use of the scanning force microscope (SFM) to visualize and analyz e chromatin fiber structures is presented. Protocols to prepare chroma tin fibers for SN imaging of fibers in air and in buffer are first dis cussed. Next, the conditions for acquiring high-quality SFM images suc h as optimal instrumental parameters, appropriate deposition substrate s, and adequate procedures of sample deposition are described. It is s hown that analysis and quantitation of the SFM images support an irreg ular, three-dimensional arrangement of nucleosomes in the native chrom atin fiber. This structure is lost in linker histone-depleted fibers, which show, instead, a beads-on-a-string structure. Molecular modeling of the chromatin fiber structures and computer simulation of the SFM imaging process indicate that the natural variability of the linker le ngth may be the major determinant of the structural irregularity of th e native chromatin fiber. Removal of linker histones (H1/H5) may chang e the amount of DNA wrapped around the histone octamer, which in turn may induce the transition from a three-dimensional irregular helix to an extended beads-on-a-string structure. Studies of trinucleosomes ind icate that both the average successive nucleosome center-to-center dis tance and the average angle between two successive linkers increase up on the removal of linker histone. (C) 1997 Academic Press.