In vitro Assembly and Electron Microscopic Analysis of 30 nm Chromatin Fibers

Genomic DNA in the eukaryotic nucleus is hierarchically packaged by histones into chromatin. The plasticity and dynamics of higher-order chromatin fiber have been widely thought as the key regulators of transcription and other biological processes inherent to DNA. Elucidating how nucleosomal arrays can be folded into higher-order chromatin fibers is essential to understand the dynamics of chromatin structure. Although the structure of nucleosomes, the fundamental repeating unit of chromatin, which comprises 147 base pairs of DNA wrapped in 1.7 superhelical turns around an octamer of histones, has been solved at the atomic resolution, there is still much controversy over the chromatin structure at the higher-order level. Here, we built an in vitro chromatin reconstitution system which adopts histone octamers and arrays of 177 bp and 200 bp repeat of the Widom 601 DNA sequence. Taking advantage of this system, we have obtained highly regular spaced and soluble nucleosome arrays, and folded the arrays into 30 rim chromatin fibers with the existence of linker histone H1 or MgCl2 respectively. Several electron microscopic techniques, including metal shadowing, negative staining and Cryo-EM, have been used to investigate the morphology of the reconstituted 30 nm chromatin fibers. Our results suggest that both histone H1 and divalent Mg2+ can help the formation of 30 nm chromatin fibers, but the resulted chromatin fibers display different topologically architectures. To investigate how the length of linker histone may affect the architecture of chromatin, we measured the diameters of the reconstituted 30 nm chromatin fibers with different nucleosome repeat lengths (NRLs) of 177 and 200 bp and found that these two classes of chromatin fibers present different diameters (P < 0.05).